ANALYSIS OF SIGNAL TRANSDUCTION PATHWAY IN BONE INDUCTION BY BONE MORPHOGENETIC PROTEIN

(1) BMP对小鼠Smad-1的磷酸化Mouse Smad-1 cDNA片段采用PCR方法从小鼠骨髓源性基质细胞ST-2的cDNA中获得1.6kb，并将具有Flag报道基因的蛋白表达载体中的cDNA转染至ST-2细胞中。用OP-1、BMP-2和TGF-β处理转化的细胞，然后用针对 Flag 蛋白的抗体。结果，在用这些生长因子处理的细胞中观察到mSmad的磷酸化。这些结果表明Smad在成骨细胞分化的信号转导途径中发挥着重要作用。(2)cbfa-1基因表达的诱导我们检测了runt家族转录因子cbfa-1的基因表达,该转录因子在骨形成中发挥着重要作用。 OP-1处理ST-2或MC3T3-E1细胞48小时后诱导的cbfa-1基因表达是对照的2至3倍。因此，结果表明BMP信号通过Smad磷酸化传递至cbfa-1并刺激成骨细胞分化。

(1) Phospholilation of mouse Smad-l by BMPMouse Smad-1 cDNA fragment of 1.6 kb was obtained from cDNA of mouse bone marrow derived stroma cell : ST-2 using PCR method, and the cDNA in protein expression vector with Flag reporter gene was transfected into ST-2 cell.. The transformed cell was treated with OP-1, BMP-2, and TGF-beta, and then cell lysate was imunoprecipitated with the antibody against Flag protein. As a result, phospholilation of mSmad was observed in the cell treated with these growth factors. These results suggest Smad play important role in the signal transudation pathway of osteoblastic differentiation.(2) Induction of cbfa-1 gene expressionWe examined the gene expression of runt family transcriptional factor, cbfa- 1, which plays important role in bone formation. The treatment of OP-1 to ST-2 or MC3T3-El cell induced induced cbfa-l gene expression two to three times by control after 48 hours. Thus, the result suggests that BMP signal is transmitted to cbfa-l byway of Smad phosphlilation and stimulates osteoblastic differentiation.