Pan Jing, Singh Ugra S, Takahashi Toshiyuki, Oka Yoshitomo, Palm-Leis Ants, Herbelin Bradley S, Baker Kenneth M
Division of Molecular Cardiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas 76504, USA.
J Cell Physiol. 2005 Feb;202(2):536-53. doi: 10.1002/jcp.20151.
Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.
包括Rho GTP酶和蛋白激酶C（PKC）在内的信号转导事件与心肌肥大有关。然而，这些信号通路在肥大过程中协同作用的机制仍不清楚。利用新生大鼠心肌细胞的体外周期性拉伸模型，我们发现，使用白屈菜红碱和佛波醇12-肉豆蔻酸酯13-乙酸酯抑制或耗尽PKC可阻止拉伸诱导的RhoA、Rac1/Cdc42激活以及Rho-鸟嘌呤核苷酸解离抑制剂（GDI）的磷酸化，这表明佛波酯敏感的PKC同工酶可能是Rho GTP酶的上游调节因子。利用腺病毒介导的PKCα和δ野生型（WT）及显性负性（DN）突变体的基因转移，我们发现拉伸诱导的Rho GTP酶激活和Rho-GDI磷酸化主要受PKCα调节。PKCδ参与Rac1激活的调节。抑制Rho GTP酶或过表达DN PKCα和δ可阻断拉伸诱导的[³H]-亮氨酸掺入增加、肌原纤维重组和细胞大小增加，这表明PKCα和δ在拉伸诱导的肥大过程中均通过Rho GTP酶介导的信号通路发挥作用。PKC和Rho GTP酶调节肥大的机制与丝裂原活化蛋白（MAP）激酶有关。过表达DN PKCα和δ可抑制拉伸刺激的MEK1/ERK1/2和MKK4/JNK磷酸化，过表达DN PKCδ可抑制MKK3/p38磷酸化。WT PKCα过表达诱导的ERK和JNK磷酸化以及WT PKCδ诱导的p38磷酸化受Rho GTP酶调节。本研究首次证明PKCα和δ是在周期性拉伸诱导的肥大过程中介导Rho GTP酶和MAP激酶激活的重要调节因子。
