Gene expressional mechanism in smooth muscle cells

原代培养的平滑肌细胞（SMCs）在含有血清的正常培养条件下迅速去分化。我们搜索了维持分化表型的SMC培养条件，发现层粘连蛋白具有延缓SMC去分化进程的效力。此外，我们发现胰岛素样生长因子（IGFI，IGFII）或胰岛素具有显着的活性，可以长时间维持分化表型，IGFI是SMC分化的最有效因子。加入抗igfi抗体可抑制层粘连蛋白的单独作用。这些结果表明，通过IGFI/IGFI受体的信号转导可能参与了SMC分化。相反，其他生长因子/细胞因子诱导SMC去分化。基于这些证据，我们结合基因表达调控研究了参与SMC表型调节的信号转导。在上述条件下培养的SMCs中，IGFI激活了磷酸肌肽3-激酶…More （PI3激酶）和蛋白激酶B (Aktl)，而丝裂原激活的蛋白激酶（MAPKs）如Erk、p38MARK和JNK未被激活。PI3激酶特异性抑制剂wortmannin和LY294002诱导SMC去分化，即使SMCs在igfi刺激条件下在层粘连蛋白上培养。这些发现表明，从IGDF/ICFI受体到PI3激酶的信号通路对于维持SMCs的分化表型至关重要。相比之下，血小板衍生生长因子（PDGF）是一种促进SMC分化的有效因子，可激活Erk和p38MAPK。添加Erk和p38MAPK特异性抑制剂PD98059和SB203580可以完全抑制pdgf诱导的SMC去分化，提示Erk和p38MAPK的协同激活可以触发诱导SMC去分化。从这些分析中，我们发现不同的信号分别参与了SMCs的分化和去分化。我们还研究了上述条件下培养的SMCs中caldesmon和alpha1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盒是这两个基因转录所必需的，而血清反应因子（SRF）是CArG盒的核心结合因子。此外，我们在α - sm肌动蛋白启动子中发现了一个新的顺式元件，作为负调节因子，并发现MSSP1是一个与该元件结合的交易因子。少

Primarily cultured smooth muscle cells (SMCs) rapidly dedifferentiate under normal culture conditions containing serum. We searched for SMC culture conditions maintaining a differentiated phenotype and found that laminin has a potency to delay the progress of SMC dedifferentiation. Furthermore, we found that insulin-like growth factors (IGFI,IGFII), or insulin possesses a remarkable activity to maintain a differentiated phenotype for a long time and IGFI is a most potent factor for SMC differentiation. Sole effect of laminin was inhibited by addition of anti-IGFI antibody. These results suggest that a signal transduction via IGFI/IGFI receptor would be involved in SMC differentiation. By contras, other growth factors/cytokines induced SMC dedifferentiation. Based on these evidences, we investigated signal transduction involved in SMC phenotypic modulation in combination with gene expressional regulations. In SMCs cultured under above conditions, IGFI activated phosphoinositide 3-kinase … More (PI3 kinase) and protein Kinase B (Aktl), whereas mitogen activated protein kinases (MAPKs) such as Erk, p38MARK and JNK were not activated. Specific inhibitors of PI3 kinase, wortmannin and LY294002, induced SMC dedifferentiation even when SMCs were cultured on laminin under IGFI-stimulated conditions. These findings suggest that a signaling pathway from IGDF/ICFI receptor to PI3 kinase is essential for maintenance of differentiated phenotype of SMCs. By contrast, platelet derived growth factor (PDGF) which is a potent factor promoting SMC defifferentiation, activated Erk and p38MAPK.PDGF-induced SMC dedifferentiation was completely inhibited by addition of both specific inhibitors of Erk and p38MAPK,PD98059 and SB203580, suggesting that coordinate activation of Erk and p38MAPK would trigger to induce SMC dedifferentiation. From these analyzes, we revealed that distinct signaling is involved in differentiation and dedifferentiation of SMCs, respectively. We also characterized transcriptional regulation of the caldesmon and the alpha1 integrin promoters in SMCs cultured under above conditions. These analyzes revealed that the CArG box within respective promoter regions are necessary for transcription of the both genes in differntiated SMCs, and the serum response factor (SRF) ia s core binding factor to the CArG box. Further, we identified a novel cis-element in the alpha-SM actin promoter which acts as a negative regulator and revealed that MSSP1 is a transacting factor bound to this element. Less