Am. J. Respir. Cell Mol. Biol., Volume 22, Number 5, May, 2000 590-596

Mechanism of Extracellular Signal-Regulated Kinase (ERK)-1 and ERK-2 Activation by Vanadium Pentoxide in Rat Pulmonary Myofibroblasts

Yi-Zhe Wang and James C. Bonner

Airway Inflammation Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Vanadium pentoxide (V₂O₅) is a cause of occupational asthma and chronic bronchitis, yet the molecular mechanisms through which V₂O₅ exerts its effects on cell function are unclear. In this study we investigated the potential of V₂O₅ to activate the extracellular signal-regulated kinases 1 and 2 (ERK-1/2) in rat pulmonary myofibroblasts. Treatment of myofibroblasts with V₂O₅ resulted in the activation of ERK-1/2, yet the inert metal titanium dioxide had no effect on ERK-1/2 activation. V₂O₅-induced ERK-1/2 activation was abolished by pretreatment with forskolin or PD98059, indicating a dependence on Raf and mitogen-activated protein (MAP) kinase kinase, respectively. Depletion of conventional protein kinase C activity with phorbol 12-myristate 13-acetate did not inhibit V₂O₅-induced ERK-1/2 activation. ERK-1/2 activation by V₂O₅ was inhibited > 70% with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478. Phosphorylation of the 170-kD EGF-R by V₂O₅ was detected after immunoprecipitation with an anti-EGF-R antibody followed by phosphotyrosine Western blotting. V₂O₅ strongly tyrosine-phosphorylated a 115-kD protein (p115) and activation of p115 was inhibited 60 to 70% by AG1478, indicating that this protein was an EGF-R substrate. Phosphorylation of p115 was also observed in EGF-stimulated cells. Immunoprecipitation of V₂O₅- or EGF-treated cell lysates with an antibody against Src homology 2 protein tyrosine phosphatase (SH-PTP2) identified p115 as a SH-PTP2-binding protein. Pretreatment of cells with the antioxidant N-acetyl-L-cysteine blocked V₂O₅-induced MAP kinase activation and p115 phosphorylation > 90%. These data suggest that V₂O₅ activation of ERK-1/2 is oxidant-dependent and mediated through tyrosine phosphorylation of EGF-R and an EGF-R substrate which we identified as a 115-kD SH-PTP2-binding protein.

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