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去分化。我们研究了利用几种细胞外基质和生长因子或细胞因子建立维持SMCs分化表型的培养系统。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SMCs分化表型的能力。此外，我们获得的证据表明，胰岛素样生长因子I （IGFI）、II （IGFII）或胰岛素具有显著的活性，可以在长时间培养中维持SMCs的分化表型，而IGFI是SMC分化的最有效因素，这表明通过层粘连蛋白和IGFI受体的信号转导可能参与了SMC的分化。使用几种激酶抑制剂的药理学研究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受体的表达是S…More MC表型依赖性的。因此，igfi受体的下调可能是血清生长因子诱导SMCs去分化的原因之一。利用我们的SMC培养系统，我们表征了SMC特异性基因的表达。在caldesmon （CaD）基因中，外显子3内两个5'剪接位点的选择性选择决定了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α -原肌球蛋白（α - tm）亚型在SMCs去分化过程中从α - tm - sm到α - tm - f1和α - tm - f2的表达变化与h- CaD到l-CaD的表达变化是协调发生的，提示SMCs中α - tm和CaD亚型的表型依赖表达存在共同的剪接机制。我们还鉴定了SMCs中CaD和α 1整合素启动子的转录调控。这些分析表明，在分化的SMCs中，各自启动子区域内的CArG box对于这两个基因的高水平转录是必需的，而血清反应因子（SRF）是CArG box结合的核心因子。我们发现- sm肌动蛋白在内脏间充质细胞中的表达与在血管间充质细胞中的表达相反；α - sm肌动蛋白在未分化和去分化的内脏SMCs中表达，但在已分化的内脏SMCs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，作为负调节因子。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