Developing a culture system of differentiated smooth muscle cells and phathological application

原代培养的平滑肌细胞（SMC）在正常培养条件下快速去分化。我们研究了使用几种细胞外基质和生长因子或细胞因子建立维持SMC分化表型的培养系统。从这些分析中，我们发现层粘连蛋白具有在无血清培养条件下维持SMC分化表型的潜能。此外，我们获得的证据表明，在几种生长因子和细胞因子中，胰岛素样生长因子I（IGFI）、II（IGPII）或胰岛素具有显著的活性，以维持SMC的分化表型，使其长时间培养，IGFI是SMC分化的最有效因子。这些结果表明，通过层粘连蛋白和IGFI受体参与SMC分化的信号转导。我们还发现IGFI受体的表达是SMC表型依赖性的。IGFI受体表达下调可能是平滑肌细胞去分化的原因之一 ...更多信息 血清生长因子。使用我们的SMC培养系统，我们的特点是钙调蛋白（CaD）和α 1整合素启动子的转录调控。这些分析表明，CArG盒在SMCs中的高水平转录中起着至关重要的作用，血清反应因子（SRF）是CArG盒结合的核心因子。我们证明，α-SM肌动蛋白在内脏SMC中的表达是相反的，在血管平滑肌细胞; 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。我们在α-SM肌动蛋白基因的启动子区发现了一个新的顺式元件，它起负调控作用。在CaD基因中，外显子3内的两个5 '-剪接位点的选择性选择决定了h-或l-CaD同种型的表达。我们发现hnRNPA 1在SMCs远端5 '剪接位点的选择中起作用。我们发现，平滑肌细胞去分化过程中α-原肌球蛋白（alpha-tropomyosin，alpha-TM）异构体的表达变化是由相互排斥的外显子2a和2b之间的选择性变化引起的，并且这种变化与CaD异构体的变化协调发生，提示alpha-TM和CaD异构体的SMC表型依赖性表达存在共同的剪接机制。少

Primarily cultured smooth muscle cells (SMCs) rapidly dedifferentiate under normal culture conditions. 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 among several growth factors and cytokines possesses a remarkable activity to maintain the differentiated phenotype of SMCs for a long culture, and IGFI is a most potent factor for SMC differentiation. These results suggest the involvement of signal transduction via laminin and IGFI receptors in SMC differentiation. We also found that the expression of IGFI-receptor is SMC phenotype-dependent. Therefore, the downregulation of IGFI-recepter might be one reason for dedifferentiation of SMCs … More by serum growth factors. Using our SMC culture system, we characterized the transcriptional regulation of the caldesmon (CaD) and the alpha1 integrin promoters. These analyzes revealed that the CArG box plays a vital role for high level transcription of the both genes in 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. We identified a novel cis-element in the promoter region of alpha-SM actin gene which acts as a negative regulator. In the CaD gene, alternative selection of two 5'-splice sites within exon 3 has determined the expression of h-or l-CaD isoform. We found functional involvement of hnRNPA1 in the selection of distal 5'-splice site in SMCs. We found that expressional change of alpha-tropomyosin (alpha-TM) isoforms during dedifferentiation of SMCs is arisen by a selectional change between mutually exclusive exons, exons 2a and 2b, and such change occurrs coordinately with CaD isoformal change, suggesting a common splicing mechanism for SMC phenotype-dependent expression of alpha-TM and CaD isoforms. Less