Epidermal growth factor, EGF, plays an essential role in corneal epithelial renewal. It is needed for proper corneal epithelial healing following wounding of this layer, which is required for normal vision. Our long-term goal is to further understand how EGF receptor-linked signaling elicits its effects on corneal epithelial homeostasis. We have found that initiation by EGF of cell cycle progression is dependent on the stimulation of ion transporters activity; EGF stimulates Na:K:2Cl cotransporter 1 (NKCC1) activity, and subsequently stimulates K+ and Cl- conductance activity, which results in a transient increase in cell volume. On the other hand, cell cycle progression is known to be associated with increases in cell volume. Therefore, we now hypothesize that in rabbit corneal epithelial cells (RCEC), EGF-induced changes in NKCC1 activity and expression is a requisite for cell cycle progression from early G1 to mitosis. We further hypothesize that modulation of activity of all ion transporters and channels involved in cell volume regulation is needed for cell proliferation and migration. Our three aims entail: 1) determining during cell cycle progression whether there is differential activity and expression of ion transporters (NKCC1, Na/K-ATPase, K+ and Cl- conductances) underlying cell volume regulation; 2) determining the interactions between the cell signaling pathways mediating EGF receptor control of the aforementioned ion transporters; 3) dissecting how EGF controls cell proliferation and migration. Cell volume and regulatory volume behavior will be characterized with video, confocal microscopy and low angle light scattering, respectively. NKCC1 gene transcription will be characterized with a reporter gene assay. NKCC1 activity will be evaluated based on measurements of 86Rb+ influx. EGF-induced activation of mitogen activated protein kinase (MAPK) signaling and crosstalk will be determined with kinase assays. Interactions of specific MAPK components with NKCC1 will be evaluated using coimmunoprecipitation. EGF mediated signaling control of epithelial proliferation and migration will be investigated using kinase inhibitors and stable MAPK gene (constitutively active and tetracycline-inducible dominant negative) expression. Rates of epithelial proliferation and migration will be characterized in an in vitro wound healing model system. These studies could provide new strategies for hastening corneal epithelial wound healing and reducing severity of losses in vision. In addition, they may provide insights on how to stimulate healing bypassing growth factor receptor control.
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