Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells
Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells
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DOI：
10.1359/jbmr.2002.17.7.1190
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发表时间：
2002-07-01
影响因子：
6.2
通讯作者：
Chihara, K
中科院分区：
文献类型：
--
作者：
Sowa, H;Kaji, H;Chihara, K
转化生长因子（TGF）β大量储存在骨基质中，并且似乎调节骨代谢。虽然Smad家族蛋白是TGF-β信号通路的关键组成部分，但Smad 3在成骨细胞表型表达中的作用仍然知之甚少。因此，本研究通过使用稳定转染Smad 3的小鼠成骨细胞系MC 3 T3-E1细胞来阐明Smad 3在成骨细胞增殖、骨基质蛋白表达和矿化中的调节作用。与空载体相比，Smad 3显著抑制[H-3]胸苷掺入和MTT-染料测定的荧光强度。北方印迹显示，Smad 3可增加I型前胶原、骨桥蛋白（OPN）和基质Gla蛋白（MGP）mRNA的表达。Smad 3的这些作用模拟了TGF-β对相同细胞的作用。另一方面，与空载体相比，Smad 3极大地增强了MC 3 T3-E1细胞的ALP活性和矿化，尽管TGF-β抑制了野生型MC 3 T3-E1细胞的ALP活性和矿化。I型胶原合成抑制剂L-azetidine-2-carboxylic acid和骨钙素（OCN）可显著拮抗Smad 3刺激的MC 3 T3-E1细胞ALP活性和矿化。总之，这项研究表明，在小鼠成骨细胞中，Smad 3抑制增殖，但它也增强ALP活性，矿化和骨基质蛋白，如I型胶原（COLI），OPN和MGP的水平。我们认为Smad 3在成骨细胞骨形成中起着重要作用，可能有助于阐明骨形成的转录机制，并可能导致骨形成药物的开发。
Transforming growth factor (TGF) beta is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-beta signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [H-3]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-beta on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-beta inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.