On the analysis and regulatory mechanism of genes responsible for mechanical stress-induced osteoblast differentiation and osteogenesis

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Neonatal mouse calvarial sutures were cultured under tensile stress (TS) and genes either upregulated or downregulated were analyzed by RT-PCR and RAP-PCR. BMP-4, α-adaptinC, tetranectin and an novel gene, designated as J9, were among many of affected genes. BMP-4 was induced in mesenchymal fibroblastic cells, which later differentiated into preosteoblasts and osteoblasts. Whereas the role of BMP-4 in osteoblast differentiation is well known, those of other three genes were not. In our study, teteranectin expression was transiently upregulated during osteoblast differentiation and diminished before calcification took place. J9 expression gradually increased with osteoblast differentiation. Very recently, tetranectin-null mice were reported to develop spinal deformity. We also identified J9 as a soluble form of IL-1 receptor associated protein, which supposed to inhibit the IL-1 signal transduction, thereby may affect apoptosis of osteoblasts. We also confirmed that J9 increases as oste … More oblasts differentiate and mature. In situ hybridization study revealed that α-adaptinC mRNA induced in mesenchymal fibroblasts as well as preosteoblasts and osteoblasts. The induction may occur earlier than 3hr TS loading, since the protein product was also detected at 3 hr. Thus the gene appears to be upregulated even earlier than BMP-4. In contrasts to these genes, alpha adaptin C expression did not change with osteoblast differentiation. Thus α-adaptinC seems to be induced by TS per se. Alpha-adaptin C is a component of clathrin-adaptor complex AP2 and plays a role in endocytosis of molecules such as epidermal growth factor receptor (EGFR). We demonstrated that overexpression of α-adaptinC in A431 cells increased endocytosis of EGFR and that TS increased phosphorylation of ERKs, the event known to be stimulated by endocytosis of EGFR. Furthermore, extracellular potassium depletion, a milieu known to be inhibitory to endocytosis, suppressed TS-induced osteoblast differentiation. Thus all four genes seem to be positively involved in the TS-induced osteogenesis. Less