GSK-3 Modulation of EGF-Induced Signaling and Proliferation in Human Corneal Epithelial Cells

Abstract

Purpose:: The mitogenic response by SV40-immortalized human corneal epithelial cells (HCEC) to epidermal growth factor (EGF) is mediated through a network of parallel signaling pathways including MAPK and PKC stimulation. In some other tissues EGF and PKC inhibit glycogen synthetase kinase 3 (GSK-3) activity by phosphorylating this kinase, which in turn suppresses ERK limb signaling. We describe in HCEC the roles of PKC and GSK-3 in controlling EGF-induced MAPK signaling and proliferation.

Methods:: Western blot analysis was used to determine the phosphorylation status of Mek1/2, Erk1/2 mitogen activated protein kinase (MAPK) and GSK-3 using inhibitors of Mek1/2 and GSK-3. The involvement of PKC and GSK-3 was determined by measuring the dose dependent inhibitory effects of 1- 5 µM bisinodylmaleimide 1 (BIM) and 30 µM SB415286 on ERK limb and GSK-3 phosphorylation status. Such an evaluation was also performed following a 24 h exposure to 1 µM PDBu to downregulate PKC. [³H] thymidine incorporation determined the mitogenic responses to 10 ng/ml EGF.

Results:: EGF (10 ng/ml) or PDBu (1 µM) for 5 min induced GSK-3 serine 21/9 phosphorylation reflective of GSK-3 inhibition. There were corresponding transient increases in Erk1/2 phosphorylation of similar magnitude. They were augmented by 60% with both EGF and PDBu. With BIM, the Erk1/2 phosphorylation status increased by 50% from the value measured with PDBu or EGF alone. Similarly, with SB415286, EGF-induced Erk1/2 phosphorylation increased by 30% relative to the effect of EGF alone. BIM completely inhibited EGF or PDBu-induced GSK-3 phosphorylation. Following PKC downregulation, BIM still blocked either EGF or PDBu-induced GSK-3 phosphorylation. EGF or PDBu increased proliferation by 75% and 38%, respectively. These mitogenic responses were fully attenuated by BIM. Such suppression also occurred following PKC downregulation. The BIM-induced decline was nontoxic since the inhibition did not fall below the baseline value.

Conclusions:: EGF-induced stimulation of HCEC proliferation is sensitive to changes in GSK-3 phosphorylation status. Direct PKC stimulation or EGF alone both inhibit GSK-3 activity by inducing GSK-3 phosphorylation. GSK-3 inhibition in turn decreases ERK branch phosphorylation whereas BIM-induced GSK-3 stimulation has the opposite effect. Therefore, the net mitogenic response to EGF is dependent on GSK-3 modulation of EGF-induced ERK limb activation.

Keywords: cornea: epithelium • cytokines/chemokines • growth factors/growth factor receptors