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

新生小鼠颅骨缝在张应力(TS)下培养，用RT-PCR和RAP-PCR分析上调或下调的基因。BMP-4、α-Adaptin C、Tetranectin和一个名为J9的新基因是许多受影响的基因之一。BMP-4在间充质成纤维细胞中被诱导，随后分化为成骨前细胞和成骨细胞。虽然BMP-4在成骨细胞分化中的作用是众所周知的，但其他三个基因的作用还不是很清楚。在我们的研究中，teteranectin的表达在成骨细胞分化过程中短暂上调，并在钙化发生之前减弱。随着成骨细胞的分化，J9的表达逐渐增强。最近，据报道，Tetranectin基因缺失的小鼠出现了脊柱畸形。我们还发现J9是一种IL-1受体相关蛋白的可溶性形式，它可能抑制IL-1信号转导，从而可能影响成骨细胞的凋亡。我们还确认J9增加为OSTE…更多的成体细胞分化和成熟。原位杂交结果显示，α-Adaptin C基因在间充质成纤维细胞、成骨前细胞和成骨细胞中均有表达。由于蛋白质产物也是在3hr检测到的，因此诱导可能发生在3hr的TS负荷之前。因此，该基因似乎比BMP-4更早上调。与这些基因相比，α-Adaptin C的表达不随成骨细胞分化而改变。因此，α-AdaptinC似乎是由TS本身诱导的。α-Adaptin C是网状蛋白-适配器复合体AP2的一种成分，在表皮生长因子受体(EGFR)等分子的内吞过程中发挥作用。我们证明，在A431细胞中过表达α-Adaptin C增加了EGFr的内吞作用，TS增加了ERKs的磷酸化，这一事件已知是由EGFr的内吞作用刺激的。此外，细胞外钾的耗竭，一种已知的抑制内吞作用的环境，抑制了TS诱导的成骨细胞分化。因此，这四个基因似乎都与TS诱导的成骨密切相关。较少

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