Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation

Abstract

Oxidative stress has been shown to increase the expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H2O2, which induces oxidative stress, could also enhance the expression of Giα proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H2O2increased the expression of Giα proteins and not of Gsα protein in a concentration- and time-dependent manner. A maximal increase of ∼40–50% was observed at 100 μM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H2O2also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H2O2-induced enhanced expression of Giα proteins to control levels. The increased expression of Giα proteins was reflected in enhanced Gifunctions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas Gsα-mediated stimulations of AC were significantly decreased. Furthermore, H2O2-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H2O2increases the expression of Giα proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.