Azar Zeina M, Mehdi Mohamad Z, Srivastava Ashok K
Laboratory of Cell Signaling, Research Centre, Centre hospitalier de l'Université de Montréal (CHUM) - Hôtel-Dieu and Department of Medicine, Université de Montréal, 3850, St. Urbain Street, Rm. 7-135, Montreal, QC H2W 1T7, Canada.
Can J Physiol Pharmacol. 2006 Jul;84(7):777-86. doi: 10.1139/y06-024.
Evidence accumulated in recent years has revealed a potential role for reactive oxygen species (ROS) in the pathophysiology of cardiovascular diseases. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully established. Previous work from our laboratory has indicated that exogenous hydrogen peroxide (H2O2) activates several signaling protein kinases, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase B (PKB) in A10 vascular smooth muscle cells (VSMC). However, the upstream elements responsible for this activation remain unclear. Although a role for epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) in H2O2-induced ERK1/2 signaling has been suggested, the contribution of this PTK or other receptor or nonreceptor PTKs to PKB activation is not well defined in VSMC. In this study, we used pharmacological inhibitors to investigate the role of receptor and Src-family-PTKs in H2O2-induced PKB phosphorylation. AG1478, a specific inhibitor of EGFR, failed to attenuate the H2O2-induced increase in PKB Ser473 phosphorylation, whereas AG1024, an inhibitor of insulin-like growth factor type1 receptor (IGF-1R)-PTK, almost completely blocked this response. H2O2 treatment also enhanced tyrosine phosphorylation of the IGF-1Rbeta subunit, which was significantly inhibited by AG1024 pretreatment of cells. Furthermore, pharmacological inhibition of Src by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole(3,4-d) pyrimidine) decreased PKB phosphorylation. Moreover, H2O2-induced PKB phosphorylation was associated with increased tyrosine phosphorylation of c-Src and Pyk2 in an AG1024- and PP2-inhibitable manner. In conclusion, these data provide evidence of the contribution of IGF-1R-PTK in initiating H2O2-evoked PKB phosphorylation in A10 VSMC, with an intermediary role for c-Src and Pyk2 in this process.
近年来积累的证据表明，活性氧（ROS）在心血管疾病的病理生理学中具有潜在作用。然而，ROS促成这些疾病发展的确切机制尚未完全明确。我们实验室之前的研究表明，外源性过氧化氢（H2O2）可激活A10血管平滑肌细胞（VSMC）中的几种信号蛋白激酶，如细胞外信号调节激酶1和2（ERK1/2）以及蛋白激酶B（PKB）。然而，负责这种激活的上游元件仍不清楚。尽管有人提出表皮生长因子受体（EGFR）蛋白酪氨酸激酶（PTK）在H2O2诱导的ERK1/2信号传导中发挥作用，但在VSMC中，这种PTK或其他受体或非受体PTK对PKB激活的贡献尚未明确界定。在本研究中，我们使用药理学抑制剂来研究受体和Src家族PTK在H2O2诱导的PKB磷酸化中的作用。EGFR的特异性抑制剂AG1478未能减弱H2O2诱导的PKB Ser473磷酸化增加，而胰岛素样生长因子1型受体（IGF-1R）-PTK的抑制剂AG1024几乎完全阻断了这种反应。H2O2处理还增强了IGF-1Rβ亚基的酪氨酸磷酸化，细胞经AG1024预处理后，这种磷酸化受到显著抑制。此外，PP2（4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶）对Src的药理学抑制降低了PKB磷酸化。此外，H2O2诱导的PKB磷酸化与c-Src和Pyk2的酪氨酸磷酸化增加有关，且这种增加可被AG1024和PP2抑制。总之，这些数据证明了IGF-1R-PTK在启动A10 VSMC中H2O2诱发的PKB磷酸化中的作用，在此过程中c-Src和Pyk2起中间作用。
