Keratinocyte Growth Factor (KGF), but Not Hepatocyte Growth Factor (HGF) Promotes Sustained Cell Survival and Long Lasting Activation PI–3K/Akt and Erk Signaling in Corneal Epithelium

Abstract

Purpose: : HGF and KGF are two important paracrine growth factors that act through different receptors to promote corneal epithelial wound healing. Our previous studies showed that both HGF and KGF stimulate PI–3K/Akt and Erk1/2 signaling, and that HGF promotes cell survival of corneal epithelial cells. (IOVS 2004, 44, 2696; Exp Eye Res 2001, 73, 191). In the current study we investigated whether there are differences in the kinetics of signaling activation and in the cell–survival capability between these two growth factors.

Methods: : Primary cultures of rabbit corneal epithelial cells grown in DMEM/F12 were stimulated with HGF or KGF (20 ng/ml) for short (0–60 min) and longer intervals (up to 24 h). Apoptosis was induced with staurosporine (10 ng/ml). Characteristic DNA fragmentation (laddering) due to apoptosis was identified by agarose gel electrophoresis. Western immunoblotting using specific antibodies was employed to identify the signaling proteins of interest.

Results: : HGF and KGF exhibited significant differences in their ability to stimulate Akt and Erk1/2. During the initial phase (0–60 min) after treatment, HGF–stimulated activation of Akt was 4 to 6 fold greater than that by KGF. Similarly, Erk1/2 phosphorylation by HGF was also several fold higher as compared to that by KGF. However, while the HGF–mediated activation of Akt and Erk declined gradually after 60 min and reached near basal levels at 24 h, KGF–dependent phosphorylation of Akt and Erk was increased. Both growth factors were able to protect cells from apoptosis 10–12 h after staurosporine treatment. While the ability of HGF to rescue the cells from apoptosis diminished markedly by 24 h, the cell–survival capability of KGF was sustained. Increased levels of cyclin–dependent kinase inhibitor p27^kip (a negative regulator of the cell cycle) were observed in epithelial cells during staurosporine–induced apoptosis. The presence of KGF, but not that of HGF, for 24 h resulted in the suppression p27^kip expression. KGF effect on p27^kip expression was blocked by PI–3K inhibitor LY294002, whereas Erk cascade inhibitor PD98059 showed little effect.

Conclusions: : KGF, unlike HGF, produces a delayed but long–lasting activation of PI–3K/Akt and Erk1/2, suggesting that this growth factor could play a pivotal role in cell–cycle control to sustain the survival of corneal epithelial cells.

Keywords: cornea: epithelium • growth factors/growth factor receptors • apoptosis/cell death