Language：English   Publishing type：Research paper (scientific journal)  

Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression. PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.

DOI： https://doi.org/10.1016/j.bbrc.2021.03.045

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Cytokinesis is initiated by the formation and ingression of the cleavage furrow. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] accumulation followed by RhoA translocation to the cleavage furrow are prerequisites for cytokinesis progression. Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PI(4,5)P₂, regulates PI(4,5)P₂-mediated cytokinesis. We found that PRIP localised to the cleavage furrow during cytokinesis. Moreover, HeLa cells with silenced PRIP displayed abnormal cytokinesis. Importantly, PI(4,5)P₂ accumulation at the cleavage furrow, as well as the localisation of RhoA and phospho-myosin II regulatory light chain to the cleavage furrow, were reduced in PRIP-silenced cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PI(4,5)P₂ levels during early cytokinesis and resulted in cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by co-expression of a PI(4,5)P₂-unbound PRIP mutant. Collectively, our results indicate that PRIP is a component at the cleavage furrow that maintains PI(4,5)P₂ metabolism and regulates RhoA-dependent progression of cytokinesis. Thus, we propose that PRIP regulates phosphoinositide metabolism correctively and mediates normal cytokinesis progression.

DOI： https://doi.org/10.1038/s41598-019-49156-3

Language：English   Publishing type：Research paper (scientific journal)  

Peripheral lipopolysaccharide (LPS) injection induces systemic inflammation through the activation of the inhibitor of nuclear factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which promotes brain dysfunction resulting in conditions including anorexia. LPS-mediated reduction of food intake is associated with activation of NF-κB signaling and phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. We recently reported phospholipase C-related catalytically inactive protein (PRIP) as a new negative regulator of phosphatidylinositol 3-kinase/AKT signaling. AKT regulates the IKK/NF-κB signaling pathway; therefore, this study aimed to investigate the role of PRIP/AKT signaling in LPS-mediated neuroinflammation-induced anorexia. PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice intraperitoneally (ip) administered with LPS exhibited increased anorexia responses compared with wild-type (WT) controls. Although few differences were observed between WT and Prip-KO mice in LPS-elicited plasma pro-inflammatory cytokine elevation, hypothalamic pro-inflammatory cytokines were significantly upregulated in Prip-KO rather than WT mice. Hypothalamic AKT and IKK phosphorylation and IκB degradation were significantly increased in Prip-KO rather than WT mice, indicating further promotion of AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was further phosphorylated in Prip-KO rather than WT mice. Furthermore, suppressor of cytokine signaling 3 (Socs3), a negative feedback regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a candidate molecule in LPS-induced anorexigenic responses, were upregulated in the hypothalamus in Prip-KO rather than WT mice. Pro-inflammatory cytokines were upregulated in hypothalamic microglia isolated from Prip-KO rather than WT mice. Together, these findings indicate that PRIP negatively regulates LPS-induced anorexia caused by pro-inflammatory cytokine expression in the hypothalamus, which is mediated by AKT-activated NF-κB signaling. Importantly, hypothalamic microglia participate in this PRIP-mediated process. Elucidation of PRIP-mediated neuroinflammatory responses may provide novel insights into the pathophysiology of many brain dysfunctions.

Language：English   Publishing type：Research paper (scientific journal)  

Mice with a knockout of phospholipase C (PLC)-related inactive protein type 1 (PRIP1
^-/-
mice) display anxiety-like behavior and altered γ-aminobutyric acid (GABA)
_A
-receptor pharmacology. Here, we examined associations between anxiety and motor-function recovery in PRIP1
^-/-
mice after a spinal cord injury (SCI) induced by a moderate contusion injury at the 10th thoracic level. Uninjured PRIP1
^-/-
mice showed less distance than wild-type (WT) mice in the center 25% in an open field test (OFT), indicating anxiety-like behavior. Anxiety behavior increased in both WT and PRIP1
^-/-
mice after SCI. WT and PRIP1
^-/-
mice were completely paralyzed on day 1 after SCI, but gradually recovered until reaching a plateau at ∼4 weeks. After SCI, the PRIP1
^-/-
mice had significantly greater motor dysfunction than the WT mice. In WT mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and velocity, and with the reaction time in a plantar pressure-sensitivity mechanical test. In PRIP1
^-/-
mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and with the latency to fall in the rotarod test. Six weeks after SCI, ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) expressions were elevated at the lesion epicenter in PRIP1
^-/-
mice, and spinal cord atrophy and demyelination were more severe than in WT mice. The axonal fiber development was also decreased in PRIP1
^-/-
mice, consistent with the poor motor-function recovery after SCI in these mice.

DOI： 10.1089/neu.2017.5492

Language：English   Publishing type：Research paper (scientific journal)  

The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] into PI(3,4,5)P₃ and the subsequent PI(3,4,5)P₃ signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P₂-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P₂ binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P₃ production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P₂ was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P₃ from PI(4,5)P₂ by PI3K, and the suppressor activity of PRIP in PI(4,5)P₂ metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.

DOI： 10.1038/s41598-017-05908-7

Language：English   Publishing type：Research paper (scientific journal)  

The GABA type A receptor (GABA_A-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABA_A-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pre-treatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phos-phorylation of cortical the GABA_A-R β3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABA_A-R β3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.

DOI： 10.1124/jpet.116.239145

Language：English   Publishing type：Research paper (scientific journal)  

Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in ther-moneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.

DOI： 10.1074/jbc.M115.705723

Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrinlabeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA -receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

DOI： 10.1242/bio.20147591

Language：English   Publishing type：Research paper (scientific journal)  

Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP -KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis. Copyright:

DOI： 10.1371/journal.pone.0100559

Language：English   Publishing type：Research paper (scientific journal)  

Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule- associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphatebinding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells. Copyright:

DOI： 10.1371/journal.pone.0098285

Language：English   Publishing type：Research paper (scientific journal)  

Background: An inositol 1,4,5-trisphosphate binding protein, comprising 2 isoforms termed PRIP-1 and PRIP-2, was identified as a novel modulator for GABA_A receptor trafficking. It has been reported that naive PRIP-1 knockout mice have hyperalgesic responses.Findings: To determine the involvement of PRIP in pain sensation, a hind paw withdrawal test was performed before and after partial sciatic nerve ligation (PSNL) in PRIP-1 and PRIP-2 double knockout (DKO) mice. We found that naive DKO mice exhibited normal pain sensitivity. However, DKO mice that underwent PSNL surgery showed increased ipsilateral paw withdrawal threshold. To further investigate the inverse phenotype in PRIP-1 KO and DKO mice, we produced mice with specific siRNA-mediated knockdown of PRIPs in the spinal cord. Consistent with the phenotypes of KO mice, PRIP-1 knockdown mice showed allodynia, while PRIP double knockdown (DKD) mice with PSNL showed decreased pain-related behavior. This indicates that reduced expression of both PRIPs in the spinal cord induces resistance towards a painful sensation. GABA_A receptor subunit expression pattern was similar between PRIP-1 KO and DKO spinal cord, while expression of K⁺-Cl^--cotransporter-2 (KCC2), which controls the balance of neuronal excitation and inhibition, was significantly upregulated in DKO mice. Furthermore, in the DKD PSNL model, an inhibitor-induced KCC2 inhibition exhibited an altered phenotype from painless to painful sensations.Conclusions: Suppressed expression of PRIPs induces an elevated expression of KCC2 in the spinal cord, resulting in inhibition of nociception and amelioration of neuropathic pain in DKO mice.

DOI： 10.1186/1744-8069-9-23

Language：English   Publishing type：Research paper (scientific journal)  

Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.

DOI： 10.1016/j.bbrc.2013.01.119

Language：English   Publishing type：Research paper (scientific journal)  

The GABA_A receptor, a pentamer composed predominantly of α, β, and γ subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABA_A receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The α subunit affects diazepam sensitivity; α1, 2, 3, and 5 subunits assemble with any form of β and the γ2 subunits to produce diazepam-sensitive receptors, whereas α4 or α6/β/γ2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the α6 subunit. The expression of α1/β/γ2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the α6 subunit were increased, the α1/β/γ2 receptors might be replaced with α6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of α6/δ receptors was decreased in cerebellar granule neurons, while that of α6/γ2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (α1/γ2) and increase in diazepam-insensitive (α6/γ2) GABA_A receptors in the cerebellar granule cells.

DOI： 10.1111/j.1471-4159.2010.06754.x

Language：English   Publishing type：Research paper (scientific journal)  

The γ-aminobutyric acid type A (GABA_A) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABA_A receptor insertion. Insulin potentiated the GABA-induced Cl^- current (I_GABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABA_A receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in I_GABA. We also revealed that PRIP recruited active Akt to the GABA_A receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABA_A receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of I_GABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABA_A receptor insertion by recruiting active Akt to the receptor complex.

DOI： 10.1074/jbc.M109.070045

Language：English   Publishing type：Research paper (scientific journal)  

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA_A receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA_A receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA_A receptors through association with receptor β subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA_A receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA_A receptor α/β/γ subunits and PRIP. PRIP was internalized together with GABA_A receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to β subunits. The clathrin heavy chain, μ2 and β2 subunits of AP2 and PRIP-1, were complexed with GABA_A receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-β2/3 GABA_A receptor antibody or by pull-down assay using β subunits of GABA_A receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA_A receptor that requires clathrin and AP2 protein complex.

DOI： 10.1111/j.1471-4159.2006.04399.x

Language：English   Publishing type：Research paper (scientific journal)  

The subunit composition of GABA_A receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABA_A receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA_A receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABA_A receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [³H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [³H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA_A receptors and GABA_A receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA_A receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABA_A receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABA_A receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

DOI： 10.1523/JNEUROSCI.3155-06.2007

Language：English   Publishing type：Research paper (scientific journal)  

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA_A receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA_A receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA_A receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA_A receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABA_A receptor clusters in cultured neurons and with recombinant GABA_A receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA_A receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA_A receptors by mediating the specific association between β subunits of these receptors with protein phosphatases.

DOI： 10.1074/jbc.M603118200

Language：English   Publishing type：Research paper (scientific journal)  

GABA_A receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABA_A receptor phospho-dependent regulation using PRIP-1^-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABA_A receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1^-/- mice. PRIP-1^-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABA_A receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABA_A receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

DOI： 10.1523/JNEUROSCI.1323-04.2004

Language：English   Publishing type：Research paper (scientific journal)  

The protein p130 was isolated from rat brain as an inositol 1, 4, 5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast two-hybrid screening identified GABARAP (GABA_A receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA_A receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABA_A receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl-current by Zn²⁺ or diazepam, both of which act at GABA_A receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA_A receptors, especially in response to the agents acting on a γ2 subunit.

DOI： 10.1093/emboj/21.5.1004

Language：English   Publishing type：Research paper (scientific journal)  

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1α (PP1cα) as a p130-binding protein. The association between p130 and PP1cα was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1cα resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1cα also detected the presence of a complex of p130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca²⁺ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1cα.

DOI： 10.1074/jbc.M009677200

Language：English   Publishing type：Research paper (scientific journal)  

The 130-kDa protein (p130) was isolated as a novel inositol 1,4,5- trisphosphate [Ins(1,4,5)P₃)-binding protein similar to phospholipase C-δ1 (PLC-δ1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525, Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M. (1996) Biochem, J. 313, 319-3251. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-δ1. Trypsin treatment of p130 produced four major polypeptides with molecular misses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were CalCulated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC- δ1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-δ1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-δ1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P₃ but the lack of catalytic activity.

DOI： 10.1046/j.1432-1327.2000.01291.x

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P₃ and shares 38% sequence identity with phospholipase C-δ₁ [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P₃. Deletion of N-terminal residues 116-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [³H]Ins(1,4,5)P₃. We also found that Ins(1,4,5,6)P₄ was as efficacious as Ins(1,4,5)P₃ in displacing [³H]Ins(1,4,5)P₃, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [³H]Ins(1,4,5,6)P₄ with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.

DOI： 10.1042/bj3180561

Language：English   Publishing type：Research paper (scientific journal)  

We have reported that two inositol 1,4,5-trisphosphate binding proteins, with molecular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the δ₁-isoenzyme of phospholipase C (PLC-δ₁) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we describe the isolation of the full-length cDNA for the 130 kDa Ins(1,4,5)P₃ binding protein, which encodes 1096 amino acids. The predicted sequence of the 130 kDa protein had 38.2% homology to that of PLC-δ₁. Three known domains of PLC-δ₁ (pleckstrin homology and putative catalytic X and Y domains) were located at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respectively. However, the protein showed no PLC activity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfection in COS-1 cells bound Ins(1,4,5)P₃ in the same way as the molecule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P₃ binding protein homologous to PLC-δ₁, but with no catalytic activity. The functional significance of the 130 kDa protein is discussed.

DOI： 10.1042/bj3130319

Language：English   Publishing type：Research paper (scientific journal)  

Rat brain membrane fractions obtained using Triton X-100 were applied to a D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P₃] immobilized column, followed by gel filtration and anion-exchange chromatography. Two proteins with molecular masses of 130 and 85 kDa, as assessed by SDS-polyacrylamide gel electrophoresis, were purified to apparent homogeneity as D-[³H] Ins(1,4,5)P₃-binding proteins with no D-Ins(1,4,5)P₃-metabolizing activity. Partial amino acid sequence determinations of these proteins revealed that the 130 kDa protein appears to be a new D-Ins(1,4,5)P₃-binding protein and the 85 kDa protein is δ₁-isozyme of phospholipase C. We have previously purified 130 and 85 kDa proteins, as D-[³H]Ins(1, 4, 5)P₃-binding proteins, from rat brain cytosol fraction. Antibodies against the 130 kDa protein from the cytosol cross-reacted with the membrane 130 kDa protein purified in this study, suggesting that the membrane 130 kDa protein is likely to be the same as the protein from the cytosol fraction. The inhibition of D-[³H]Ins(1,4,5)P₃ binding by D-isomers of inositol phosphates available clarified that the 130 kDa protein has a similar affinity for D-Ins(1,4,5,6)P₄ to that for D-Ins(1,4,5)P₃, while the 85 kDa protein is specific to D-Ins(1,4,5)P₃.

DOI： 10.1093/oxfordjournals.jbchem.a124447

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In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P₃ affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P₃ affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P₃-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P₃- binding proteins but with no Ins(1,4,5)P₃ metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the δ-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P₃-recognizing proteins hitherto examined. Ins(1,4,5)P₃ at concentrations greater than 1 μM strongly inhibited 85-kDa phospholipase Cδ activity, without changing its dependence on the concentrations of free Ca²⁺ and H⁺. Among inositol phosphates examined, Ins(3,4,5,6)P₄ inhibited the binding of [³H]Ins(1,4,5)P₃ to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P₃. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P₃-binding proteins in the rat brain, one of which is the δ isozyme of phospholipase C.

Publisher：Current Oral Health Reports  

Purpose of Review: There is an increasing interest in the use of dietary supplements and natural products for the treatment and prevention of obesity and periodontitis. Epicatechin (EC), a flavonoid abundant in cocoa, green tea, and red wine, possesses excellent anti-inflammatory and antioxidant properties. This review summarizes preclinical and clinical findings on the effects of EC on obesity and periodontitis. Recent Findings: Obesity not only leads to weight gain but also causes chronic low-level inflammation in adipose tissue. Periodontitis is an infectious disease caused by periodontal pathogens that chronically destroy periodontal tissues. Chronic inflammation leads to lifestyle-related diseases, such as diabetes, hypertension, and arteriosclerosis. Current literature suggests that EC can reduce inflammatory responses and improve obesity-related complications and periodontal disease status. Summary: These findings highlight the potential use of EC in the management of obesity and periodontitis. However, direct evidence for the use of EC supplementation in the treatment of obesity and periodontitis in humans remains limited. Further well-designed controlled studies are required to confirm its efficacy.

DOI： 10.1007/s40496-024-00390-3

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Publisher：Journal of Functional Foods  

Periodontitis is a prevalent chronic inflammatory disease requiring the development of drug-based therapies. Epicatechin is a major polyphenol in cocoa extract and possesses numerous pharmacological properties. This study aimed to investigate the suppressive effect of epicatechin on C-C motif chemokine ligand 19 (CCL19)-mediated periodontitis using a mouse model. CCL19 expression was high in mouse gingiva with ligature-induced periodontitis. CCL19 expression was increased by lipopolysaccharide stimulation in murine gingival fibroblasts ESK-1 cells, and CCL19 induced the production of tumor necrosis factor alpha and interleukin-1 beta in mouse macrophage-like RAW264.7 cells. Importantly, lipopolysaccharide-enhanced CCL19 expression in ESK-1 cells was significantly suppressed by pretreatment with epicatechin. In addition, epicatechin administration significantly reduced alveolar bone resorption, and expression of Ccl19 and proinflammatory cytokines in the inflamed gingiva in high-fat diet-fed mice. These results demonstrate that epicatechin protects against periodontitis by reducing CCL19 levels, which may be a promising countermeasure for periodontitis in clinical practice.

DOI： 10.1016/j.jff.2024.106512

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

The skeletal muscle is a pivotal organ involved in the regulation of both energy metabolism and exercise capacity. There is no doubt that exercise contributes to a healthy life through the consumption of excessive energy or the release of myokines. Skeletal muscles exhibit insulin sensitivity and can rapidly uptake blood glucose. In addition, they can undergo non-shivering thermogenesis through actions of both the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and small peptide, sarcolipin, resulting in systemic energy metabolism. Accordingly, the maintenance of skeletal muscles is important for both metabolism and exercise. Prolyl isomerase Pin1 is an enzyme that converts the cis-trans form of proline residues and controls substrate function. We have previously reported that Pin1 plays important roles in insulin release, thermogenesis, and lipolysis. However, the roles of Pin1 in skeletal muscles remains unknown. To clarify this issue, we generated skeletal muscle-specific Pin1 knockout mice. Pin1 deficiency had no effects on muscle weights, morphology and ratio of fiber types. However, they showed exacerbated obesity or insulin resistance when fed with a high-fat diet. They also showed a lower ability to exercise than wild type mice did. We also found that Pin1 interacted with SERCA and elevated its activity, resulting in the upregulation of oxygen consumption. Overall, our study reveals that Pin1 in skeletal muscles contributes to both systemic energy metabolism and exercise capacity.

DOI： 10.1016/j.bbrc.2024.150001

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DOI： 10.1177/20416695241254016

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DOI： 10.1177/20416695241249945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEBS Letters  

GPRC5C is an orphan G protein-coupled receptor (GPCR) that belongs to the class C GPCR family. Although GPRC5C is expressed in various organs, its function and ligand are still undetermined. We found that GPRC5C is expressed in mouse taste cells, enterocytes, and pancreatic α-cells. In functional imaging assays, HEK293 cells heterologously expressing GPRC5C and the chimeric G protein α subunit Gα16-gust44 showed robust intracellular Ca2+ increases in response to monosaccharides, disaccharides, and a sugar alcohol, but not an artificial sweetener or sweet-tasting amino acid. Notably, Ca2+ increases occurred after washout, not during stimulation. Our findings suggest that GPRC5C has receptor properties which lead to novel ‘off’ responses to saccharide detachment and may work as an internal or external chemosensor specifically tuned to natural sugars.

DOI： 10.1002/1873-3468.14695

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Language：Japanese   Publisher：The Japanese Pharmacological Society  

DOI： 10.1254/fpj.23016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

The expression profiles of exosomal microRNAs (miRNAs) are regulated by the microenvironment, and appropriate priming with mesenchymal stem cells (MSCs) is one of the strategies to enhance the paracrine potency of MSCs. Our previous work demonstrated that exosomes from tumor necrosis factor (TNF)-α-primed human gingiva-derived MSCs (GMSCs) could be a therapeutic tool against periodontitis, and that TNFα-inducible exosomal miR-1260b is essential for the inhibition of alveolar bone loss. However, the precise molecular mechanism underlying miR-1260b-mediated inhibition of osteoclastogenesis is not yet fully understood. Here, we found that the activating transcription factor (ATF)-6β, a novel miR-1260b-targeting gene, is critical for the regulation of osteoclastogenesis under endoplasmic reticulum (ER) stress. An experimental periodontal mouse model demonstrated that induction of ER stress was accompanied by enhanced ATF6β expression, and local administration of miR-1260b and ATF6β siRNA using polyethylenimine nanoparticles (PEI-NPs) significantly suppressed the periodontal bone resorption. In periodontal ligament (PDL) cells, the ER stress inducer, tunicamycin, enhanced the expression of the receptor activator of NF-κB ligand (RANKL), while miR-1260b-mediated downregulation of ATF6β caused RANKL inhibition. Furthermore, the secretome from miR-1260b/ATF6β-axis-activated PDL cells inhibited osteoclastogenesis in human CD14^+ peripheral blood-derived monocytes. These results indicate that the miR-1260b/ATF6β axis mediates the regulation of ER stress, which may be used as a novel therapeutic strategy to treat periodontal disease.

DOI： 10.3389/fcell.2022.1061216

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DOI： 10.3389/icell.2022.1061216

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ejphar.2022.175273.

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Endocrinology  

Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.

DOI： 10.1530/JOE-21-0424

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100-1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

DOI： 10.1016/j.neulet.2022.136467

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The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100-1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

DOI： https://doi.org/10.1016/j.neulet.2022.136467

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cell Science  

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.

DOI： 10.1242/jcs.258584

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Animals suffering from uncontrollable stress sometimes show low effort to escape stress (learned helplessness). Changes in serotonin (5-hydroxytryptamine) signalling are thought to underlie this behaviour. Although the release of 5-hydroxytryptamine is triggered by the action potential firing of dorsal raphe nuclei 5-hydroxytryptamine neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-hydroxytryptamine neurons in naïve rats, learned helplessness rats and rats resistant to inescapable stress (non-learned helplessness). Five-week-old male Sprague Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as learned helplessness or non-learned helplessness. Activity-dependent 5-hydroxytryptamine release induced by the administration of high-potassium solution was slower in free-moving learned helplessness rats. Subthreshold electrophysiological properties of 5-hydroxytryptamine neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in learned helplessness rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 μM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels produced afterhyperpolarization. Additionally, dendrotoxin-I, a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that dendrotoxin-I significantly enhanced the spike firing of 5-hydroxytryptamine neurons in learned helplessness rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of dendrotoxin-I. These results suggest that the upregulation of dendrotoxin-I-sensitive Kv1 channels underlies the firing attenuation of 5-hydroxytryptamine neurons in learned helplessness rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and non-learned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels. The dendrotoxin-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-hydroxytryptamine neurons.

DOI： https://doi.org/10.1093/braincomms/fcab285

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1016/j.jbc.2021.100274

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In the spinal cord, γ-aminobutyric acid (GABA) interneurons play an essential role in antinociception. However, not all actions of GABA favor antinociception at the supraspinal level. We previously reported that gabaculine, which increases endogenous GABA in the synaptic clefts, induces loss of the righting reflex (LORR) that is one indicator of hypnosis, but not immobility in response to noxious stimulus. A slow pain is transmitted to the spinal cord via C fibers and evokes substance P (SP) release from their terminals. However, the antinociceptive effects of gabaculine are still unknown. Our study examined whether the analgesic effects of the opioid morphine or the α2-adrenoceptor agonist dexmedetomidine, whose actions are mediated through facilitation of the descending analgesic pathway, are affected by gabaculine-induced LORR. We also explored the effects of GABA receptor agonists on SP release from cultured dorsal root ganglion (DRG) neurons. All drugs were administered systemically to mice. To assess antinociception, loss of nociceptive response (analgesia) and immobility were evaluated. DRG cells were dissected from rats. Gabaculine produced no analgesia. Either morphine or dexmedetomidine in combination with gabaculine induced immobility; however, the doses of each drug required to induce immobility were much higher than those required to induce analgesia. Capsaicin significantly increased SP release from DRG cells, but a high concentration (1 mM) of the GABA receptor agonist muscimol, propofol, gaboxadol, or baclofen did not inhibit the capsaicin-induced SP release, suggesting that their antinociceptive effects were not through this mechanism. Thus, the gabaculine-induced LORR may inhibit the descending analgesic pathway.

DOI： 10.1016/j.pbb.2020.173034.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Periodontal disease (PD) is a risk factor for systemic diseases, including neurodegenerative diseases. The role of the local and systemic inflammation induced by PD in neuroinflammation currently remains unclear. The present study investigated the involvement of periodontal inflammation in neuroinflammation and blood-brain barrier (BBB) disruption. Methods: To induce PD in mice (c57/BL6), a ligature was placed around the second maxillary molar. Periodontal, systemic, and neuroinflammation were assessed based on the inflammatory cytokine mRNA or protein levels using qPCR and ELISA. The BBB permeability was evaluated by the mRNA levels and protein levels of tight junction-related proteins in the hippocampus using qPCR and immunofluorescence. Dextran tracing in the hippocampus was also conducted to examine the role of periodontal inflammation in BBB disruption. Results: The TNF-α, IL-1β, and IL-6 levels markedly increased in gingival tissue 1 week after ligation. The IL-6 serum levels were also increased by ligature-induced PD. In the hippocampus, the IL-1β mRNA expression levels were significantly increased by ligature-induced PD through serum IL-6. The ligature-induced PD decreased the claudin 5 expression levels in the hippocampus, and the neutralization of IL-6 restored its levels. The extravascular 3-kDa dextran levels were increased by ligature-induced PD. Conclusions: These results suggest that the periodontal inflammation-induced expression of IL-6 is related to neuroinflammation and BBB disruption in the hippocampus, ultimately leading to cognitive impairment. Periodontal therapy may protect against neurodegenerative diseases.

DOI： 10.3390/brainsci10100679.

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MicroRNA (miRNA) plays an important role in diverse cellular biological processes such as inflammatory response, differentiation and proliferation, and carcinogenesis. miR-146a has been suggested as a negative regulator of the inflammatory reaction. Although, it has been reported as expressed in inflamed adipose and periodontal tissues, however, miR-146a's inhibitory effects against inflammatory response in both the tissues, are not well understood. Therefore, in this study, the inhibitory effects of miR-146a on both adipose and periodontal inflammation, was investigated. In vitro study has revealed that miR-146a transfection into either adipocytes or gingival fibroblasts, has resulted in a reduced cytokine gene expression, observed on co-culturing the cells with macrophages in the presence of lipopolysaccharides (LPS), in comparison to the control miRNA transfected. Similarly, miR-146a transfection into macrophages resulted in a reduced expression of TNF-α gene and protein in response to LPS stimulation. In vivo study revealed that a continuous intravenous miR-146a administration into mice via tail vein, protected the mice from developing high-fat diet-induced obesity and the inflammatory cytokine gene expression was down-regulated in both adipose and periodontal tissues. miR-146a appeared to be induced by macrophage-derived inflammatory signals such as TNF-α by negative feed-back mechanism, and it suppressed inflammatory reaction in both adipose and periodontal tissues. Therefore, miR-146a could be suggested as a potential therapeutic molecule and as a common inflammatory regulator for both obesity-induced diabetes and related periodontal diseases.

DOI： https://doi.org/10.1016/j.bbrep.2020.100757

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Purpose: The general anesthetics propofol and etomidate mainly exert their anesthetic actions via GABA A receptor (GABA_A-R). The GABA_A-R activity is influenced by phospholipase C-related inactive protein type-1 (PRIP-1), which is related to trafficking and subcellular localization of GABA_A-R. PRIP-1 deficiency attenuates the behavioral reactions to propofol but not etomidate. However, the effect of these anesthetics and of PRIP-1 deficiency on brain activity of CNS are still unclear. In this study, we examined the effects of propofol and etomidate on the electroencephalogram (EEG). Methods: The cortical EEG activity was recorded in wild-type (WT) and PRIP-1 knockout (PRIP-1 KO) mice. All recorded EEG data were offline analyzed, and the power spectral density and 95% spectral edge frequency of EEG signals were compared between genotypes before and after injections of anesthetics. Results: PRIP-1 deficiency induced increases in EEG absolute powers, but did not markedly change the relative spectral powers during waking and sleep states in the absence of anesthesia. Propofol administration induced increases in low-frequency relative EEG activity and decreases in SEF95 values in WT but not in PRIP-1 KO mice. Following etomidate injection, low-frequency EEG power was increased in both genotype groups. At high frequency, the relative power in PRIP-1 KO mice was smaller than that in WT mice. Conclusions: The lack of PRIP-1 disrupted the EEG power distribution, but did not affect the depth of anesthesia after etomidate administration. Our analyses suggest that PRIP-1 is differentially involved in anesthetic EEG activity with the regulation of GABA_A-R activity.

DOI： 10.1007/s00540-019-02663-z

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Background: The chemokine receptor CCR7, expressed on various immune cells, is associated with cell migration and lympho-node homing. Mice lacking Ccr7 are protected from diet-induced obesity and subsequent insulin resistance. We evaluated the mechanism underlying these protective effects from the standpoint of energy expenditure. Methods: Wild-type and Ccr7 null mice were fed a high-fat diet, and the regulation of energy metabolism and energy metabolism-related molecules, e.g., Ucp1, Cidea, and Pgc1α, were evaluated. Results: Food intake did not differ between groups. O₂ consumption and CO₂ production were higher in Ccr7 null mice than in wild-type mice, despite a similar respiratory quotient and glucose and lipid utilization, suggesting that energy expenditure increased in Ccr7 null mice via enhanced metabolism. In white adipose tissues of Ccr7 null mice, Prdm16, Cd137, Tmem26, Th, and Tbx1 expression increased. Similarly, in brown adipose tissues of Ccr7 null mice, Dio2, Pgc1α, Cidea, Sirt1, and Adiponectin expression increased. In both white and brown adipose tissues, Ucp1 gene and protein expression levels were higher in null mice than in wild-type mice. Conclusions: In Ccr7 null mice, browning of white adipocytes as well as the activation of brown adipocytes cause enhanced energy metabolism, resulting in protection against diet-induced obesity.

DOI： 10.1186/s12986-019-0372-5

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Background: Overweight and obesity are defined as excessive or abnormal fat accumulation in adipose tissues, and increase the risk of morbidity in many diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, and stroke, through pathophysiological mechanisms. There is strong evidence that weight loss reduces the risk of metabolic syndrome by limiting blood pressure and improving the levels of serum triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol. To date, several attempts have been made to develop effective anti-obesity medication or weight-loss drugs; however, satisfactory drugs for clinical use have not yet been developed. Therefore, elucidation of the molecular mechanisms driving fat metabolism (adipogenesis and lipolysis) represents the first step in developing clinically useful drugs and/or therapeutic treatments to control obesity. Highlight: In our previous study on intracellular signaling of phospholipase C-related catalytically inactive protein (PRIP), we generated and analyzed Prip-double knockout (Prip-DKO) mice. Prip-DKO mice showed tolerance against insulin resistance and a lean phenotype with low fat mass. Here, we therefore reviewed the involvement of PRIP in fat metabolism and energy expenditure. We conclude that PRIP, a protein phosphatase-binding protein, can modulate fat metabolism via phosphoregulation of adipose lipolysis-related molecules, and regulates non-shivering heat generation in brown adipocytes. Conclusion: We propose PRIP as a new therapeutic target for controlling obesity or developing novel anti-obesity drugs.

DOI： 10.1016/j.job.2019.04.002

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Non-shivering thermogenesis in adipocytes provides defense against low temperatures and obesity development, but the underlying regulatory mechanism remains to be fully clarified. Based on both markedly increased Pin1 expression in states of excess nutrition and resistance to obesity development in Pin1 null mice, we speculated that adipocyte Pin1 may play a role in thermogenic programs. Adipose-specific Pin1 knockout (adPin1 KO) mice showed enhanced transcription of thermogenic genes and tolerance to hypothermia when exposed to cold. In addition, adPin1 KO mice were resistant to high-fat diet-induced obesity and glucose intolerance. A series of experiments revealed that Pin1 binds to PRDM16 and thereby promotes its degradation through the ubiquitin-proteasome system. Consistent with these results, Pin1 deletion in differentiated adipocytes showed enhancement of thermogenic programs in response to the β3 agonist CL316243 through the upregulation of PRDM16 proteins. These observations indicate that Pin1 is a negative regulator of non-shivering thermogenesis.

DOI： 10.1016/j.celrep.2019.02.066

Language：English   Publishing type：Research paper (scientific journal)  

Patients with major depressive disorder have elevated peripheral inflammation; the degree of this increase correlates with the severity of the disorder. Chronic psychological stress increases pro-inflammatory cytokines and promotes microglial activation, leading to stress vulnerability. Epigenetics, including DNA methylation and histone modification, are also related to the pathophysiology of major depressive disorder. Sodium butyrate (SB), a histone deacetylase inhibitor, exerts an antidepressant effect by altering gene expression in the hippocampus. In this study, we investigated whether lipopolysaccharide (LPS)-induced depressive-like behaviors in mice are affected by the repeated treatment with SB. Intraperitoneal injection of LPS (5 mg/kg) induced cytokines and ionized calcium-binding adaptor molecule 1(Iba1), a marker of microglial activation, in the hippocampus. It also increased the immobility time in a forced swim test, without changing locomotion. Repeated treatment with SB reduced LPS-induced alterations. These findings suggested that epigenetic regulation exist in hippocampal microglial activation, and is involved in depressive-like behaviors associated with neuro-inflammation. Further, using cDNA microarray analyses, we examined whether LPS and SB treatment affected the microglial gene profiles. Our results indicated 64 overlapping genes, between LPS-increased genes and SB-decreased genes. Among these genes, EF hand calcium binding domain 1 was a particularly distinct candidate gene. Altogether, our findings indicated that microglial activation mediated through epigenetic regulation may be involved in depressive-like behaviors. In addition, we demonstrated the effect of SB on gene information in hippocampal microglia under neuroinflammatory conditions.

DOI： 10.1016/j.brainres.2017.12.004

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Purpose: The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABA
_A
receptor signaling. Methods: PRIP regulates the intracellular trafficking of β subunit-containing GABA
_A
receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via β subunit-containing GABA
_A
receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. Results: The cell surface expression of GABA
_A
receptor β3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABA
_A
receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. Conclusion: PRIP regulates the cell surface expression of the GABA
_A
receptor β3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABA
_A
receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.

DOI： 10.1007/s00540-017-2350-2

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Objectives We have previously demonstrated that phospholipase C-related catalytically inactive protein (PRIP) is involved in fat metabolism and energy consumption. However, whether PRIP participates in body energy metabolism in vivo remains to be determined. Therefore, we examined whether PRIP deficiency affects whole-body energy homeostasis, which is modulated by non-shivering thermogenesis in brown adipose tissue, using a cold exposure animal model. Methods Fasting plasma triacylglycerol levels were measured to evaluate fat metabolism in wild-type and Prip-KO mice. In addition, a glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. To determine changes in energy consumption, mice were exposed to a cold environment for 7 days, and expression of uncoupling protein 1 (UCP1) in brown adipose tissue was analyzed via western blotting. Results Fasting plasma levels of triacylglycerols were significantly higher in Prip-KO mice than in wild-type mice. However, Prip-KO mice showed a healthy phenotype based on GTT and ITT. UCP1 expression was significantly upregulated in the brown and white adipose tissues of Prip-KO mice exposed to cold conditions. Conclusion Prip-KO mice exhibit greater ability to consume lipids as an energy source, indicating that PRIP modulates of systemic energy expenditure. Our findings provide increased understanding of PRIP-mediated non-shivering thermogenic mechanisms and offers important insights for the treatment and control of obesity.

DOI： 10.1016/j.job.2017.04.001

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Background and aims Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. Methods and results DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (C–C motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. Conclusions EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.

DOI： 10.1016/j.numecd.2016.11.008

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Obesity is defined as abnormal or excessive fat accumulation. Chronic inflammation in fat influences the development of obesity-related diseases. Many reports state that obesity increases the risk of morbidity in many diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, stroke, sleep apnea, and breast, prostate and colon cancers, leading to increased mortality. Obesity is also associated with chronic neuropathologic conditions such as depression and Alzheimer's disease. However, there is strong evidence that weight loss reduces these risks, by limiting blood pressure and improving levels of serum triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol. Prevention and control of obesity is complex, and requires a multifaceted approach. The elucidation of molecular mechanisms driving fat metabolism (adipogenesis and lipolysis) aims at developing clinical treatments to control obesity. We recently reported a new regulatory mechanism in fat metabolism: a protein phosphatase binding protein, phospholipase C-related catalytically inactive protein (PRIP), regulates lipolysis in white adipocytes and heat production in brown adipocytes via phosphoregulation. Deficiency of PRIP in mice led to reduced fat accumulation and increased energy expenditure, resulting in a lean phenotype. Here, we evaluate PRIP as a new therapeutic target for the control of obesity.

DOI： 10.1016/j.jdsr.2016.06.001

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DOI： 10.1254/fpj.146.93

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We previously demonstrated that the deletion of phospholipase C-related catalytically inactive protein-1/2 (PRIP-1/2) enhances the desensitization of GABA_A receptors (GABA_ARs), while it facilitates their resensitization at the offset of GABA puff, causing a hump-like tail current (tail-I) in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex. In the present study, we investigated whether inhibitory synaptic transmission in L3 PCs in the barrel cortex is altered in the PRIP-1/2 double-knockout (PRIP-DKO) mice, and if so, how the interaction between excitation and inhibition is subsequently modified. PRIP-1/2 deletion resulted in the prolongation of the decay phase of inhibitory postsynaptic currents/potentials (IPSCs/IPSPs) in L3 PCs evoked by stimulation of L3, leaving the overall features of miniature IPSCs unchanged. An optical imaging revealed that the spatiotemporal profile of a horizontal excitation spread across columns in L2/3 caused by L4 stimulation in the barrel cortex was more restricted in PRIP-DKO mice compared to the wild type, while those obtained in the presence of bicuculline were almost identical between the two genotypes. These findings suggest that PRIP-1/2 deletion enhances the lateral inhibition by prolonging inhibitory synaptic actions to limit the intercolumnar integration in the barrel cortex. Considering the present findings together with our previous study including a mathematical simulation, the prolongation of inhibitory synaptic actions is likely to result from an enhancement of desensitization followed by an enhanced resensitization in GABA_ARs.

DOI： 10.1007/s00424-014-1592-1

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Rationale: The selective N-methyl-D-aspartate (NMDA) channel blocker MK-801 is known to induce no loss of the righting reflex (LORR) and to stimulate catecholaminergic (CAergic) neurons in rodents, playing a crucial role in arousal. Objectives: We examined whether MK-801 in combination with CA receptor ligands, which inhibit CAergic neuronal activities, could induce anesthesia including LORR. Methods: All drugs were administered systemically to mice. To assess anesthesia, three different behaviors were used: loss of nociceptive response (analgesia in the free-moving state without LORR), LORR, and loss of movement in response to noxious stimulation (immobility under LORR). Results: A very large dose of MK-801 (50 mg/kg) induced neither analgesia nor LORR. In contrast, MK-801 in combination with a small dose of the dopamine (DA) receptor antagonist haloperidol (0.2 mg/kg) dose-dependently produced LORR with a 50 % effective dose (ED₅₀) of 1.6 (0.9-3.0; 95 % confidence limit) mg/kg, but not immobility. The α₂-adrenoceptor agonist dexmedetomidine induced not only analgesia, but also immobility in animals treated with MK-801 (5 mg/kg) plus haloperidol (0.2 mg/kg), which then lost their righting reflex. The ED₅₀ value of 0.26 (0.10-0.66) mg/kg (various doses of dexmedetomidine plus a fixed dose of MK-801 and haloperidol) for immobility was approximately three-fold larger than that of 0.09 (0.03-0.23) mg/kg (dexmedetomidine plus vehicle saline) for analgesia. This may occur, as LORR induced by MK-801 plus haloperidol inhibits the pain suppression system. The other ligands had little or no effect. Conclusions: The DAergic stimulant actions of MK-801 may mask its LORR effects by NMDA channel blockade.

DOI： 10.1007/s00213-014-3634-y

Language：English   Publishing type：Research paper (scientific journal)  

Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients. Copyright:

DOI： 10.1371/journal.pone.0091746

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Phospholipase C-related catalytically inactive proteins (PRIP-1/2) are previously reported to be involved in the membrane trafficking of GABA_A receptor (GABA_AR) and the regulation of intracellular Ca²⁺ stores. GABA_AR-mediated currents can be regulated by the intracellular Ca²⁺. However, in PRIP-1/2 double-knockout (PRIP-DKO) mice, it remains unclear whether the kinetic properties of GABA_ARs are modulated by the altered regulation of intracellular Ca²⁺ stores. Here, we investigated whether GABA_AR currents (I_GABA) evoked by GABA puff in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex are altered in PRIP-DKO mice. The deletion of PRIP-1/2 enhanced the desensitization of I_GABA but induced a hump-like tail current (tail-I) at the GABA puff offset. I_GABA and the hump-like tail-I were suppressed by GABA_AR antagonists. The enhanced desensitization of I_GABA and the hump-like tail-I in PRIP-DKO PCs were mediated by increases in the intracellular Ca²⁺ concentration and were largely abolished by a calcineurin inhibitor and ruthenium red. Calcium imaging revealed that Ca²⁺-induced Ca²⁺ release (CICR) and subsequent store-operated Ca²⁺ entry (SOCE) are more potent in PRIP-DKO PCs than in wild-type PCs. A mathematical model revealed that a slowdown of GABA-unbinding rate and an acceleration of fast desensitization rate by enhancing its GABA concentration dependency are involved in the generation of hump-like tail-Is. These results suggest that in L3 PCs of the barrel cortex in PRIP-DKO mice, the increased calcineurin activity due to the potentiated CICR and SOCE enhances the desensitization of GABA_ARs and slows the GABA-unbinding rate, resulting in their unusual resensitization following removal of GABA.

DOI： 10.1007/s00424-014-1511-5

Language：English   Publishing type：Research paper (scientific journal)  

Background: Recent studies have revealed the antinociceptive effects of glycine transporter (GlyT) inhibitors in neuropathic pain models such as sciatic nerve-injured and diabetic animals. Bone cancer can cause the most severe pain according to complex mechanisms in which a neuropathic element is included. Bone cancer modifies the analgesic action of opioids and limits their effectiveness, and thus novel medicament for bone cancer pain is desired.
Methods: For the femur bone cancer model, NCTC 2472 tumor cells were injected into the medullary cavity of the distal femur of C3H/HeN mice. Effects of GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitors, ORG 25935, and knockdown of the expression of spinal GlyTs protein by GlyTs siRNA on pain-like behaviors, such as allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality, were examined in the femur bone cancer model mice. Effects of morphine in combination with GlyT inhibitor were examined.
Results: GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitor ORG 25935 by IV or oral administration or knockdown of the expression of spinal GlyTs protein improved painlike behaviors at 11 days after tumor transplantation. The pain-relief activity was potent and long lasting. Morphine at a dose with no analgesic activity combined with ORG 25543 further promoted the ORG 25543-induced pain-relief activity. Injection of ORG 25543 on the second day after tumor implantation caused 3 phases of pain responses; pain-like behaviors were initially accelerated (at 2-4 days) and subsequently almost disappeared (5-7 days) and then reappeared. Intrathecal injection of strychnine 1 day after injection of ORG 25543 transiently antagonized the pain-relief activity of ORG 25543. In control mice, strychnine improved pain-like behaviors 4 days after tumor implantation and aggravated the behaviors between 4 and 5 days. The evidence suggests that the different mechanisms are phase-dependently involved.
Conclusions: GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.

DOI： 10.1213/ANE.0000000000000388

Language：English   Publishing type：Research paper (scientific journal)  

Background: Platelet-activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF-R) antagonists have varied anti-allodynia effects by route of administration and nerve injury models in rats. Methods: The present study elucidated the effectiveness of PAF antagonists against neuropathic pain in four different models of peripheral nerve injury and provided insights into the mode of anti-allodynia action. Results: PAF antagonists, TCV-309, BN 50739 and WEB 2086 by intravenous (i.v.) and oral administration have potent and long-lasting anti-allodynia action in mice neuropathic pain models. Treatment with PAF antagonists before surgery delayed the initiation of allodynia until the effects of these treatments were abolished. Intrathecal (i.t.) injection of the PAF antagonists and siRNA against PAF receptor ameliorated allodynia. I.t. injection of the glycine receptor (GlyR)α3 siRNA reduced the anti-allodynia effect of PAF antagonists. This evidence suggests that the anti-allodynia effect of PAF antagonists is at least in part mediated by spinal relief of PAF-induced dysfunction of GlyRα3. An analysis of the mode of anti-allodynia action of TCV-309 in vivo revealed a competitive action against PAF shortly after the injection of TCV-309, converting to a non-competitive action later. Conclusions: The present results revealed the effectiveness in anti-allodynia of PAF antagonists in different nerve injury models, and the unique mode of action; long-lasting anti-allodynia effects mediated by spinal GlyRa3 with a competitive manner at the initial stage and the following non-competitive manner of inhibition.

DOI： 10.1002/j.1532-2149.2013.00289.x

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PRIP, phospholipase C (PLC)-related but catalytically inactive protein is a protein with a domain organization similar to PLC-δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatase 1 and 2A (PP1c and PP2Ac), depending on the phosphorylation of PRIP. We also found that Akt was precipitated along with PRIP by anti-PRIP antibody from neuronal cells. In this article, we summarize our current reach regarding the interaction of PRIP with Akt and protein phosphatases, in relation to the cellular phospho-regulations. PP1 and PP2A are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled-down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but in close proximity. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the reduced binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the increased binding of PP2A in invitro experiments. This binding regulation of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling.

DOI： 10.1016/j.jbior.2013.07.001

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Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABA type A receptor (GABA _A-R) β-subunit and several other proteins involved in the trafficking of GABA _A-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse showed an epileptic phenotype, confirmed by electroencephalogram. These ictal seizures were completely suppressed by diazepam (DZP), but the interictal discharges could not be abolished. We studied the electrophysiological properties of GABAergic transmission in hippocampal CA1 pyramidal neurons, using a slice patch-clamp technique. There was no difference in the effect of up to 1 μM DZP on the amplitude and frequency of miniature inhibitory postsynaptic currents between PRIP-1 knockout neurons versus wild-type neurons. In contrast, the amplitude of the tonic GABA current in PRIP-1 knockout neurons was markedly reduced compared with that in wild-type neurons. Consequently, the effect of DZP on PRIP-1 knockout mice was reduced. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 knockout mice.

DOI： 10.1124/jpet.111.182386

Language：English   Publishing type：Research paper (scientific journal)  

Phospholipase C-related inactive protein (PRIP) plays important roles in trafficking to the plasma membrane of GABA_A receptor, which is involved in the dominant inhibitory neurotransmission in the spinal cord and plays an important role in nociceptive transmission. However, the role of PRIP in pain sensation remains unknown. In this study, we investigated the phenotypes of pain behaviors in PRIP type 1 knockout (PRIP-1 ^-/- ) mice. The mutant mice showed hyperalgesic responses in the second phase of the formalin test and the von Frey test as compared with those in wild-type mice. In situ hybridization studies of GABA_A receptors revealed significantly decreased expression of γ2 subunit mRNA in the dorsal and ventral horns of the spinal cord in PRIP-1 ^-/- mice, but no difference in α1 subunit mRNA expression. β2 subunit mRNA expression was significantly higher in PRIP-1 ^-/- mice than in wild-type mice in all areas of the spinal cord. On the other hand, the slow decay time constant for the spontaneous inhibitory current was significantly increased by treatment with diazepam in wild-type mice, but not in PRIP-1 ^-/- mice. These results suggest that PRIP-1 ^-/- mice exhibit the changes of the function and subunits expression of GABA_A receptor in the spinal cord, which may be responsible for abnormal pain sensation in these mice.

DOI： 10.1186/1744-8069-7-79

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PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.

DOI： 10.1074/jbc.M111.235903

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Potentiation of inhibitory γ-aminobutyric acid subtype A (GABA_A) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABA_A receptors containing the γ2 subunit on the neuronal cell surface. Immunohistochemistry for GABA_A receptor subunits demonstrated reduced expression of γ2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the α1 and β3 subunits even though there were no apparent differences in the immunoreactivities for the β2 subunits between wild-type and DKO mice. The tail-clamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07 ± 0.01% versus 1.36 ± 0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABA_A receptor γ2 subunit affects the composition and function of spinal GABA_A receptors and potentiates the immobilizing action of isoflurane.

DOI： 10.1016/j.neulet.2010.01.031

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Orofacial movements are regulated by D₁-like dopamine receptors interacting with additional mechanisms. Phospholipase C-related catalytically inactive protein (PRIP) regulates cell surface expression of GABA_A receptors containing a γ2 subunit. Mutant mice with double knockout of PRIP-1 and PRIP-2 were used to investigate aspects of GABAergic regulation of orofacial movements and interactions with D₁ mechanisms. Vertical jaw movements, tongue protrusions and movements of the head and vibrissae were reduced in PRIP-1/2 double knockouts. The GABA_Aergic agent diazepam reduced movements of the head and vibrissae; these effects were unaltered in PRIP-1/2 double knockouts. The D₁-like agonist SKF 83959 induced vertical jaw movements, incisor chattering, and movements of the head and vibrissae that were unaltered in PRIP-1/2 double knockouts. However, SKF 83959-induced tongue protrusions were reduced in PRIP-1/2 double knockouts. PRIP-mediated regulation of GABA_Aergic receptor mechanisms influences topographically distinct aspects of orofacial movement and interacts with D₁ receptor systems.

DOI： 10.1002/syn.20798

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The transmission of sensory information to the central nervous system during oral functions such as mastication and swallowing is well regulated by neural networks, including 7gamma;-aminobutyric acid (GABA)-mediated inhibitory interneurons. Therefore, an understanding of the mechanism of GABAergic transmission might provide new insight into theclinical treatment of eating and swallowing disorders. Type A GABA receptors are ligand-gated Cl^- channels composed pentamerically of two a(1-6), two y3(1-3), and one γ(1-3) subunit. One major issue regarding the efficacy of GABAergic transmissionis how to control receptor numbers on neuronal surfaces, determining synaptic strength. The number of receptors on the postsynaptic membrane is maintained by a balance between receptor insertion and endocytosis. Insulin increases the number of GABA_A receptors by stimulating insertion into the surface membrane via Akt-mediated phosphorylation of the subunits. Conversely, brain-derived neurotrophic factor decreases the number by stimulating endocytosis. Here we show that PRIP (phospholipase C-related but catalytically inactive protein), isolated in our laboratory, actively participates in these two events by regulating the phosphorylation of/J subunits and controlling the functions of binding partners such as Akt and protein phosphatases.

DOI： 10.2330/joralbiosci.52.322

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Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and PRIP-2 [officially designated PLCL1 and PLCL2]) was first identified in our laboratory, but the biological functions have remained elusive. Therefore, we generated Plcll and Plcl2 double-knockout mice (Plcl1 ^tm1Mh; Plcl2^tm1Tta) to gain insight into the biological function. Double-knockout mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments to discrim-inate whether males or females were defective indicated that the cause appeared to be on the female side. Mutant female mice had an apparently smaller uterus by gross anatomical observation and had more estrous days during the cycles. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone were measured for 5-6 consecutive days and were significantly higher in the mutant, which was also confirmed by examining the secretion of LH from the explant culture of anterior pituitary glands of wild-type and double-knockout mice. These results suggest that through gonadotropin secretion, PRIP plays an important role in female reproduction.

DOI： 10.1095/biolreprod.109.076760

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A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-δ1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P₂ present in the plasma membrane. A chimeric molecule of PLC-δ1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P₂ revealed that the PH domain of PLC-δ1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P₂ were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca²⁺-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P₂ present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca²⁺, suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P₃.

DOI： 10.1016/j.cellsig.2009.03.008

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GABA_A receptors are heteropentameric ligand-gated chloride channels composed of a variety of subunits, including α1 - 6, β1 - 3, γ1 - 3, δ, ε, θ, and π, and play a key role in controlling inhibitory neuronal activity. Modification of the efficacy of the synaptic strength is produced by changes in both the number of neuronal surface receptors and pentameric molecular assembly, leading to differences of sensitivity to neurotransmitters and neuromimetic drugs. Therefore, it is important to understand the molecular mechanisms regulating the so-called "life cycle of GABA_A receptors" including sequential pentameric assembly at the site synthesized, intracellular transport through the Golgi apparatus and the cytoplasm, insertion into the cell membrane, functional modulation at the cell surface, and finally internalization, followed by either recycling back to the surface membrane or lysosomal degradation. This review is focused on events related to the surface expression of the receptor containing the γ2 subunit and clathrin /AP2 complex-mediated phospho-regulated endocytosis of the receptor, with special reference to the function of novel GABA_A receptor modulators, GABARAP (GABA_A receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein).

DOI： 10.1254/jphs.CP0070063

Language：English   Publishing type：Research paper (scientific journal)  

The K⁺ Cl^- cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H₂O₂) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg²⁺) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl^-]_i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.

DOI： 10.1523/JNEUROSCI.3104-06.2007

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The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and-2. Regarding its ability to bind Ins (1,4,5)-P₃ via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P₃-mediated Ca²⁺ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABA_A receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABA_A receptor signaling. PRIP-1 and-2 double knock-out (DKO) mice were analyzed for GABA_A receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors; sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABA_A receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABA_A receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP.

DOI： 10.2330/joralbiosci.49.244

Language：English   Publishing type：Research paper (scientific journal)  

GABA_A receptors are a family of ligand-gated ion channels that are pentamer composed pre-dominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter (GABA, γ-aminobutyric acid) and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABA_A receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABA_A receptors. We have first isolated PRIP (phospholipase C-related, but catalytically inactive protein) as a novel inositol 1,4,5-trisphosphate binding protein, and subsequently found GABARAP (GABA_A receptor associated protein) as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c (catalytic subunit of protein phosphatase 1). These findings have prompted us to explore the possible involvement of PRIP in modulation of GABA_A receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABA_A receptor signaling on the basis of the characteristics of these interacting molecules.

DOI： 10.2330/joralbiosci.49.105

Language：English   Publishing type：Research paper (scientific journal)  

Although conjugation of overexpressed GABARP to phospholipid has been reported during starvation-induced autophagy, it is unclear whether endogenous GABARAP-phospholipid conjugation is also activated under starvation conditions. We observed little accumulation of GABARAP-phospholipid conjugate (GABARAP-PL) in mouse liver and kidney under starvation conditions, whereas endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart, independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II, together with inactivation of the mTor kinase-signaling pathway. In HeLa and C2C12 cells, GABARAP-PL accumulation in the presence of lysosomal protease inhibitors was independent of starvation-induced autophagy, whereas LC3-II accumulation was significant during starvation-induced autophagy. Interestingly, we observed activation of lysosomal turnover of GABARAP-PL during the differentiation of C2C12 cells to myotubes, along with increased lysosomal turnover of LC3-II. Under these conditions, S6 ribosomal protein was still phosphorylated, suggesting that the mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, in contrast to starvation-induced autophagy. These results indicated that lysosomal turnover of GABARAP-PL was activated during the differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, whereas little lysosomal turnover of GABARAP-PL was activated during starvation-induced autophagy.

DOI： 10.4161/auto.2871

Language：English   Publishing type：Research paper (scientific journal)  

PRIP, phospholipase C related, but catalytically inactive protein was first identified as a novel inositol 1,4,5-trisphosphate binding protein. It has a number of binding partners including protein phosphatase (PP1 and 2A), GABA_A receptor associated protein, and the β subunits of GABA_A receptors, in addition to inositol 1,4,5-trisphosphate. The identification of these molecules led us to examine the possible involvement of PRIP in the phospho-regulation of the β subunits of GABA_A receptors using hippocampal neurons prepared from PRIP-1 and 2 double knock-out (DKO) mice. Experiments were performed with special reference to the dephosphorylation processes of the β subunits. The phosphorylation of β3 subunits by the activation of protein kinase A in cortical neurons of the control mice continued for up to 5 min, even after washing out of the stimulus, followed by a gradual dephosphorylation. That of DKO mice gradually increased in spite of the lower phosphorylation levels induced by the stimulation. There was little difference in the amount of cellular cyclic AMP and protein kinase A activity between the control and mutant mice, indicating that phosphatases such as PP1 and PP2A are primarily involved in the difference. The time course of PP1 activity changes in the vicinity of the receptors in control mice corresponded to the phosphorylation of PRIP, while that of the mutant mice decreased with the period of the incubation. This is a good agreement with the suggestion that PRIP binds to and inactivates PP1, which is regulated by the phosphorylation of PRIP at threonine 94. These results suggest that PRIP plays an important role in controlling the dynamics of GABA_A receptor phosphorylation by through PP1 binding and, therefore, the efficacy of synaptic inhibition mediated by these receptors.

DOI： 10.1016/j.advenzreg.2006.01.006

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Investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins(1,4,5)P₃ via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P₃-mediated Ca²⁺ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knockout mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP (GABA_A receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in GABA_A receptor signaling. For this purpose PRIP knock-out mice were analyzed for GABA_A receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the β-subunit of GABA_A receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in Ins(1,4,5)P3-mediated Ca²⁺ signaling and GABA_A receptor signaling based on the characteristics of binding molecules.

Language：English   Publishing type：Research paper (scientific journal)  

1. Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFκB pathway, the major cell survival pathway. 2. The PI3K-Akt-NFκB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-α or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P ₃), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor κB (IκB) kinase (IKK) and finally phosphorylation of IκB and its subsequent degradation. 3. Pretreatment with alendronate at 100 μM for 24 h prior to the stimulation with tumor necrosis factor-α or insulin partially inhibited the IκB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IκB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P ₃. 4. The inhibitory effect of alendronate on IκB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5. The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFκB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

DOI： 10.1038/sj.bjp.0706373

Language：English   Publishing type：Research paper (scientific journal)  

GABA_A receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of α, β, and γ subunits. It has been shown that GABA_A receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABA _A receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395-415 of the receptor β subunits and co-localizes with GABA_A receptors in hippocampal neurons. We also established that NSF can regulate GABA_A receptor cell surface expression depending upon residues 395-415 in the β3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABA_A receptors.

DOI： 10.1016/j.mcn.2005.07.006

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Microtubule-associated protein (MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8), which is essential for autophagy. MAP-LC3 is cleaved by a cysteine protease to produce LC3-I, which is located in cytosolic fraction. LC3-I, in turn, is converted to LC3-II through the actions of E1- and E2-like enzymes. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. We determined the solution structure of LC3-I and found that it is divided into N- and C-terminal subdomains. Additional analysis using a photochemically induced dynamic nuclear polarization technique also showed that the N-terminal subdomain of LC3-I makes contact with the surface of the C-terminal subdomain and that LC3-I adopts a single compact conformation in solution. Moreover, the addition of dodecylphosphocholine into the LC3-I solution induced chemical shift perturbations primarily in the C-terminal subdomain, which implies that the two subdomains have different sensitivities to dodecylphosphocholine micelles. On the other hand, deletion of the N-terminal subdomain abolished binding of tubulin and microtubules. Thus, we showed that two subdomains of the LC3-I structure have distinct functions, suggesting that MAP-LC3 can act as an adaptor protein between microtubules and autophagosomes.

DOI： 10.1074/jbc.M413565200

Language：English   Publishing type：Research paper (scientific journal)  

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [ins(1,4,5)P₃] binding protein with a domain organization similar to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P₃. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P₃-mediated Ca²⁺ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1 ^-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca²⁺ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1^-/- mice than in those from wild type mice. The relative amounts of [³H]Ins(1,4,5)P₃ measured in neurons labeled with [³H]inositol was also lower in cells from PRIP-1 ^-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1^-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,S)P₃ is enhanced in cells from PRIP-1^-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1 PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P ₃ to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P₃-mediated Ca²⁺ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P₃.

DOI： 10.1002/jcp.20136

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we investigated the effects of pentylenetetrazol (PTZ), a chemical convulsant that interacts with the GABA _A receptor, in mice lacking the phospholipase C (PLC)-related inactive protein-1 (PRIP-1). PRIP-1 knockout mice did not develop spontaneous behavioral seizure. PRIP-1 knockout mice had markedly shorter latencies until the first clonic convulsion (CL) and tonic extensor (TE) following PTZ administration and increased incidence of convulsion compared to those in wild-type mice. Furthermore, the mortality rate by PTZ in mice lacking the PRIP-1 was also significantly increased in comparison with that in wild-type mice. These findings suggested that mice lacking the PRIP-1 were hypersensitive to PTZ-induced convulsion, and PRIP-1 might play roles in suppressing excessive excitability via interactions with the GABA _A receptor.

DOI： 10.1016/j.brainres.2004.08.009

Language：English   Publishing type：Research paper (scientific journal)  

γ-Aminobutyric acid_A (GABA_A) receptors mediate most of the fast inhibitory neurotransmission in the central nervous system. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. This review highlights recent findings on the regulation of GABA_A-receptor expression and function, focusing on the mechanisms of sorting, targeting, synaptic clustering, and endocytic events of GABA_A receptors, all which are regulated by their associated proteins. Now these topics are an area of active interest in studies on inhibitory neurotransmission.

DOI： 10.1254/fpj.123.105

Language：English   Publishing type：Research paper (scientific journal)  

The number of postsynaptic γ-aminobutyric acid type A (GABA _A) receptors is a fundamental determinant of the variability of inhibitory synaptic responses in the central nervous system. In rat visual cortex, [³H] SR-95531 binding assays revealed that brain-derived neurotrophic factor (BDNF), one of the neurotrophins, induced a rapid increase in the total number of cell surface GABA_A receptors, through the activation of Trk B receptor tyrosine kinases. We also demonstrated that BDNF rapidly induced a sustained potentiation of GABA_A receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons freshly isolated from P14 rats. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and phospholipase C-γ. In addition, intracellular Ca²⁺ was important for the potentiation of GABA_A responses induced by BDNF. The selective increase in mean miniature inhibitory postsynaptic (mIPSC) current amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABA _A receptors in visual cortical pyramidal neurons. These results suggest that BDNF could alter the number of cell surface GABA_A receptors in a region-specific manner.

DOI： 10.1074/jbc.M305872200

Language：English   Publishing type：Research paper (scientific journal)  

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP₅ and InsP ₆. We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates - both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP₆ levels in ATDC5 cells. However, InsP₅ levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP₅ and InsP₆ levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP₅ and InsP ₆. Thus, substantial changes in MINPP expression can occur without a net effect upon InsP₅ and InsP₆ turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.

DOI： 10.1016/S0014-4827(03)00337-9

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

A family of phospholipase C-related, catalytically inactive proteins (designated PRIP) have been identified as a group of novel inositol 1,4,5-trisphosphate binding proteins with a domain organization similar to phospholipase C-δ but lacking the enzymatic activity. The PRIP family consists of at least two types of proteins (PRIP-1 and PRIP-2 subfamilies). In the present study, we examined the tissue distribution of PRIP-2, its expression in rat brain at the mRNA level, and the characteristics of its binding to inositol compounds, protein phosphatase 1, and γ-amino butyric acid receptor associated protein. We also compared these characteristics with those of PRIP-1. Northern blot analysis and reverse-transcription polymerase chain reaction showed that PRIP-1 was present mainly in the brain, whereas PRIP-2 was expressed ubiquitously. In situ hybridization studies using rat brain revealed that the mRNA for both PRIP-1 and PRIP-2 was similarly expressed; it was detected in the granular cell and Purkinje cell layers in the cerebellum, and in the hippocampal pyramidal cells, dentate granule cells, and pyramidal and/or granule cells of the cerebral cortex in the cerebrum. PRIP-2 bound inositol 1,4,5-trisphosphate and its parent lipid, phosphatidylinositol 4,5-bisphosphate, with a similar affinity, while PRIP-1 preferentially bound the former ligand by about 10-fold. PRIP-1 and PRIP-2 interacted with protein phosphatase 1 and γ-amino butyric acid receptor associated protein in a similar manner. These results indicate that, similar to PRIP-1, PRIP-2 may be involved in both inositol 1,4,5-trisphosphate-mediated and γ-amino butyric acid-related signaling.

DOI： 10.1016/S0024-3205(02)02275-0

Language：English   Publishing type：Research paper (scientific journal)  

Tumour necrosis factor (TNF) is known to induce apoptosis, but recently, TNF was shown to promote cell survival, a process regulated by phosphatidylinositol-3-OH kinase (PI3K) and the NFκB pathway. In this study, we investigated the relationship between the molecules implicated in regulating TNF-induced cell survival and apoptosis induced by TNF in a human head and neck squamous cell carcinoma cell line (SAS), with special reference to the Akt pathway, one of the pathways related to cell survival. In SAS cells, TNF induced the phosphorylation of Akt at both Ser473 and Thr308, causing the activation of Akt, and also induced the phosphorylation and degradation of IκB (inhibitor of NFκB). This phosphorylation and degradation was inhibited by pretreating the cells with the PI3K inhibitors, wortmannin or LY294002. The apoptosis of SAS cells induced by TNF was dependent on the concentration: a high concentration of TNF, but not a low concentration, induced apoptosis within 30 h. However, a low concentration of TNF in the presence of wortmannin or LY294002 induced apoptosis. Furthermore, expression of the kinase-negative form of Akt, IKKα or IKKβ, and the undegradable mutant of IκB, also induced apoptosis at low concentrations of TNF. When the SAS cells expressed constitutively activated Akt, apoptosis was not induced, even by high concentrations of TNF. These observations suggest that, in the SAS cell line, the PI3K-NFκB pathway contributes to TNF-induced cell survival and that inhibition of this pathway accelerates apoptosis.

DOI： 10.1016/S0898-6568(02)00025-6

Language：English   Publishing type：Research paper (scientific journal)  

Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins (1,4,5) P₃ binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABA_A-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by β-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the γ2 subunit of the GABA_A receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABA_A receptors, especially in response to the agents acting on the γ2 subunit.

DOI： 10.1254/fpj.119.241

Language：English   Publishing type：Research paper (scientific journal)  

Ameloblastoma is a unique tumor in the oral and maxillofacial region with various levels of proliferative activity in each type. p53 is most commonly found to be mutated in human cancer and sometimes is overexpressed also in other lesions, such as ameloblastoma. Murine Double Minute 2 (MDM2) is able to physically associate with the p53 tumor suppressor and therefore block the growth suppressive functions of p53. In the present study, immunohistochemistry, western blotting and enzyme-linked immunosorbent assay for p53 mutant selective test were done. MDM2 was overexpressed in ameloblastoma and the results showed different numbers of MDM2 labeling index based on both WHO classification and cytological pattern of outer layer cells. Basal ameloblastoma, which has a high proliferative activity, had the highest MDM2 labeling index. We suggest MDM2 protein caused the high proliferative activity of ameloblastoma, especially in basal cell ameloblastoma.

DOI： 10.1016/S1368-8375(01)00036-7

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1023/A:1013884402033

Language：English   Publishing type：Research paper (scientific journal)  

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 μM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.

DOI： 10.1016/S1357-2725(01)00067-X

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The protein p130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin.

DOI： 10.2220/biomedres.22.157

Language：English   Publishing type：Research paper (scientific journal)  

p130 was originally identified as an Ins(1,4,5)P₃-binding protein similar to phospholipase C-δ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P₃-mediated Ca²⁺ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P₃ with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P₃. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca²⁺ signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca²⁺ concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P₃; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P₃ could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P₃-mediated Ca²⁺ signalling.

DOI： 10.1042/0264-6021:3490357

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Two inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) binding proteins, having molecular masses of 85 kDa and 130 kDa, were purified from rat brain using an Ins(1,4,5)P₃ affinity column; the former protein was found to be the δ₁-isozyme of phospholipase C (PLC-δ₁) and the latter protein was an unidentified novel protein. Subsequent molecular biology studies isolated the full-length cDNA for the 130 kDa-Ins(1,4,5)P₃ binding protein (p130) which encoded 1,096 amino acids. The predicted amino acid sequence of p 130 had 38.2% homology to that of PLC-δ₁. The region of p130 responsible for InS(1,4,5)P₃ binding was mapped in a pleckstrin homology (PH) domain of the molecule. The PH domain of p130 could also bind to Ins(1,4,5,6)P₄ with a similar affinity to Ins(1,4,5)P₃. The 85 kDa protein (PLC-δ₁) was also analyzed for the binding site by molecular biological, peptide synthetic chemical and immunological studies: the sequence 30-43 of PLC-δ₁ was primarily involved in binding, which was mapped in the N-terminal of the PH domain of the molecule. Experiments as to the effect of Ins(1,4,5)P₃ on PLC-δ₁ activity showed that Ins(1,4,5)P₃ at concentrations over 1 μM strongly inhibited PLC activity of PLC-δ₁. The PH domains derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and p130, were analyzed for the capability and specificity of binding of inositol phosphates and derivatives of inositol lipids. We concluded from these studies that inositol phosphates and/or inositol lipids might be common ligands for the PH domains, and therefore inositol phosphates/inositol lipids might be involved in more aspects of cellular functions than originally thought, because more than 90 proteins to date are known to include PH domain. Which ligands are physiologically relevant for the PH domain, would depend on binding affinities and their cellular abundance.

Language：English   Publishing type：Research paper (scientific journal)  

The pleckstrin homology (PH) domains of phospholipase C (PLC)-δ1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] by PLC-δ1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca²⁺-dependent activation of PLC-δ1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-δ1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to Ptdlns(4,5)P₂ is regulated by changes in free Ca²⁺ concentration within the physiological range. A Ca²⁺ dependent increase in the binding to PtdIns(4,5)P₂ was observed with a full-length PLC-δ1, while the isolated PH domain did not show any Ca²⁺ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca²⁺ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-δ1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca²⁺ dependent increase in the binding to PtdIns(4,5)P₂. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P₂ showed no Ca²⁺ dependent enhancement of binding. However, when present within a chimera also containing PLC-δ1 EF-hand motifs, the Ca²⁺ dependent binding was again observed. These results suggest that the binding of Ca²⁺ to the EF-hand motifs can modulate binding to PtdIns(4,5)P₂ mediated by the PH domain.

DOI： 10.1046/j.1432-1327.1999.00786.x

Language：English   Publishing type：Research paper (scientific journal)  

The pleckstrin homology domain (PH domain) is now well known as a structural module for the binding of inositol compounds. In the present study, polyclonal antibodies against the peptide KVKSSSWRRERFYK, derived from the N-terminal of the PH domain of phospholipase C-δ1 (PLC-δ1), were raised in rabbits. These were then tested for their ability to inhibit the binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] to the binding proteins including the receptor molecule. The Fab fragment of the antibodies but not the whole molecule inhibited the binding of Ins(1,4,5)P₃ not only to PLC-δ1 but also to the Ins(1,4,5)P₃ receptor, indicating that the antibodies raised recognized the binding site for Ins(1,4,5)P₃ in the receptor. Rat basophilic leukemic cells were permeabilized with saponin and assayed for Ins(1,4,5)P₃mediated Ca²⁺ release. Pretreatment of permeabilized RBL cells with the Fab fragment of the antibodies diminished the release of Ca²⁺ caused by Ins(1,4,5)P₃, and further absorption experiments using a variety of synthetic peptides suggested that the tripeptide KVK is the epitope of the antibodies. Structural information about KVK will help in screening for Ins(1,4,5)P₃ antagonists.

DOI： 10.1006/bbrc.1999.0869

Language：English   Publishing type：Research paper (scientific journal)  

The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) binding protein from rat brain and was molecularly cloned to be found similar to phospholipase C-δ₁ (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-δ₁, Biochem. J. 313, 319-325). The 130-kDa protein and its deleted protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length protein and other deleted proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg²⁺ to homogenates of COS-1 cells caused the translocation of expressed proteins from the cytosol to the membrane fraction, yet further addition of AlF₄^- which induced the activation of GTP binding proteins did not cause a further translocation. The protein translocated to the membrane by the addition of Mg²⁺ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P₃ or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa protein, (ii) the activation of GTP binding protein does not appear to cause the translocation of the 130-kDa protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa protein to a very small extent, probably through the binding site in the PH domain. Copyright (C) 1999 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0009-3084(99)00016-X

Language：English   Publishing type：Research paper (scientific journal)  

Rat brain cytosol was applied to a heparin column and eluted with 0.9 M-NaCl. The total binding activity of [³H] inositol 1,4,5-trisphosphate to the eluate was increased about 6-fold compared with the original cytosol. When the eluate was mixed with a flow-through fraction from the heparin column, however, the activity returned to the original lever, suggesting that the flow-through fraction contained an inhibitory factor(s) which prevented the binding. The factor(s) was purified by sequential column chromatography using gel permeation, a hydrophobic gel, and finally, a hydroxylapatite gel. Silver staining of sodium dedecyl sulfate gel electrphoresis of the sample thus purified showed a broad band located between the authentic molecular weight markers of 580 and 390 k. A carbohydrate staining method showed that the factor is a glycoprotein.

Language：English   Publishing type：Research paper (scientific journal)  

We have isolated a novel inositol 1,4,5-trisphosphate binding protein with molecular mass of 130 kDa (p130), homologous to phospholipase C-δ1 in amino acid sequence but with no catalytic activity. Here we report the expression and localization of p130 at the mRNA level in rat brain. Northern blotting showed that gene expression encoding p130 was most abundant in brain. Brain localization of p130-mRNA using an in situ hybridization technique revealed that in the cerebellum, the mRNA was detected in the granular cell and Purkinje cell layers, and cerebellar nuclei. In the cerebrum, the mRNA was localized in hippocampal pyramidal cells, dentate granule cells and pyramidal and/or granule cells of the cerebral cortex. The brain localization of p130-mRNA was similar to that of the β-subtype of phospholipase C, indicating that p130 may be mainly involved in phospholipase Cβ-mediated signaling.

DOI： 10.1016/S0304-3940(98)00810-6

Language：English   Publishing type：Research paper (scientific journal)  

We examined whether a phosphotyrosine binding (PTB) domain from the human insulin receptor substrate-1 (hIRS-1) is capable of binding inositol phosphates/phosphoinositides. The binding specificity was compared with that of the pleckstrin homology (PH) domain derived from the same protein because the three dimensional structure was found to be very similar to that of the PH domain, despite the lack of sequence similarity. We also attempted to locate the site of binding of the inositol compounds. The PTB domain bound [³H]Ins(1,4,5)P₃, which was displaced most strongly by Ins(1,3,4,5,6)P₅ and InsP₆, indicating that these inositol polyphosphates show the highest affinity. The PTB domain bound to liposomes containing PtdIns(4,5)P₂, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, but not phosphatidylinositol. In contrast, the PH domain showed a preference for Ins(1,4,5)P₃, the polar head of PtdIns(4,5)P₂. Site-directed mutagenesis studies were performed to map the binding site for inositol phosphates in the PTB domain. Mutation of K169Q, K171Q or K177Q, located in the loop connecting the β1 and β2 strands, which is partially responsible for binding inositol phosphates/phosphoinositides in the PH domains of several other proteins, reduced binding activity, probably because of a reduction in affinity. Mutation of R212Q or R227Q, shown to be involved in the binding of a phosphotyrosine, had little effect on the binding capacity. These results indicate that the PTB domain of hIRS-1 can bind inositol phosphates/phosphoinositides. Therefore signalling through the PTB domain could be regulated by the binding not only of proteins with phosphotyrosine but also of inositol phosphates/phosphoinositides, implying that PTB domains could be involved in a myriad of interconnections between intracellular signalling pathways.

DOI： 10.1042/bj3340211

Language：English   Publishing type：Research paper (scientific journal)  

Many of the proteins that participate in cell signalling contain structural modules involved in regulatory interactions between components of signal transduction cascades. One of such modules is the pleckstrin homology (PH) domain, a region of approximately 120 amino acids that can form an electrostatically polarized tertiary structure. Several molecules such as inositol 1,4,5-trisphosphate/phosphatidylinositol 4,5-bisphosphate, the βγ-subunits of heterotrimeric G proteins and protein kinase C have been proposed as common ligands for the PH domain. Through these potential interactions, the PH domain has been proposed to play a role in membrane recruitment of proteins containing the PH domain, thus targeting them to appropriate cellular compartment or enabling them to interact with other components of the signal transduction pathway. In this review, we mainly focus on membrane targeting through the binding to inositol phosphates/phosphoinositides.

DOI： 10.1254/jjp.76.255

Language：English   Publishing type：Research paper (scientific journal)  

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domain from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6- tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6- tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphonoisitide derivative. However, all PH domains had similar affinity inositol 1,3,4,5- tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important lipids could be important ligands for the PH domain, and therefore inositol phosphate/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

DOI： 10.1016/S0167-4889(97)00109-2

Language：English   Publishing type：Research paper (scientific journal)  

Studies in the laboratory using the recombinant pleckstrin homology (PH) domains from several proteins have confirmed our proposal that inositol phosphates/inositol lipids would be physiologically relevant ligands for the PH domain. In this study, we examined whether a phospho-tyrosine binding (PTB) domain is capable of binding specific inositol phosphates, and compared the specificity with that seen for the PH domain originated from the same protein, human insulin receptor substrate (IRS)-l. The PTB domain, the 3D structure of which is similar to that of PH domain, bound D[3H]Ins(l,4,5)P3 and the binding was displaced by Hns(l,4,5)P3 and oIns(l,3,4)P3 to the lesser extent than that by D-[ns(l,4,5)P3, indicating the specific binding. D-Ins(l,3,4,5)P4 and D-Ins(l,3,4,5,6)P5 as well as Ins?6 had the same affinity as D-Ins(l,4,5)P3. In contrast, the PH domain showed the specific preference to u-Ins(l,4,5)P3, the polar head of phosphatidylinositol 4,5-bisphosphate (PIP2). Therefore, IRS-1 is possible to be recruited for the function to the plasma membrane, in which PlPa is present, by the binding via the PH domain. On the other hand, the binding of the PTB domain to PIP2 in the plasma membrane or phosphotyrosine on the receptor molecules or adaptor molecules may be inhibited by the binding to D-Ins(l,3,4,5,6)P5 as well as InsPe which are the most abundant inositol compounds in cells.

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d-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) analogues derived at 3-OH with a bulky substituent were chemically synthesized and structural features of vicinity surrounding the 3-OH of Ins(1,4,5)P₃, recognized by metabolic enzymes and by the receptor were explored. 3-Benzoyl-, 3-methylbenzoyl- and 3-para-aminobenzoyl-Ins(1,4,5)P₃ inhibited the dephosphorylation of [³H]Ins(1,4,5)P₃ by the 5-phosphatase present in erythrocyte ghosts, but the potency varied. The inhibitory potency for the former two compounds was slightly lower than that for Ins(1,4,5)P₃, while that for the latter compound was higher. Transfer of the amino group to the meta-position of the benzoyl group led to a less potent analogue. In an assay of [³H]Ins(1,4,5)P₃ 3-kinase at a low Ca²⁺ concentration, catalyzed by rat brain cytosol, 3-meta-aminobenzoyl-Ins(1,4,5)P₃ was the most potent among compounds examined, including Ins(1,4,5)P₃ in inhibiting the phosphorylation, whereas both 3-benzoyl- and 3-methylbenzoyl-Ins(1,4,5)P₃ at concentrations up to 30 μM, were without effect. All analogues examined were effective in inhibiting [³H]Ins(1,4,5)P₃ binding to purified Ins(1,4,5)P₃ receptor, but all 3-derived analogues were less potent and 3-benzoyl-Ins(1,4,5)P₃ was the least potent. It would thus appear that the space in the vicinity surrounding the 3-hydroxyl group of Ins(1,4,5)P₃ is sterically restrictive with regard to recognition by metabolic enzymes and the receptor, whereas the amino group providing arms for either the electrostatic interaction or the hydrogen bond, makes the analogues more potent.

DOI： 10.1016/0304-4165(95)00043-B

Language：English   Publishing type：Research paper (scientific journal)  

It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃)-immobilized matrix was the δ₁ isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-δ₁ in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P₃ binding. The lysate was applied to Ins(1,4,5)P₃-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P₃ binding. The lysate was also purified to near homogeneity using a glutathione- Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-δ₁ consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P₃ binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-δ₁ which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P₃ binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-δ₁, which contains 6 basic amino acids, binds to an Ins(1,4,5)P₃-immobilized matrix. Moreover, Ins(1,4,5)P₃ binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-δ₁ is important for the binding of both Ins(1,4,5)P₃ and phosphatidylinositol 4,5-bisphosphate.

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Synthetic DL-myo-inositol 1,2,4,5-tetrakisphosphate (DL-Ins-(1,2,4,5)P₄) functioned as a full agonist, with only 3-fold less potency than D- Ins(1,4,5)P₃ in eliciting the release of Ca²⁺ from nonmitochondrial pools of permeabilized rat basophilic leukemic cells. DL-Ins(1,2,4,5)P₄ inhibited the binding of D-[³H]Ins(1,4,5)P₃ to the purified D-Ins(1,4,5)P₃ receptor with almost the same potency as seen for the Ca²⁺ release. This compound inhibited the hydrolysis of D-[³H]Ins(1,4,5)P₃ to D-[³H]Ins(1,4)P₂ catalyzed by erythrocyte ghosts, with a K(i) value of as low as 1.4 μM, but it could not serve as a substrate for the same enzyme. D-Ins(1,4,5)P₃ 3- kinase in rat brain cytosol did not recognize the compound at concentrations up to 30 μM. Thus, it would appear that DL-Ins(1,2,4,5)P₄ can serve as a potent and long lasting experimental and pharmacological tool for stimulating D-Ins(1,4,5)P₃-mediating processes.

Language：English   Publishing type：Research paper (scientific journal)  

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) binding domain of phospholipase C-δ1 (PLC-δ1) was determined by examining binding activity of the synthetic peptide corresponding to residues 30-43 of PLC-δ1. The peptide coupled with carrier proteins such as keyhole limpet hemocyanin or bovine serum albumin bound Ins(1,4,5)P₃, whereas a scrambled peptide with the same amino acids did not do so. Polyclonal antibody against the peptide was examined to determine whether it would cause inhibition of the Ins(1,4,5)P₃ binding to PLC-δ1. Fab fragment of antibody to the peptide did inhibit binding to PLC-δ1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-δ1 is responsible for the binding of Ins(1,4,5)P₃.

DOI： 10.1006/bbrc.1994.2845

Language：English   Publishing type：Research paper (scientific journal)  

A series of myo-inositol 1,4,5-trisphosphate analogues with the 2-acyl substituents p-aminobenzoyl (7), p-azidobenzoyl (8), 4-{5-[2-(benzamido)ethyl]-2-hydroxyphenylazo}benzoyl (9), and cis,trans-4-aminocyclohexylcarbonyl (10) were synthesised and examined for their effects on the 5-phosphatase, the 3-kinase, the tritiated trisphosphate-binding activity, and the Ca²⁺-releasing activity. Each analogue inhibited the hydrolysis of d-[5-³²P]Ins(1,4,5)P₃ and the phosphorylation of d-[³H]Ins(1,4,5)P₃, catalysed by erythrocyte ghosts and brain cytosol, respectively. The analogues acted as full agonists in releasing Ca²⁺ from permeabilised cells and also inhibited the binding of d-[³H]Ins(1,4,5)P₃ to cerebellum microsomes. The analogues 7 and 10 were utilised for immobilisation of the trisphosphate on Sepharose^TM and the subsequent affinity chromatography effected purification of the above proteins. A photoaffinity probe, the appendage of which acted as the photoaffinity probe as well as a non-radioactive molecular marker, was also derived from the analogue 7.

DOI： 10.1016/0008-6215(92)85048-5

Language：English   Publishing type：Research paper (scientific journal)  

Binding activity of [³H]inositol 1,3,4,5-tetrakisphosphate (InsP₄) was characterized with rat cerebellar membranes. Two types of InsP₄ analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP₄ have been synthesized and their effects on the binding activity were also examined. [³H]InsP₄ binding was gradually displaced by increasing amounts of unlabeled InsP₄, with an IC₅₀ of 60-170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca²⁺, this being in clear contrast with [³H]InsP₃ binding noted in the same species of tissue. Heparin inhibited the binding, with an IC₅₀ of 1.7, 3 or 20 μg/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [³H]InsP₃ binding. InsP₄ analogs were as effective as InsP₄ in displacing [³H]InsP₄ from rat cerebellar membranes, thereby indicating that the 2nd hydroxl group may not be involved in recognition of InsP₄ by its binding sites.

DOI： 10.1016/0005-2736(91)90127-T

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Venue：web開催   Country：Japan  

肥満で増大した脂肪組織は、炎症性サイトカインを分泌し、脂肪組織や肝臓などに慢性炎症を引き起こす。この肥満による慢性炎症反応は、生活習慣病の病態の基盤となる。閉経後の女性は、内臓脂肪型肥満をきたしやすく生活習慣病リスクが増大するが、その発症機序には未だ解明の余地がある。さて、炎症反応で中心的な役割を担う転写因子のNF-Bは、receptor activator of NF-B ligand (RANKL)によっても活性化される。我々は、閉経後にRANKLの血中濃度が上昇することに着目し、RANKL-NF-B経路の活性化が閉経後の脂肪蓄積の一因ではないかとの仮説を立て、本研究を行った。
　野生型マウスの骨髄細胞をRANKLで刺激すると、TNFのmRNA発現は２相性に上昇した。このTnfaの発現変化は、第１相が古典的経路に第２相が非古典的経路に依存することをNF-B古典的経路阻害剤を用いて確認し、経路依存的に炎症反応が制御される可能性を示した。本研究ではNF-Bの非古典的経路に焦点をあて、閉経後肥満の発症機構を解析するために、非古典的経路の活性化が障害されているaly/alyマウスを用いて実験を行なった。野生型およびaly/alyマウスの卵巣を摘出（OVX）した閉経モデルマウスを高脂肪・高ショ糖食の自由摂餌下で飼育した。野生型マウスでは、OVX後10週の間に脂肪細胞の肥大、脂肪組織の炎症、肝臓への異所性脂肪蓄積が起こり、インスリン抵抗性、耐糖能異常を示した。一方、OVX施行で野生型同様にaly/alyマウスの血清RANKL濃度は上昇したにも関わらず、野生型で認めた脂肪蓄積・炎症は抑制され、全身の糖代謝の異常も抑制された。これらの結果は、RANKL-NF-B の非古典的経路の活性化が、閉経後の肥満を引き起こす要因となることを示している。

Event date： 2021.10

Language：Japanese  

Venue：web開催   Country：Japan  

Event date： 2021.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：web開催   Country：Japan  

Event date： 2020.9 - 2020.10

Language：Japanese  

Venue：鹿児島大学（Web開催）   Country：Japan  

Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京歯科大学   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：広島大学   Country：Japan  

Event date： 2019.8

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：東京医科歯科大学   Country：Japan  

Event date： 2019.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：広島大学   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪国際会議場   Country：Japan  

Phospholipase C-related catalytically inactive protein modulates cytokinesis progression

Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：横浜市   Country：Japan  

Implication of PRIP in constitutive internalization of GABA(A) receptor

Presentation type：Symposium, workshop panel (public)  

Venue：Beijing   Country：China  

Presentation type：Symposium, workshop panel (public)  

Venue：Beijing   Country：China  

Presentation type：Symposium, workshop panel (public)  

Venue：名古屋市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：仙台市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：名古屋市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Dream Center, Gyeongju   Country：Korea, Republic of  

Presentation type：Oral presentation (general)  

Venue：Dream Center, Gyeongju   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：札幌市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：札幌市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京都   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：横浜市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Snowbird, Utah   Country：United States  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

Language：Japanese  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：3

Number of peer-reviewed articles in Japanese journals：0

Type：Competition, symposium, etc. 

Number of participants：1,000

Type：Competition, symposium, etc. 

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：2

Number of peer-reviewed articles in Japanese journals：4

Type：Academic society, research group, etc. 

Type：Peer review 

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Type：Competition, symposium, etc. 

Number of participants：500

Type：Competition, symposium, etc. 

Number of participants：100

Type：Academic society, research group, etc. 

Type：Peer review 

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：3

Type：Academic society, research group, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Competition, symposium, etc. 

Number of participants：500

Type：Competition, symposium, etc. 

Number of participants：80

Type：Academic society, research group, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Scientific advice/Review 

Type：Academic society, research group, etc. 

Type：Scientific advice/Review 

Type：Academic society, research group, etc. 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Number of participants：150

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Number of participants：100

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Number of participants：120

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc.