Mechanism of extracellular signal-regulated kinase (ERK)-1 and ERK-2 activation by vanadium pentoxide in rat pulmonary myofibroblasts

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-I/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.