Molecular and cell biolobical analysis of the smooth muscle cell differentiation

原代培养的平滑肌细胞(SMCs)在含血清的正常培养条件下迅速脱分化。我们研究了使用几种细胞外基质和生长因子或细胞因子来建立一种保持SMC分化表型的培养体系。从这些分析中，我们发现层粘连蛋白在无血清培养条件下具有维持SMC分化表型的效力。此外，我们还发现胰岛素样生长因子I、II(IGFII)或胰岛素具有维持SMC长期分化表型的显著活性，且IGFI是SMC分化最有效的因子，提示层粘连蛋白和IGFI受体的信号转导可能参与SMC的分化。使用几种激酶抑制剂的药理学研究结果表明，酪氨酸磷酸化和PI3激酶参与了这种信号转导。我们还发现IGFI受体的表达为S…更多地依赖于MC表型。因此，IGFI受体下调可能是血清生长因子诱导的SMC去分化的原因之一。利用我们的SMC培养系统，我们鉴定了SMC特异性基因的表达。在钙调蛋白(CAD)基因中，外显子3内两个5‘-剪接位点的交替选择决定了h-或L-CAD的表达。我们发现hnRNPA1在功能上参与了远端5‘-剪接位点的选择。我们发现在SMC去分化过程中，α-原肌球蛋白(α-TM)亚型的表达发生了从α-TM-SM到α-TM-F1和α-TM-F2的表达变化，并且这种转换与从h-到L-CAD的表达变化相一致，这表明在SMC中α-TM和CAD亚型的表达具有共同的剪接机制。我们还研究了血管内皮细胞中CAD和α1整合素启动子的转录调控。这些分析表明，在分化的SMC中，各自启动子区域的Carg box是这两个基因高水平转录所必需的，而血清反应因子(SRF)是Carg box结合的核心因素。我们证明了α-SM肌动蛋白在内脏SMC中的表达与在血管SMC中的表达相反；α-SM肌动蛋白在未分化和去分化的内脏SMC中表达，但在分化的内脏SMC中不表达，并在α-SM肌动蛋白的启动子区域发现了一个新的顺式元件，它起负调控作用。较少

Primarily cultured smooth muscle cell (SMCs) rapidly dedifferentiate under normal culture conditions containing serum. We investigated to establish a culture system maintaining a differentiated phenotype of SMCs using several extracellular matrices and growth factors or cytokines. From these analyzes, we found that laminin has a potency to maintain a differentiated phenotype of SMCs under serum-free culture conditions. Furthermore, we obtained evidence that insulin-like growth factor I (IGFI), II (IGFII), or insulin possesses the remarkable activity to maintain a differentiated phenotype of SMCs for a long culture, and IGFI is a most potent factor for SMC differentiation, suggesting that signal transduction via laminin and IGFI receptors would be involved in SMC differentation. Pharmacological studies using several kinase inhibitors have revaled that tyrosine phosphorylation and PI3 kinase are involved in such signal transduction. We also found that the expression of IGFI-recepter is S … More MC phenotype-dependent. Therefore, the downregulation of IGFI-recepter might be one reason for dedifferentation of SMCs induced by serum growth factors. Using our SMC culture system, we characterized the SMC-specific gene expressions. In the caldesmon (CaD) gene, alternative selection of two 5'-splice sites within exon 3 determined h- or l-CaD expression. We found that hnRNPA1 is functionally involved in the selection of distal 5'-splice site. We found that expressional change of alpha-tropomyosin (alpha-TM) isoforms from alpha-TM-SM to alpha-TM-F1 and alpha-TM-F2 during dedifferentiation of SMCs and such conversion occurrs coordinately with the expressional change from h- to l-CaD,suggesting a common splicing mechanism for the phenotype-dependent expression of alpha-TM and CaD isoforms in SMCs. We also characterized transcriptional regulation of the CaD and the alpha1 integrin promoters in SMCs. These analyzes revealed that the CArG box within respective promoter regions are necessary for high level transcription of the both genes in differentiated SMCs, and the serum response factor (SRF) is a core factor for the CArG box binding. We demonstrated that the expression of alpha-SM actin in visceral SMCs is opposite to that in vascular SMCs ; alpha-SM actin is expressed in undifferentiated and dedifferentiated visceral SMCs, but not in differentiated visceral SMCs, and identified a novel cis-element in the promoter region of alpha-SM actin which acts as a negative regtulator. Less