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Bone is an important tissue contributing to the regulation of homeostasis in the body and bone remodeling is stringently regulated by a balance between bone resorption by osteoclasts and bone formation by osteoblasts.
Phospholipase C (PLC)-related, but catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate binding protein, which is conserved among different species, but its biological function remains elusive. We therefore generated PRIP gene-deficient (KO) mice to gain insights into the biological function. Base on the expression pattern of PRIP, we initially focused on its function in the inhibitory signaling in the central nervous system and in the regulation of protein phosphorylation in signal transduction. However, we unexpectedly found an increased bone mass in KO mice and therefore have been studying the involvement of PRIP in bone metabolism. Our results indicate that PRIP positively regulates osteoclast differentiation through calcium-calcineurin-NFAT signaling.
We thus revealed the involvement of PRIP in bone metabolism through reverse genetics, however we never imagined of this possibility when we generated the KO mice. Reverse genetics is one of the best approaches to identify diverse function of a gene/protein. Furthermore, it all started when we noticed a thing in KO mice.
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Miho Matsuda, Ayako Murakami, Masato Hirata, Involvement of phospholipase C-related but catalytically inactive protein in the early stage of osteoclast differentiation, cell biology 2016 ASCB Annual Meeting, 2016.12, Phospholipase C-related but catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient (PRIP-KO) mice and reported that PRIP-KO mice exhibited the upregulation of the bone mineral density and trabecular bone volume, due to the enhancement of bone formation while decreased bone resorption. In this study, we investigated the possible mechanisms by which PRIP regulates bone resorption. Orthodontic tooth movement was performed using wild type (WT) and PRIP-KO mice equipped with orthodontic devices and analyzed by micro-computed tomography and TRAP staining. In the mutant mice, tooth movement was reduced compared with wild type (WT) mice, and osteoclasts were observed at the lesser amount on pressure side in KO maxillary bones. In vitro osteoclast formation assay and flow cytometric analysis were also performed and revealed the decreased differentiation for osteoclast in bone marrow cells isolated from KO mice. The expression of genes implicated in osteoclast differentiation and bone resorption was decreased in KO mice. Immunoblot analysis using cultured bone marrow cells indicated the lower expression of calcineurin in KO cells, and the phosphatase activity by calcineurin was decreased in KO cells. The expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), which is an essential factor for osteoclast differentiation and the activity is regulated by calcineurin, was also decreased in KO cells in the early stage of osteoclast differentiation. These results suggest that PRIP positively regulates osteoclast differentiation, especially in the early stage, probably through calcium-calcineurin-NFAT signaling. . |
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Phospholipase C-related catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel inositol 1,4,5-trisphosphate binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and -2 double knockout (DKO) mice and found their reduced fertility: DKO mice exhibited reduced fertility in the female such as decreased number of pups, longer estrous days, increased secretion of gonadotropins, etc. This time we examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were remarkably decreased in DKO female mice, indicating that PRIP plays an important role in follicle maturation and/or ovulation, but not fertilization or implantation. Microarray and quantitative real-time PCR analyses revealed the increased expressions of molecules involved in ovulation including Has2, Ptgs2, PTX3, Tnfaip6 and Areg in both genotype. We examined the ability of COC expansion in both genotypes but little difference was observed. On the other hand, histological analysis revealed more immature follicles and fewer mature follicles or corpus lutea in DKO ovaries. Immunological analysis showed the expression of luteinizing hormone receptors (LHR) at earlier stages of follicle maturation in DKO ovary, and elevated level of ERK phosphorylation in DKO granulosa cells. These results suggest that the appearance of LHR at immature follicle stages leads to suppress follicle maturation by excessive LH signaling in DKO ovary, indicating that PRIP may be involved in positive regulation of ovarian follicle maturation through LH signaling. . |
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Phospholipase C-related catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel inositol 1,4,5-trisphosphate binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and –2 double knockout (DKO) mice to gain insight into the biological function. DKO mice exhibited reduced fertility in the female such as decreased number of pups, longer estrous days, increased secretion of gonadotropins, etc. We examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were remarkably decreased in DKO female mice, indicating that PRIP plays an important role in follicle maturation and/or ovulation, but not fertilization or implantation. Microarray and quantitative real-time PCR analyses revealed the increased expressions of molecules involved in ovulation including Has2, Ptgs2, PTX3, Tnfaip6 and Areg. We examined the ability of COC expansion in both genotypes but little difference was observed. On the other hand, histological analysis of ovaries from both genotypes revealed more immature follicles and fewer mature follicles or corpus lutea in DKO ovaries. Immunofluorescence analysis showed the expression of luteinizing hormone receptors (LHR) at earlier stages of follicle maturation in DKO ovary and more increase of antral follicles. These results are summarized as below: the increased LH secretion from pituitary gland and the appearance of LHR at immature follicle stages lead excessive LH signaling to suppress follicle maturation in DKO females, indicating that PRIP may be involved in positive regulation of ovarian follicle maturation through LH signaling. . |
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Miho Matsuda, Masato Hirata, Involvement of a novel signaling molecule, PRIP in the regulation of reproduction system
, The 22th IUBMB, 37st FEBS Congress, 2012.09, PRIP (PLC-Related, but catalytically Inactive Protein) は、Ins(1,4,5)P3 結合性の分子として見いだされた新規分子であり、種を越えて広く存在するタンパク質である。２つのサブタイプが存在し、PRIP-1は脳に多く発現しているのに対し、PRIP-2は比較的ユビキタスな発現パターンを示す。我々は、PRIP-1及びPRIP-2のダブルノックアウトマウスを作製し、本変異マウスにおいて総出産仔数の減少、血中性腺刺激ホルモン量の増加などの表現型を見いだし、性周期制御にPRIPが強く関与することを明らかにした。排卵数を調べるため、PMSG（妊馬血清性ゴナドトロピン）、HCG（人絨毛生性腺刺激ホルモン）を用いた排卵誘発を行ったところ、変異型で激減していた。このことから、出産仔数の減少は、排卵後の授精や着床における異常によるものではなく、卵成熟を含めた排卵に至るまでの過程における異常によるものであることが示唆された。そこで、まず排卵前後における遺伝子発現の変化を検討するために、卵巣RNAを用いたマイクロアレイ解析を行った。その結果、PMSG投与後48時間でHCGを投与した卵巣において、排卵前の卵胞で発現するHas2 (hyaluronan synthase 2)、Ptgs2 (cyclooxygenase 2)、PTX3 (pentraxin 3)、Tnfαip6 (TNFα-induced protein 6)などの発現量が、変異型で増加していた。また、同時期の卵巣の組織学的解析より、変異型において卵母細胞が残ったまま黄体化したような所見があり、これらのことから排卵時の制御におけるPRIPの関与が示唆された。そこで現在、野生型及び変異型マウスにおいて、排卵直前に起こる急激な変化（卵丘細胞-卵母細胞複合体のexpansionやヒアルロン酸の発現量及び局在等）について解析を進めている。. |
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Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and –2 double knockout (DKO) mice to gain insight into the biological function. DKO mice showed dysfunction of the reproductive system in the female. We examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were significantly decreased in mutant female mice, compared to that seen with wild-type, which was also confirmed by histological analysis of the ovaries after ovulation. These results suggested that PRIP plays an important role in not fertilization or implantation but follicle maturation and/or ovulation. Microarray analysis indicated the increased expressions of ovulation related molecules such as Has2, Ptgs2, PTX3, Tnfαip6 and Ereg. Historogical analysis of ovaries, just before ovulation, showed some abnormal follicles in the DKO. We also examined the ability of COC expansion in both genotypes and the significantly difference was not observed. On the other hand, TUNEL assay was performed to detect apoptotic follicles related to follicle maturation. There were some differences between in WT and DKO ovaries, depending on the follicle stages. Further analyses are underway to reveal the role of PRIP on follicle maturation and ovulation. . |
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Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and –2 double knockout (DKO) mice to gain insight into the biological function. DKO 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 indicated that the cause appeared to be on the female side. The observation of the estrous cycle in mice by histological analysis of vaginal smears showed that the estrous days were apparently increased in DKO mice. Levels of serum LH and FSH 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 DKO mice. We also examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were significantly decreased in mutant female mice, compared to that seen with wild-type, which was also confirmed by histological analysis of the ovaries after ovulation. These results suggest that PRIP plays an important role in female reproductive system, especially in gonadotropin secretion, ovarian follicle maturation and ovulation.. |
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Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and -2) was first identified as a novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein, but the biological functions have remained elusive. We therefore generated PRIP-1 and –2 double knockout (DKO) mice to gain insight into the biological function. DKO 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 indicated that the cause appeared to be on the female side. The observation of the estrous cycle in mice by histological analysis of vaginal smears showed that the estrous days were apparently increased in DKO mice. Levels of serum LH and FSH 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 DKO mice. We also examined the ability of the ovaries in response to gonadotropins: the total number of the ovulated oocytes were significantly decreased in mutant female mice, compared to that seen with wild-type, which was also confirmed by histological analysis of the ovaries after ovulation. The analysis also showed that the numbers of follicles originally were not significantly different between WT and DKO ovaries. These results suggest that PRIP plays an important role in female reproductive system, especially in gonadotropin secretion and ovarian follicle maturation.. |
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PRIP (phospholipase C related, but catalytically inactive protein) was identified as a novel inositol 1,4,5-trisphosphate binding protein, whose domain organization is similar to that of phospholipase C-_1 but is catalytically inactive, comprizing two isoforms, PRIP-1 and PRIP-2. To get insight into the biological functions, we generated the double knockout (DKO) mice of both PRIP-1 and PRIPﾐ2, followed by the phenotypic analyses. DKO mice grew normally and became fertile. However DKO female exhibited the decreased litter occasion and litter size, indicating the dysfunction of female reproductive system. Then we examined the estrus cycle by cytological analysis of daily vaginal smears, resulting in the irregular cycle in DKO mice. We further noticed that the mutant mice had apparently smaller sized uterus by gross anatomical observation. Basal levels of serum leuteinizing hormone and follicle stimulating hormone were significantly higher in the mutant, the event of which was also confirmed by examining the secretion from the tissue culture of anterior pituitary glands. These results suggest that PRIP is involved in maternal reproduction, through the gonadotropine secretion.. |
| 10. |
PRIP (phospholipase C related, but catalytically inactive protein) was identified as a novel inositol 1,4,5-trisphosphate binding protein, whose domain organization is similar to that of phospholipase C-_1 but is catalytically inactive, comprizing two isoforms, PRIP-1 and PRIP-2. To get insight into the biological functions, we generated the double knockout (DKO) mice of both PRIP-1 and PRIPﾐ2, followed by the phenotypic analyses. DKO mice grew normally and became fertile. However DKO female exhibited the decreased litter occasion and litter size, indicating the dysfunction of female reproductive system. Then we examined the estrus cycle by cytological analysis of daily vaginal smears, resulting in the irregular cycle in DKO mice. We further noticed that the mutant mice had apparently smaller sized uterus by gross anatomical observation. Basal levels of serum leuteinizing hormone and follicle stimulating hormone were significantly higher in the mutant, the event of which was also confirmed by examining the secretion from the tissue culture of anterior pituitary glands. These results suggest that PRIP is involved in maternal reproduction, through the gonadotropine secretion.. |
