| Project/Area Number |
05454157
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
SOBUE Kenji Osaka Univ.Med.Sch.Prof., 医学部, 教授 (20112047)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Kenichiro Osaka Univ.Med.Sch.Assistant.Prof., 医学部, 助手 (90238105)
INUI Makoto Osaka Univ.Med.Sch.Associate.Prof., 医学部, 助教授 (70223237)
田中 潤也 大阪大学, 医学部, 助手 (70217040)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1994: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1993: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | Smooth muscle cells / Phenotypic modulation / Actomyosin system / Caldesmon / Genomic structure / Splicing / Promoter / Transcriptional regulation / 接着因子 / 平滑筋細胞分化 / アクトミオシン系制御 / エンハンサー |
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| Research Abstract |
Caldesmon (CaD), which plays a vital role in the actomyosin system, is distributed in smooth muscle and nonmuscle cells, and its isoformal interconversion between h (high Mr form)- and l (low Mr form)-CaDs is a favorable molecular event for studying phenotypic modulation of smooth muscle cells (SMCs). This study has focused on the molecular mechanism of SMCs differentiation using such CaD isoformal interconversion as a molecular marker. Genomic analysis of CaD revealed that the expressional change in the isoforms is regulated at both levels, transcription and unique splicing ; the expression of h- and l-CaDs was regulated by alternative selection of the two distinct 5'-splice sites withn exon 3. We also found that the signal transduction mediated alpha1beta1 integrin retards the oneset of dedifferentiation of cultured SMCs and that alpha1beta1 integrin is located in cell adheadion of differentiated SMCs. Under this culture system, h-CaD and other molecular marker such as high Mr tropom
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yosin and metavinculin were maintained for several days in SMCs. Therefore, we have carried out the ptomoter anlysis of the CaD gene in SMCs using this culture system. Transient transfection assays in primary cultured SMCs, mouse skeletal muscle cell line (C2C12 cells), and HeLa cells revealed that the CaD promoter activity was high levels in SMCs, but was extremely low in other cells. In addition, the promoter activity and the protein levels of CaD indifferentiated SMCs were higher than those in dedifferentiated SMCs. High levels of the promoter activity in SMCs depended on a unique CArG box-like motif, CCAAAAAAGG,located at -309 to -300 upstream from the transcriptional starting site, and this motif in addition to its 5'-snd 3'-flanking 6 uncleotide sequences (CArG1) were essential for enhancement of the promoter activity. These results suggest that the CArG1 is an essential cis-element for cell type-specific high expression of the CaD gene and that the function of of the CArG1 might be controled under phenotypic modulation of SMCs. Less
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