| Budget Amount *help |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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
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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
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