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

Abstract

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling

“…Contrary to our expectation, we found increased bone mineral density and trabecular bone volume in these mice, indicating that PRIP deficiency triggers bone mass increase as a result of increased bone formation and/or decreased bone resorption, independent of the reproductive hormone imbalance (26). We further determined that PRIP deficiency stimulates bone morphogenetic protein signaling, resulting in increased bone mass (27,28). However, the role of PRIP in osteoclastogenesis is not yet fully clarified.…”

Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts

“…Culture medium was changed every 3 days. Primary osteoblasts isolated from the calvaria of newborn mice [37] were grown under 5% CO 2 in α-Minimum Essential medium containing 20% fetal bovine serum, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin.…”

The role of biomechanical forces and MALAT1/miR‐329‐5p/PRIP signalling on glucocorticoid‐induced osteonecrosis of the femoral head

“…In 2015, Japanese researchers 17 demonstrated that PRIP was implicated in BMP‐induced osteoblast differentiation through negative regulation of Smad phosphorylation, which resulted from methylation of inhibitory Smad6. In 2017, these researchers demonstrated that PRIP deficiency impaired osteoclast differentiation, particularly at the early stages, and that PRIP stimulated osteoclast differentiation though calcium‐calcineurin‐NFATc1 signalling via regulation of intracellular Ca2+ in PRIP‐KO osteoclasts 18 .…”

“…Additional evidence is provided by osteoclast differentiation stimulated through calcium-calcineurin-NFATc1 signalling by regulating intracellular Ca 2+ in PRIP-KO osteoclasts. [15][16][17] Based on recent advances in elucidating the pathogenic mechanisms of GIONFH, we investigated the effects of biomechanical forces, bone metabolism and non-coding RNA in glucocorticoidinduced osteonecrosis of the femoral head. In this study, we observed the microstructure of femoral head in a rat model of GIONFH, before and after exercise on a treadmill or the tail suspension test that results in skeletal unloading.…”

“…This similarity is implicated in BMP‐induced osteoblast differentiation by the negative regulation of Smad phosphorylation through the methylation of inhibitory Smad6. Additional evidence is provided by osteoclast differentiation stimulated through calcium‐calcineurin‐NFATc1 signalling by regulating intracellular Ca 2+ in PRIP‐KO osteoclasts 15‐17 …”