Cell Cycle Controlling Proteins cdc42, p21cip and p27kip Expression is Differentially Regulated by HGF, KGF, EGF and Extracellular Matrix Proteins (ECMs) in Corneal Epithelium

Abstract

Purpose: : Growth factors- and ECM-activated signals are necessary for corneal epithelial cell regeneration. While growth factors control the cell cycle (Chandrasekher et al, abstract #1964, ARVO 2010) it is not clear how ECM signals influence the cell cycle. The purpose of this study is to compare the effects of hepatocyte, keratinocyte and epidermal growth factors (HGF, KGF and EGF) and ECMs (laminin, LM; fibronectin, FN; collagen I, Col I; collagen IV, Col IV;) on the expression of cell cycle controlling proteins p21^cip, p27^kip and cdc42 (GTP binding cell division cycle protein).

Methods: : Epithelial cells from rabbit corneas were cultured in DMEM-F12/10% FCS. Primary cultures were treated with HGF/KGF/EGF (20ng/ml) and LM/FN/Col I/Col IV (10-50µg/ml). Protein expression was analyzed by immunoblotting and immunofluorescence. Activity of cdc42 was assayed by precipitating cdc42 bound to p21 activated kinase linked to agarose beads. Proliferation was determined by assaying for DNA with CyQuant reagent.

Results: : Treatment of cells with HGF, KGF and EGF increased the expression of cdc42 after 24hr. Immunofluorescence studies employing anti-cdc42 also revealed increased expression in the presence of growth factors. The level of active cdc42 (GTP-bound form) was elevated within 10 min after EGF and HGF stimulation. However, in the presence of FN, Col I and Col IV, cdc42 expression decreased. While HGF, KGF and EGF increased the expression of p21^cip, its expression decreased with FN, Col I and Col IV. Growth factors caused a decrease in the levels of p27^kip and promoted cell proliferation, whereas ECMs up-regulated the expression of p27^kip but showed no effect on cell proliferation. However, EGF, KGF and HGF as well as ECMs caused a significant increase in tyrosine phosphorylation of 100-125 kDa range proteins in corneal epithelial cells.

Conclusions: : Our studies suggest that growth factors and ECMs modulate cell cycle by exerting different effects on the expression of p21^cip, p27^kip and cdc42. While growth factors-mediated cell cycle control up-regulates proliferation, ECMs could influence cell cycle arrest to facilitate cell migration and tissue regeneration during wound healing. Increase in protein tyrosine phosphorylation (100-125kDa) in the presence of ECMs and growth factors suggests possible cross-talk between growth factors and ECM receptor-mediated signals.

Keywords: cornea: epithelium • growth factors/growth factor receptors • extracellular matrix