| Co-Investigator(Kenkyū-buntansha) |
SHIOJIMA Ichiro University of Tokyo, Third Department of Internal Medicine, Medical Staff, 医学部(病), 医員
KOMURO Issei University of Tokyo, Third Department of Internal Medicine, Research Associate, 医学部(病), 助手 (30260483)
YAZAKI Yosio University of Tokyo, Third Department of Internal Medicine, Professor, 医学部(病), 教授 (20101090)
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| Research Abstract |
We have previously reported by using deformarable silicone dishes that stretching of cardiac myocytes evokes the phosphorylation cascade of protein kinases such as extracellular signal-regulated kinases (ERKs) and 90-kD ribosomal S6 kinase followed by an increase in protein synthesis, and that activation of ERKs is critical for hypertrophic responses. To further elucidate stretch-induced signal transduction pathways leading to ERK activation in cardiac myocytes, the role of presumable upstream kinases such as Src.Ras, Raf-1 kinase (Raf-1) and protein kinase C (PKC) was examined by using inhibitory proteins. Active mutant of CSK (the recently isolated tyrosine kinase which inhibits Src family kinases), dominant-negative Ras mutant or dominat-negative Raf-1 mutant was co-transfected into cardiac myocytes of neonatal rats with HA-tagged ERK2. After transfection, cardiac myocytes cultured on deformable silicone dishes were stretched by 20% for 8 min. HA-tagged ERK2 was immunoprecipitated using an anti-HA monoclonal antibody and the activity of ERK2 was assayd by using myelin basic protein as a substrate. Stretching of cardiac myocytes rapidly increased the activity of ERK2. Co-transfection of either CSK or dominant-negative Ras with HA-tagged ERK2 showed no inhibitory efects on ERK2 activation by stretch, while the co-transfection of dominant-negative Raf-1 completly inhibited mechanical stretch-induced ERK2 activation. Futhermore, when PKC was inhibited by calphostin C (10^<-6>M for 60 min) or long exposure of TPA (10^<-7>M for 24 hours), Raf-1 and ERK activation induced by mechanical stretch was almost completely surpressed. PKC activators such as TPA (10^<-7>M) activated Raf-1 and ERK in cultured cardiac myocytes. These results suggest that PKC,but not Src or Ras, plays a vital role in mechanical stress-induced ERK activation through Raf-1.
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