Elucidation of molecular mechanism of mechanical stretch-induced cardiac hypertrophy

我们以前曾报道过，通过使用可变形的硅胶盘，心肌细胞的拉伸引起蛋白激酶的磷酸化级联反应，如细胞外信号调节激酶（ERK）和90 kD核糖体S6激酶，随后增加蛋白质合成，ERK的激活是肥大反应的关键。为了进一步阐明牵张诱导的心肌细胞ERK激活的信号转导途径，我们用抑制蛋白检测了可能的上游激酶如Src、Ras、Raf-1激酶（Raf-1）和蛋白激酶C（PKC）的作用。将CSK（最近分离的抑制Src家族激酶的酪氨酸激酶）的活性突变体、显性负性Ras突变体或显性负性Raf-1突变体与HA标记的ERK 2共转染到新生大鼠的心肌细胞中。转染后，将在可变形硅胶皿上培养的心肌细胞拉伸20%，持续8分钟。使用抗HA单克隆抗体免疫沉淀HA标记的ERK 2，并以髓鞘碱性蛋白为底物测定ERK 2的活性。心肌细胞的拉伸迅速增加ERK 2的活性。CSK或显性负性Ras与HA标记的ERK 2共转染对牵张诱导的ERK 2活化没有抑制作用，而显性负性Raf-1共转染完全抑制机械牵张诱导的ERK 2活化。当用钙磷蛋白C（10 μ <-6>M，60 min）或TPA（10 μ <-7>M，24 h）长期作用抑制PKC时，机械牵张诱导的Raf-1和ERK激活几乎完全被抑制。PKC激活剂如TPA（10 μ <-7>M）在培养的心肌细胞中激活Raf-1和ERK。这些结果表明，PKC，而不是Src或Ras，在机械应力诱导的ERK激活通过Raf-1起着至关重要的作用。

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.