Role of EGFR Transactivation in preventing Apoptosis in Pseudomonas aeruginosa–Infected human corneal epithelial Cells

Abstract

Abstract: : Purpose: To elucidate the mechanisms underlying epidermal growth factor (EGF) receptor (EGFR) transactivation in response to bacterial infection and to determine the role of EGFR–mediated signaling pathways in preventing infection induced apoptosis in human corneal epithelial cells. Methods: Pseudomonas aeruginosa (PA, PAO1 strain) were spun down onto an epithelial monolayer of a telomerase–immortalized HCE cell line, HUCL in the presence of an EGFR inhibitor tyrphostin AG1478, extracellular signal–regulated kinase (ERK) inhibitor U0126, PI3K inhibitor LY294002, HB–EGF antagonist CRM197, HB–EGF neutralizing antibody, or matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies and/or Western blotting with phosphotyrosine antibody. Phosphorylation of ERK and AKT and generation of cleaved caspase–3 and PARP were determined by Western blotting. Apoptotic cells were characterized by stained positive with active caspase–3 antibody, lack of mitochondrial cytochrome C staining, and condensed chromosomes. Apoptosis was confirmed through measuring caspase–3 activity and assessing the generation of cleaved caspase–3 and PARP. Results: PA infection of HUCL cells results in EGFR activation and EGFR–dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2 or PI3K activities with kinase specific inhibitors (AG1478, U0126 and LY294002, respectively) greatly increased the number of apoptotic cells in PA–infected HUCL cells. This increased apoptosis was accompanied by increasing cellular caspase–3 activity and generating cleaved caspase–3 (active form) and PARP. Blocking HB–EGF ectodomain shedding by inhibition of MMP–mediated protolysis, down–regulation HB–EGF, or neutralizing its activity, retarded infection induced EGFR transactivation and as a consequence, increased infection induced HUCL apoptosis. Conclusions:Bacterial infection of corneal epithelial cells induces EGFR transactivation through HB–EGF ectodomain shedding and EGFR and its downstream ERK and PI3K signaling pathways play a role in preventing epithelial apoptosis in the early stage of bacterial infection of corneal epithelial cells.

Keywords: apoptosis/cell death • cornea: epithelium • Pseudomonas