|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1995 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1994 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Caldesmon (CaD) , which plays a vital role in the actomyosin system, is distributed in smooth muscle and nonmuscle cells, and its isoformal interconversion between high Mr form (h-CaD) and low Mr form (1-CaD) is a favorable molecular marker for phenotypic modulation of smooth muscle cells (SMCs). The expressional level of CaD gene is up-regulated in developing SMCs. We found that primary cultured gizzard SMCs on laminin maintain differentiated phenotype. This study has focused on the transcriptional mechanism in SMCs using the present culture syetem. Here, we have characterized the transcriptional tegulation of the SMC-type promoter in CaD gene which shows the high activity in SMCs. Transient transfection of CAT constructs in both phenotypes of SMCs, differentiated and dedifferentiated cells, revealed that the high level of CaD promoter activity depends on a unique CArG box-like motif, CCAAAAAAGG,located at-309 to-300 upstream from the transcriptional starting site. This CArG box-like motif is similar to serum response element (SRE) , while this motif in the CaD gene is not able to exhibit serum responsibility in SMCs. We also identified a specific nuclear protein factor interacting with this motif by gel shift assay, and found that the amount of this protein factor is much higer in the nuclear extracts from differentiated SMCs than those from dedifferentiated SMCs. Gel shift assay using anti-serum response factor (SRF) IgG resulted in a supershifted complex, suggesting that the protein factor interacting with this CArG box-like motif might be SRF or SRF-related nuclear protein. CArG box-like motif are also localized in the promoter regions of other SMC-specific cytoskeletal proteins, such as alpha-SM actin, SM22, and alpha1 integrin. From these results, SMC-specific transcriptional regulation would seem to be controled by a common mechanism, in which CArG box-like motif and SRF or SRF-related protein factor are involved.