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 bro nchitis, 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 V2O 5 resulted in the activation of ERK-1/2 yet the inert metal titanium dioxid e had no effect on ERK-I/2 activation. V2O5-induced ERK-1/2 activation was abolished by pretreatment with forskolin or PD98059, indicating a dependenc e on Raf and mitogen-activated protein (MAP) kinase kinase, respectively. D epletion of conventional protein kinase C activity with phorbol 12-myristat e 13-acetate did not inhibit V2O5-induced ERK-1/2 activation. ERK-1/2 activ ation by V2O5 was inhibited > 70% with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478. Phosphorylation of the 170-kD EG F-R by V2O5 was detected after immunoprecipitation with an anti-EGF-R antib ody followed by phosphotyrosine Western blotting. V2O5 strongly tyrosine-ph osphorylated a 115-kD protein (p115) and activation of p115 was inhibited 6 0 to 70% by AG1478, indicating that this protein was an EGF-R substrate. Ph osphorylation of p115 was also observed in EGF-stimulated cells. Immunoprec ipitation 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 phosphorylati on > 90%. These data suggest that V2O5 activation of ERK-1/2 is oxidant-dep endent and mediated through tyrosine phosphorylation of EGF-R and an EGF-R substrate which we identified as a 115-kD SH-PTP2-binding protein.