| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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
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(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
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