Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Akiyuki Nishimura Last modified date:2019.06.19

Lecturer / Department of Translational Pharmaceutical Sciences / Department of Pharmaceutical Health Care and Sciences / Faculty of Pharmaceutical Sciences


Papers
1. Akiyuki Nishimura, Tsukasa Shimauchi, Tomohiro Tanaka, Kakeru Shimoda, Takashi Toyama, Naoyuki Kitajima, Tatsuya Ishikawa, Naoya Shindo, Takuro Numaga-Tomita, Satoshi Yasuda, Yoji Sato, Koichiro Kuwahara, Yoshito Kumagai, Takaaki Akaike, Tomomi Ide, Akio Ojida, Yasuo Mori, Motohiro Nishida, Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence, Science Signaling, 10.1126/scisignal.aat5185, 11, 556, 2018.11, Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission-associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding-dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1-filamin A interaction by cilnidipine suppressed mitochondrial hyperfission-associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development..
2. Caroline Sunggip, Kakeru Shimoda, Sayaka Oda, Tomohiro Tanaka, Kazuhiro Nishiyama, Supachoke Mangmool, Akiyuki Nishimura, Takuro Numaga-Tomita, Motohiro Nishida, TRPC5-eNOS axis negatively regulates ATP-induced cardiomyocyte hypertrophy, Frontiers in Pharmacology, 10.3389/fphar.2018.00523, 9, MAY, 2018.05, Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation..
3. Warisara Parichatikanond, Akiyuki Nishimura, Motohiro Nishida, Supachoke Mangmool, Prolonged stimulation of β2-adrenergic receptor with β2-agonists impairs insulin actions in H9c2 cells, Journal of Pharmacological Sciences, 10.1016/j.jphs.2018.09.007, 2018.01, Insulin resistance is a condition in which there is a defect in insulin actions to induce glucose uptake into the cells. Overstimulation of β2-adrenergic receptors (β2ARs) is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which β2-agonists affect insulin resistance in the heart are incompletely understood. The β2-agonists are used for treatment of asthma due to bronchodilating effects. We also investigated the effects of β2-agonists in human bronchial smooth muscle (HBSM) cells. In this study, we demonstrate that chronic treatment with salbutamol, salmeterol, and formoterol inhibited insulin-induced glucose uptake and GLUT4 synthesis in H9c2 myoblast cells. Sustained β2AR stimulation also attenuated GLUT4 translocation to the plasma membrane, whereas short-term stimulation had no effect. In HBSM cells, prolonged treatment with β2-agonists had no effect on insulin-induced glucose uptake and did not alter insulin-induced expressions of GLUT1, GLUT4, and GLUT10. In addition, genetic polymorphisms at amino acid positions 16 and 27 of β2AR are linked to insulin resistance by significant suppression of GLUT4 translocation compared to wild-type. Thus, prolonged β2AR stimulation by β2-agonists impairs insulin actions through suppression of GLUT synthesis and translocation only in H9c2 cells..
4. Takuro Numaga-Tomita, Sayaka Oda, Kazuhiro Nishiyama, Tomohiro Tanaka, Akiyuki Nishimura, Motohiro Nishida, TRPC channels in exercise-mimetic therapy, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-018-2211-3, 2018.01, Physical exercise yields beneficial effects on all types of muscle cells, which are essential for the maintenance of cardiovascular homeostasis and good blood circulation. Daily moderate exercise increases systemic antioxidative capacity, which can lead to the prevention of the onset and progression of oxidative stress-related diseases. Therefore, exercise is now widely accepted as one of the best therapeutic strategies for the treatment of ischemic (hypoxic) diseases. Canonical transient receptor potential (TRPC) proteins are non-selective cation channels activated by mechanical stress and/or stimulation of phospholipase C-coupled surface receptors. TRPC channels, especially diacylglycerol-activated TRPC channels (TRPC3 and TRPC6; TRPC3/6), play a key role in the development of cardiovascular remodeling. We have recently found that physical interaction between TRPC3 and NADPH oxidase (Nox) 2 under hypoxic stress promotes Nox2-dependent reactive oxygen species (ROS) production and mediates rodent cardiac plasticity, and inhibition of the TRPC3-Nox2 protein complex results in enhancement of myocardial compliance and flexibility similar to that observed in exercise-treated hearts. In this review, we describe current understanding of the roles of TRPC channels in striated muscle (patho)physiology and propose that targeting TRPC-based protein complexes could be a new strategy to imitate exercise therapy..
5. Takaaki Akaike, Tomoaki Ida, Fan Yan Wei, Motohiro Nishida, Yoshito Kumagai, Md Morshedul Alam, Hideshi Ihara, Tomohiro Sawa, Tetsuro Matsunaga, Shingo Kasamatsu, Akiyuki Nishimura, Masanobu Morita, Kazuhito Tomizawa, Akira Nishimura, Satoshi Watanabe, Kenji Inaba, Hiroshi Shima, Nobuhiro Tanuma, Minkyung Jung, Shigemoto Fujii, Yasuo Watanabe, Masaki Ohmuraya, Péter Nagy, Martin Feelisch, Jon M. Fukuto, Hozumi Motohashi, Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics, Nature Communications, 10.1038/s41467-017-01311-y, 8, 1, 2017.12, Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction..
6. Akiyuki Nishimura, Caroline Sunggip, Sayaka Oda, Takuro Numaga-Tomita, Tsuda Makoto, Motohiro Nishida, Purinergic P2Y receptors
Molecular diversity and implications for treatment of cardiovascular diseases, Pharmacology and Therapeutics, 10.1016/j.pharmthera.2017.06.010, 180, 113-128, 2017.12, Purinergic signaling, mediated mainly by G protein-coupled P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR-mediated biological functions are predominantly activated by mononucleotides released from non-adrenergic, non-cholinergic nerve endings or non-secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand-independent P2YR function through receptor-receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein-coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide-responsive P2Y6R and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2Y6R antagonist MRS2578 interrupts heterodimerization between P2Y6R and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2Y6R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases..
7. Sayaka Oda, Takuro Numaga-Tomita, Naoyuki Kitajima, Takashi Toyama, Eri Harada, Tsukasa Shimauchi, Akiyuki Nishimura, Tatsuya Ishikawa, Yoshito Kumagai, Lutz Birnbaumer, Motohiro Nishida, TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice, Scientific Reports, 10.1038/s41598-017-07903-4, 7, 1, 2017.12, Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6..
8. Motohiro Nishida, Akiyuki Nishimura, Tetsuro Matsunaga, Hozumi Motohashi, Shingo Kasamatsu, Takaaki Akaike, Redox regulation of electrophilic signaling by reactive persulfides in cardiac cells, Free Radical Biology and Medicine, 10.1016/j.freeradbiomed.2017.01.024, 109, 132-140, 2017.08, Maintaining a redox balance by means of precisely controlled systems that regulate production, and elimination, and metabolism of electrophilic substances (electrophiles) is essential for normal cardiovascular function. Electrophilic signaling is mainly regulated by endogenous electrophiles that are generated from reactive oxygen species, nitric oxide, and the derivative reactive species of nitric oxide during stress responses, as well as by exogenous electrophiles including compounds in foods and environmental pollutants. Among electrophiles formed endogenously, 8-nitroguanosine 3’,5’-cyclic monophosphate (8-nitro-cGMP) has unique cell signaling functions, and pathways for its biosynthesis, signaling mechanism, and metabolism in cells have been clarified. Reactive persulfide species such as cysteine persulfides and polysulfides that are endogenously produced in cells are likely to be involved in 8-nitro-cGMP metabolism. These new aspects of redox biology may stimulate innovative and multidisciplinary research in cardiovascular physiology and pathophysiology. In our review, we focus on the redox-dependent regulation of electrophilic signaling via reduction and metabolism of electrophiles by reactive persulfides in cardiac cells, and we include suggestions for a new therapeutic strategy for cardiovascular disease..
9. Tsukasa Shimauchi, Takuro Numaga-Tomita, Tomoya Ito, Akiyuki Nishimura, Ryosuke Matsukane, Sayaka Oda, Sumio Hoka, Tomomi Ide, Norimichi Koitabashi, Koji Uchida, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy, JCI insight, 10.1172/jci.insight.93358, 2, 15, 2017.08, Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress-mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load-induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy..
10. Caroline Sunggip, Akiyuki Nishimura, Kakeru Shimoda, Takuro Numaga-Tomita, Tsuda Makoto, Motohiro Nishida, Purinergic P2Y6 receptors
A new therapeutic target of age-dependent hypertension, Pharmacological Research, 10.1016/j.phrs.2017.03.013, 120, 51-59, 2017.06, Aging has a remarkable effect on cardiovascular homeostasis and it is known as the major non-modifiable risk factor in the development of hypertension. Medications targeting sympathetic nerve system and/or renin-angiotensin-aldosterone system are widely accepted as a powerful therapeutic strategy to improve hypertension, although the control rates remain unsatisfactory especially in the elder patients with hypertension. Purinergic receptors, activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in many biological processes, including platelet aggregation, neurotransmission and hormone release, and regulation of cardiovascular contractility. Since clopidogrel, a selective inhibitor of G protein-coupled purinergic P2Y12 receptor (P2Y12R), achieved clinical success as an anti-platelet drug, P2YRs has been attracted more attention as new therapeutic targets of cardiovascular diseases. We have revealed that UDP-responsive P2Y6R promoted angiotensin type 1 receptor (AT1R)-stimulated vascular remodeling in mice, in an age-dependent manner. Moreover, the age-related formation of heterodimer between AT1R and P2Y6R was disrupted by MRS2578, a P2Y6R-selective inhibitor. These findings suggest that P2Y6R is a therapeutic target to prevent age-related hypertension..
11. Sarawuth Phosri, Ajaree Arieyawong, Kwanchai Bunrukchai, Warisara Parichatikanond, Akiyuki Nishimura, Motohiro Nishida, Supachoke Mangmool, Stimulation of adenosine A2B receptor inhibits endothelin-1-induced cardiac Fibroblast proliferation and α-smooth muscle actin synthesis through the cAMP/Epac/PI3K/Akt-signaling pathway, Frontiers in Pharmacology, 10.3389/fphar.2017.00428, 8, JUN, 2017.06, Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified. Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA. Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation. Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis..
12. Tsukasa Shimauchi, Akiyuki Nishimura, Tatsuya Ishikawa, Motohiro Nishida, Eco-pharma of approved drug focused on mitochondria fission, Folia Pharmacologica Japonica, 10.1254/fpj.149.269, 149, 6, 269-273, 2017.01.
13. Akiyuki Nishimura, Motohiro Nishida, Purinergic signaling in cardiovascular system, Folia Pharmacologica Japonica, 10.1254/fpj.149.84, 149, 2, 84-90, 2017.01.
14. Takuro Numaga-Tomita, Naoyuki Kitajima, Takuya Kuroda, Akiyuki Nishimura, Kei Miyano, Satoshi Yasuda, Koichiro Kuwahara, Yoji Sato, Tomomi Ide, Lutz Birnbaumer, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis, Scientific Reports, 10.1038/srep39383, 6, 2016.12, Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca 2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca 2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca 2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-Associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-Associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation..
15. Naoyuki Kitajima, Takuro Numaga-Tomita, Masahiko Watanabe, Takuya Kuroda, Akiyuki Nishimura, Kei Miyano, Satoshi Yasuda, Koichiro Kuwahara, Yoji Sato, Tomomi Ide, Lutz Birnbaumer, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling, Scientific Reports, 10.1038/srep37001, 6, 2016.11, Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca 2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca 2+-dependent Nox2 activation through TRPC3-mediated background Ca 2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca 2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca 2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities..
16. Akiyuki Nishimura, Caroline Sunggip, Hidetoshi Saitoh, Tsukasa Shimauchi, Takuro Numaga-Tomita, Katsuya Hirano, Tomomi Ide, Jean Marie Boeynaems, Hitoshi Kurose, Tsuda Makoto, Bernard Robaye, Kazuhide Inoue, Motohiro Nishida, Purinergic P2Y6 receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension, Science Signaling, 10.1126/scisignal.aac9187, 9, 411, 2016.01, The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR), promoted Ang II-induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduced b-arrestin-dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II-induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascularsmoothmuscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin-dependent proliferation to G protein- dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II..
17. Leanna R. Gentry, Akiyuki Nishimura, Adrienne D. Cox, Timothy D. Martin, Denis Tsygankov, Motohiro Nishida, Timothy C. Elston, Channing J. Der, Divergent roles of CAAX motif-signaled posttranslational modifications in the regulation and subcellular localization of Ral GTPases, Journal of Biological Chemistry, 10.1074/jbc.M115.656710, 290, 37, 22851-22861, 2015.09, The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxylterminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB..
18. Masaki Torii, Daisuke Kojima, Akiyuki Nishimura, Hiroshi Itoh, Yoshitaka Fukada, Light-dependent activation of G proteins by two isoforms of chicken melanopsins, Photochemical and Photobiological Sciences, 10.1039/c5pp00153f, 14, 11, 1991-1997, 2015.01, In the chicken pineal gland, light stimuli trigger signaling pathways mediated by two different subtypes, Gt and G11. These G proteins may be activated by any of the three major pineal opsins, pinopsin, OPN4-1 and OPN4-2, but biochemical evidence for the coupling has been missing except for functional coupling between pinopsin and Gt. Here we investigated the relative expression levels and the functional difference among the three pineal opsins. In the chicken pineal gland, the pinopsin mRNA level was significantly more abundant than the others, of which the OPN4-2 mRNA level was higher than that of OPN4-1. In G protein activation assays, Gt was strongly activated by pinopsin in a light-dependent manner, being consistent with previous studies, and weakly activated by OPN4-2. Unexpectedly, illuminated OPN4-2 more efficiently activated G protein(s) that was endogenously expressed in HEK293T cells in culture. On the other hand, Gq, the closest analogue of G11, was activated only by OPN4-1 although the activity was relatively weak under these conditions. These results suggest that OPN4-1 and OPN4-2 couple with Gq and Gt, respectively. Two melanopsins, OPN4-1 and OPN4-2, appear to have acquired mutually different functions through the evolution..
19. Hiroshi Itoh, Akiyuki Nishimura, Yusuke Nagai, [New research initiatives in the regulatory mechanisms of heterotrimeric G protein signaling]., Seikagaku. The Journal of Japanese Biochemical Society, 85, 7, 531-542, 2013.07.
20. Akiyuki Nishimura, Maurine E. Linder, Identification of a novel prenyl and palmitoyl modification at the CaaX motif of Cdc42 that regulates RhoGDI binding, Molecular and Cellular Biology, 10.1128/MCB.01398-12, 33, 7, 1417-1429, 2013.04, Membrane localization of Rho GTPases is essential for their biological functions and is dictated in part by a series of posttranslational modifications at a carboxyl-terminal CaaX motif: prenylation at cysteine, proteolysis of the aaX tripeptide, and carboxymethylation. The fidelity and variability of these CaaX processing steps are uncertain. The brain-specific splice variant of Cdc42 (bCdc42) terminates in a CCIF sequence. Here we show that brain Cdc42 undergoes two different types of posttranslational modification: classical CaaX processing or novel tandem prenylation and palmitoylation at the CCaX cysteines. In the dual lipidation pathway, bCdc42 was prenylated, but it bypassed proteolysis and carboxymethylation to undergo modification with palmitate at the second cysteine. The alternative postprenylation processing fates were conserved in the GTPases RalA and RalB and the phosphatase PRL-3, proteins terminating in a CCaX motif. The differentially modified forms of bCdc42 displayed functional differences. Prenylated and palmitoylated brain Cdc42 did not interact with RhoGDI and was enriched in the plasma membrane relative to the classically processed form. The alternative processing of prenylated CCaX motif proteins by palmitoylation or by endoproteolysis and methylation expands the diversity of signaling GTPases and enables another level of regulation through reversible modification with palmitate..
21. Mohamed Aittaleb, Akiyuki Nishimura, Maurine E. Linder, John J G Tesmer, Plasma membrane association of p63 Rho guanine nucleotide exchange factor (p63RhoGEF) is mediated by palmitoylation and is required for basal activity in cells, Journal of Biological Chemistry, 10.1074/jbc.M111.273342, 286, 39, 34448-34456, 2011.10, Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a Gα q-regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by Gα q. Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocate sp63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation- deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-δ1 pleckstrin homology domains or by co-expression with wild-type Gα q but not with palmitoylation-deficient Gα q.Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gα q, as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity..
22. Akiyuki Nishimura, Ken Kitano, Jun Takasaki, Masatoshi Taniguchi, Norikazu Mizuno, Kenji Tago, Toshio Hakoshima, Hiroshi Itoh, Structural basis for the specific inhibition of heterotrimeric G q protein by a small molecule, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1003553107, 107, 31, 13666-13671, 2010.08, Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered G q-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of Gq protein (Gαq) by inhibiting the GDP release from Gαq. X-ray crystal structure analysis of the Gαqβγ-YM- 254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gαq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Gα subunit and an insight into the molecular mechanism of G protein activation..
23. Yusuke Nagai, Akiyuki Nishimura, Kenji Tago, Norikazu Mizuno, Hiroshi Itoh, Ric-8B stabilizes the α subunit of stimulatory G protein by inhibiting its ubiquitination, Journal of Biological Chemistry, 10.1074/jbc.M109.063313, 285, 15, 11114-11120, 2010.04, The α subunit of stimulatory G protein (Gαs) activates adenylyl cyclase, which catalyzes cAMP production, and regulates many physiological aspects, such as cardiac regulation and endocrine systems. Ric-8B (resistance to inhibitors of cholinesterase 8B) has been identified as the Gαs-binding protein; however, its role in Gs signaling remains obscure. In this study, we present evidence that Ric-8B specifically and positively regulates Gs signaling by stabilizing the Gαs protein. An in vitro biochemical study suggested that Ric-8B does not possess guanine nucleotide exchange factor activity. However, knockdown of Ric-8B attenuated β-adrenergic agonist-induced cAMP accumulation, indicating that Ric-8B positively regulates Gs signaling. Interestingly, overexpression and knockdown of Ric-8B resulted in an increase and a decrease in the Gαs protein, respectively, without affecting the Gαs mRNA level.We found that the Gαs protein is ubiquitinated and that this ubiquitination is inhibited by Ric-8B. This Ric-8B-mediated inhibition of Gαs ubiquitination requires interaction between Ric-8B and Gαs because Ric-8B splicing variants, which are defective for Gαs binding, failed to inhibit the ubiquitination. Taken together, these results suggest that Ric-8B plays a critical and specific role in the control of Gαs protein levels by modulating Gαs ubiquitination and positively regulates Gs signaling..
24. Akiyuki Nishimura, Miyuki Okamoto, Yo Sugawara, Norikazu Mizuno, Junji Yamauchi, Hiroshi Itoh, Ric-8A potentiates Gq-mediated signal transduction by acting downstream of G protein-coupled receptor in intact cells, Genes to Cells, 10.1111/j.1365-2443.2006.00959.x, 11, 5, 487-498, 2006.05, RIC-8 was originally found by genetic studies on in vitro as a guanine nucleotide exchange factor for G protein α subunits. However, the physiological role of a mammalian homolog Ric-8A on G protein-coupled receptor signaling in intact cells is largely unknown. We isolated Ric-8A using a yeast two-hybrid system with Gαq and examined the role of Ric-8A on Gq-mediated signaling. The small interfering RNA of Ric-8A diminished the Gq-coupled receptor-mediated ERK activation and intracellular calcium mobilization in 293T cells. Ric-8A was translocated to the cell membrane in response to the Gq-coupled receptor stimulation. The expression of the myristoylation sequence-conjugated Ric-8A mutant was located in the membranes and shown to enhance the Gq-coupled receptor-mediated ERK activation. Moreover, this enhancement on ERK activation and the guanine nucleotide exchange activity of Ric-8A for Gαq were inhibited by Gq selective inhibitor YM-254890. These results suggested that Ric-8A potentiates Gq-mediated signal transduction by acting as a novel-type regulator in intact cells..
25. Norikazu Mizuno, Hiroshi Kokubu, Maiko Sato, Akiyuki Nishimura, Junji Yamauchi, Hitoshi Kurose, Hiroshi Itoh, G protein-coupled receptor signaling through Gq and JNK negatively regulates neural progenitor cell migration, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0506101102, 102, 35, 12365-12370, 2005.08, In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated. Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Gαq but not Gαi. The expression of constitutively active mutant of Gαq, GαqR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Gαq caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK..