Fig. 5.

Stimulation of EGFR signaling is attenuated by selective EGFR kinase inhibition. Total EGFR protein levels were measured by Western blot analysis and normalized with β-actin in peripheral lung lysates prepared from control rats and rats treated for 14 days with MCT in the presence or absence of the EGFR kinase inhibitor, gefitinib (30 mg/kg by oral gavages, daily). No differences in EGFR protein levels were observed between groups (A, left panel). The levels of SDS-resistant EGFR dimer were also evaluated by performing Western blot analysis. The level of SDS-resistant EGFR dimer was significantly increased in lung tissues of MCT treated rats and this increase was not significantly attenuated by gefitinib (A, right panel). However, the increased ratio of autophosphorylated EGFR dimer/autophosphorylated EGFR monomer in lung tissues of MCT treated rats was significantly attenuated by treatment with gefitinib (B). The level of EGFR signaling was evaluated by immunopreciptating EGFR and probing for Grb2 and Ras. EGFR/Grb2 (C) and EGFR/Ras (D) interactions were significantly increased in MCT-treated rats and this was attenuated by gefitinib. The total level of proliferation in peripheral lung tissue was evaluated by measuring PCNA protein levels by Western blot analysis. The levels of PCNA were normalized to β-actin. The levels of PCNA are significantly increased in MCT-treated rats and this is attenuated by gefitinib (E). The proliferation of pulmonary SMC was also evaluated by measuring the levels of the SMC marker, caldesmon by Western blot analysis. The levels of caldesmon were normalized to β-actin. The levels of caldesmon are significantly increased in MCT-treated rats and this is attenuated by gefitinib (F). Results are expressed as mean±SEM; N=3–7. *P<0.05 vs. control group. †P<0.05 vs. MCT alone.