PERK is essential for neonatal skeletal development to regulate osteoblast proliferation and differentiation

Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk-/- mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology.     

Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype

Although a number of secreted factors have been demonstrated to be bone regulators, none of these are unique to bone. Using a viral-based signal-trap strategy we have identified a novel gene we have termed "osteocrin." A 1280-bp mRNA encodes osteocrin producing a mature protein of 103 amino acids with a molecular mass of 11.4 kDa. Osteocrin shows no homology with any known gene except for two conserved sequence motifs reminiscent of dibasic cleavage sites found in peptide hormone precursors. Immunofluorescence and Western blot analysis confirmed the secretory nature of osteocrin. Two protein species were identified in the medium of cells overexpressing osteocrin, a full-length 11.4 kDa species and a processed approximately 5 kDa species. Mutation of the 76KKKR79 dibasic cleavage site abolished the appearance of this smaller osteocrin fragment. By in situ hybridization in mouse embryos, osteocrin was expressed specifically in Cbfa-1-positive, osteocalcin-negative osteoblasts. Immunohistochemistry on adult mouse bone showed osteocrin localization in osteoblasts and young osteocytes. By Northern blot analysis, osteocrin expression was only detected in bone, expression peaking just after birth and decreasing markedly with age. In primary osteoblastic cell cultures osteocrin expression coincided with matrix formation then decreased in very mature cultures. Treatment of cultures with 1,25-dihydroxyvitamin D3 resulted in a rapid dose-dependent down-regulation of osteocrin expression, suggesting direct regulation. Chronic treatment of primary cultures with osteocrin-conditioned media inhibited mineralization and reduced osteocalcin and alkaline phosphatase expression. These results suggest that osteocrin represents a novel, unique vitamin D-regulated bone-specific protein that appears to act as a soluble osteoblast regulator.     

脉冲电磁场促进大鼠成骨细胞成熟与矿化依赖于NO/cGMP信号途径

脉冲电磁场(pulse electromagnetic fields, PEMFs)能够促进大鼠成骨细胞（rat osteoblast cells, ROB）成熟矿化,但是其作用机制并不明确。本实验主要研究PEMFs促进大鼠成骨细胞成熟矿化与NO/cGMP信号途径的关系,进而阐明PEMFs促进成骨细胞成熟矿化的机理。首先将成骨细胞经50Hz、0.6mT脉冲电磁场作用不同时间后,检测细胞培养液中一氧化氮(Nitric Oxide, NO)和细胞内3?-5?-环鸟苷一磷酸(3?-5?-cyclic-GMP, cGMP)的含量,以探明电磁场是否影响NO和cGMP的合成;其次,提取总蛋白,应用蛋白质印迹检测细胞内eNOS、iNOS和PKG-1的蛋白表达量;最后,利用NOS的阻断剂L-NAME抑制NO信号通路后,检测成骨性相关指标,包括碱性磷酸酶（ALP）活性、钙化结节数量、成骨性基因Bmp-2、Collagen-1、Osterix及破骨细胞调节因子Rankl基因的表达量。结果发现经PEMFs处理后,NO含量及cGMP含量均有明显升高;细胞内eNOS、iNOS和PKG-1蛋白表达量较空白对照组均有显著升高,说明PEMFs能够激活NO/cGMP信号途径。且经PEMFs处理的成骨细胞,ALP活性升高,BMP-2、Collagen-1和Osterix基因表达量显著增加,Rankl基因表达量下降,成骨细胞形成钙化结节的能力增强,当加入L-NAME,PEMFs引起的ALP活性增加、成骨性基因表达升高和钙化结节形成能力增强的趋势均被显著抑制。上述结果表明,经PEMFs处理成骨细胞成熟矿化过程中 NO/cGMP信号通路被激活;如该通路被抑制,则电磁场促成骨作用被抵消。说明脉冲电磁场促进成骨细胞成熟矿化依赖于NO/cGMP信号通路。     

HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.     

Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype.

Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH.