Involvement of PRIP, Phospholipase C-related, but Catalytically Inactive Protein, in Bone Formation

Abstract

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.Phospholipase C-related, but catalytically inactive protein (PRIP) 4 was first identified as a novel D-myo-inositol-1,4,5-trisphosphate-binding protein and tentatively named p130 based on molecular size (1). Subsequent molecular cloning studies revealed that the molecule is similar to phospholipase C-␦1 but is catalytically inactive, which is the reason for its revised name, and is expressed predominantly in the brain (2-5). Later, an isoform with relatively broad tissue distribution, including the brain, was reported (6, 7), indicating that PRIP is composed of types 1 and 2. PRIP has a number of binding partners, including GABARAP (␥-aminobutyric acid type A (GABA A ) receptorassociated protein) (8), protein phosphatase-1␣ (9 -11) and -2A (12), and GABA A receptor ␤ subunits (12-14), besides Ins(1,4,5)P 3 (1-5). The finding of these binding partners led us to examine the possible involvement of PRIP in Ins(1,4,5)P 3 -mediated Ca 2ϩ signaling (15, 16) and GABA A receptor signaling (17-20), using cloned culture cells reconstituted with the gene of interest or the gene-silencing technique, and PRIP-1 alone and/or PRIP-1 and -2 gene-deficient mice were also analyzed for GABA A receptor signaling from multiple aspects, including isolated neuron cultures, electrophysiological analysis of brain slices, and behavioral analysis. So far, it is known that there is no great difference between types 1 and 2 regarding the capability of binding to partners described above, therefore, we have mainly used PRIP-1 and -2 gene-deficient mice for further analysis, but not single gene-deficient mice.During the course of mutant animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, i...

Involvement of PRIP (Phospholipase C‐Related But Catalytically Inactive Protein) in BMP‐Induced Smad Signaling in Osteoblast Differentiation

“…We reported that the increased bone mass observed in PRIP-KO mice was caused by enhanced bone formation, and BMP stimulation of newborn calvaria cells caused the prolonged phosphorylation of Smad1/5, probably explaining the enhanced bone formation as one of the many underlying mechanisms [Tsutsumi et al, 2011]. The present study was undertaken to investigate the mechanisms of how PRIP is implicated in the BMP signaling pathway.…”

“…Smad1/5 is a main factor for BMP signaling and the phosphorylation of Smad1/5 C-terminal is an initial step to transmit the BMP signaling cascade following BMP binding to the receptors [Massague et al, 2005;Katagiri, 2008]. We previously reported that Smad1/5 phosphorylation by BMP stimulation in KO calvaria cells was more evident than that in WT cells; particularly after the peak than at a peak, the results led us to assume that PRIP might be involved in the dephosphorylation process [Tsutsumi et al, 2011].…”

“…Three‐dimensional analysis of the femurs of female mice, histomorphometrical assay of bone formation parameters, including the bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number, and biochemical analysis using primary preosteoblast cell cultures were performed. All these results indicated increased bone mass in KO mice, caused by enhanced bone formation, indicating that PRIP is implicated in the regulation of bone properties, independently of general hormonal regulation [Tsutsumi et al, ].…”

Phospholipases of Mineralization Competent Cells and Matrix Vesicles: Roles in Physiological and Pathological Mineralizations

“…PRIP has a number of binding partners, including the catalytic subunit of protein phosphatase 1α (PP1α) and PP2A [512,513] and the phosphorylated (active) form of Akt [514] in addition to IP 3 and PIP 2 [515]. PRIP gene-deficient mice ( prip −/− ), genetically deficient in type 1 or type 2 isoforms or both, brought light the physiological functions of PRIP: modulation of GABAA receptor signaling [516], dysfunction of reproduction, negative regulation of multiple-hormone secretion [517] and bone properties [518]. PRIP is involved in the phosphorylation-dependent modulation of exocytosis in PC-12 cells [519].…”

“…Exocytosis of various peptide hormones, such as gonadotropins and insulin, was up-regulated in prip −/− mice, indicating that PRIP is likely to be involved in dense core vesicle exocytosis in a negative manner. PRIP is implicated in the regulation of bone formation in a negative manner, partly through the regulation of SMAD phosphorylation [518]. Since PC-PLC contributes to the progression of atherosclerosis, it was proposed that pharmacological blockade of PC-PLC is a possible approach to atherosclerosis therapy.…”