Abstract
Purpose: :
Cdc42, the Rho family small GTPase is known to control cellular processes such as migration, cytokinesis and growth by regulating the rearrangement of actin cytoskeleton. During these processes Cdc42 oscillates between inactive (GDP bound) and active (GTP bound) forms. Certain serine residues of Cdc42 also become phosphorylated during cellular reactions. Growth factors as well as extracellular matrix (ECM) proteins produced immediately after injury to the cornea help in wound healing. In our earlier studies (Pothula et al, abstract # 2038 ARVO 2011) we found that HGF, KGF, EGF and ECM proteins modulate the expression of various cell cycle proteins. Here, we report their influence on Cdc42 ser71 activation and upregulation of Cdc42 expression through Erk signaling.
Methods: :
Epithelial primary cultures from rabbit corneas were grown in DMEM-F12/10% FCS were treated with HGF or KGF (20ng/ml) and laminin (10-50µg/ml). Cdc42 expression and its phosphorylation at ser71site were analyzed by immunoblotting. Involvement of PI3K/Akt or Erk signaling is assessed after treating the cell cultures with PI-3K inhibitors (wortmanin and LY294002) and Erk- kinase inhibitor (PD98059) in the presence and absence of growth factors and ECM proteins.
Results: :
Treatment of cells with HGF, KGF, EGF and laminin increased the expression of Cdc42 after 24hr. In the presence of growth factors and laminin the ser71 phosphorylation of Cdc42 is also enhanced considerably within 10-30 minutes. Growth factors and laminin although caused the increase in protein tyrosine phosphorylation, Cdc42 is not found to be associated with tyrosine phosphorylated proteins. While the presence of Erk inhibitor caused the upregulation of Cdc42 expression, the inhibition of PI-3k signaling had minimal or no effect on the upregulation of Cdc42.
Conclusions: :
Our studies suggest that growth factors and laminin proteins-mediated regulation of Cdc42 may be important for the corneal epithelial wound healing. Phosphorylation of Cdc42 at serine 71 could modulate its signaling by translocating the activated Cdc42 to cell membrane which assists in cell migration. Up regulation of Cdc42 expression in the presence of Erk inhibitors suggests that signaling pathways upstream of Erk could play an important role in modulating Cdc42 function..
Keywords: cornea: epithelium • signal transduction • growth factors/growth factor receptors