The long-term goal of the present study is to investigate growth factor-regulated cell proliferation during the corneal epithelial renewal process and wound healing. We recently found that CTCF, an important nuclear protein that binds to CCCTC sequence, activated by epidermal growth factor (EGF) mediates the regulation of homeobox Pax6 gene expression. Undoubtedly, EGF-induced activation of CTCF controlling Pax6 expression plays a central role in regulation of proliferation/differentiation during corneal epithelial renewal and wound healing. We also found in corneal epithelial cells that EGF regulates CTCF expression and phosphorylation through the Erk (MAP kinase) signaling pathway. CTCF functions as a repressor in regulating expression of Pax6 gene by interacting with a specific element in the 5' flanking region upstream from the Pax6 P0 promoter. Our results strongly support the notion that EGF-induced corneal epithelial proliferation requires the activation of CTCF whereas Pax6 activity has to be inhibited because it has been suggested that Pax6 may promote corneal epithelial cell early differentiation. We hypothesize that the effectiveness of EGF-induced corneal epithelial cell proliferation is dependent on the ability of the growth factor to increase CTCF activity and to decrease Pax6 gene expression. To test this hypothesis, we will undertake three specific aims including: 1) investigation of the effects of EGF-induced CTCF activation and Pax6 suppression on cell proliferation, 2) delineation of the interaction between Erk and CTCF that enables EGF to regulate CTCF activity, and 3) elucidation of the regulatory mechanism that enables CTCF to regulate Pax6 gene expression. Such studies will provide the first step towards testing the physiological significance of CTCF in mediating growth factor-induced corneal epithelial cell proliferation. In addition, the results will also reveal novel regulatory mechanisms that describe why Pax6 is retained in the mature corneal epithelia and how Pax6 expression affects corneal epithelial cell fate. Furthermore, the new insights will be obtained into the mechanisms that mediate growth factor control of cellular proliferation and differentiation.
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