Am. J. Respir. Cell Mol. Biol., Volume 19, Number 2, August, 1998 324-332

Hydrogen Peroxide Stimulates Extracellular Signal-regulated Protein Kinases in Pulmonary Arterial Smooth Muscle Cells

Jiahui Zhang, Najia Jin, Yi Liu, and Rodney A. Rhoades

Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana

Hydrogen peroxide (H₂O₂) has emerged as an important intracellular signaling molecule and has been shown to stimulate the growth of vascular smooth muscle cells. Activation of p44 and p42 extracellular signal-regulated protein kinases (ERK₁ and ERK₂) is an important step in the cascade leading to cell growth and proliferation. In the present study, we investigated the effects and mechanisms of H₂O₂ on activation of ERK₁ and ERK₂ in pulmonary arterial smooth muscle cells (PASMC). Assays of immune-complex kinase activity revealed that exposure of PASMC to H₂O₂ stimulated myelin basic protein (MBP) phosphorylation in a concentration- and time-dependent manner. Western blot analysis done with phospho-specific mitogen-activated protein (MAP) kinase antibodies demonstrated that H₂O₂ stimulated the phosphorylation of p42, p44, p46, and p38 MAP kinases. H₂O₂ also increased the expression of the early immediate genes c-jun and fra-1. Activation of ERK₁ and ERK₂ by H₂O₂ was significantly reduced by downregulation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by a PKC inhibitor, calphostin C. In addition, removal of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ pool by thapsigargin, or pretreatment of PASMC with the calmodulin antagonist N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or with calmidazolium chloride also decreased H₂O₂-induced ERK₁ and ERK₂ activation. Furthermore, stimulation of ERK₁ and ERK₂ activity by H₂O₂ was partly attenuated by genistein, a tyrosine kinase inhibitor. Taken together, these data suggest that H₂O₂ activates ERK₁, ERK₂, p46 JNK, and p38 MAP kinases in PASMC. The activation of ERK₁ and ERK₂ appears to be primarily dependent on PKC, and to be partly modulated by Ca²⁺/calmodulin and by activation of tyrosine kinases.

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