EGF–induced PKC translocation to the plasma membrane through ERK1/2/cPLA2/15(S)–HETE pathway in human corneal epithelial cells (HCEC)

Abstract

Abstract: : Purpose: Recent studies have suggested that 12(S)–HETE regulates epithelial cell proliferation and wound healing in corneal epithelial cells (Ottino et al. Exp Eye Res. 2003) but the underlying mechanism is not yet fully understood. 15(S)–HETE is the major arachidonic acid (AA) metabolite in HCEC and this eicosanoid significantly accelerates and potentiates EGF–mediated PKCα translocation to the plasma membrane (Sharma et al. ARVO. 2003). Here we investigated the possible mechanism of PKCα translocation to the plasma membrane in HCEC. Methods: HCEC were labeled with ³H–AA overnight in KBM (without growth supplements) and treated with EGF (10 ng/ml) for different times. Medium was collected and eicosanoids and AA were extracted. Lipids from the cytosolic fraction of the cells were also extracted. The extracted HETEs and AA were separated on reverse–phase HPLC and the fractions collected and counted in a liquid scintillation counter. Phospho–ERK1/2 (p–ERK1/2) and phospho–cPLA₂ (p–cPLA₂) expressions were analyzed using Western blotting. HCEC were also analyzed with immunofluorescence staining for EGF–induced activation and cellular localization of p–cPLA₂. Results: EGF stimulation for 5 min induced a significant conversion of AA to 15(S)–HETE. Similarly, we found a significant up–regulation in 12(S)–HETE synthesis upon EGF treatment at 5 min. When medium was analyzed for the release of labeled 15(S)–HETE and 12(S)–HETE, we found significantly higher release at 5 and 15 min of EGF stimulation. At the same time, the total ³H–AA pool in the cytosolic fractions was significantly depleted upon EGF stimulation. Western blot shows activation of ERK1/2 upon EGF stimulation from 1 min to 15 min and of cPLA₂ between 2 and 15 min of EGF stimulation. PD98059 (a specific inhibitor of MEK) blocked the EGF–induced ERK1/2 and cPLA₂ activation. Immunofluorescence showed a significant increase in p–cPLA₂ staining upon EGF treatment localized in the plasma membrane as early as 1 and 2 min after stimulation. Conclusions: EGF quickly activated both ERK1/2 and cPLA₂ and translocated p–cPLA₂ to the plasma membrane, which may thereby involve in AA release from membrane lipids and conversion to HETEs. These HETEs induced PKCα translocation to the plasma membrane, which upon activation may contribute to cellular proliferation. Therefore, the mechanism by which EGF regulates proliferation of corneal epithelial cells and promotes wound healing involves an ERK1/2 /cPLA₂ /HETEs pathway (Supported by NIH–NE1 EY06635 and EY04928).

Keywords: signal transduction • eicosanoids • growth factors/growth factor receptors