Role of CTCF in EGF-Induced Corneal Epithelial Growth

描述（由申请人提供）：本研究的长期目标是研究角膜上皮更新过程和伤口愈合过程中生长因子调节的细胞增殖。我们最近发现CTCF是一个结合CCCTC序列的重要核蛋白，被表皮生长因子（EGF）激活介导同源盒Pax6基因的表达调控。毫无疑问，egf诱导的CTCF激活控制Pax6的表达在角膜上皮更新和伤口愈合过程中的增殖/分化调控中起着核心作用。我们还发现，在角膜上皮细胞中，EGF通过Erk （MAP激酶）信号通路调节CTCF的表达和磷酸化。CTCF作为一种抑制因子，通过与Pax6 P0启动子上游5'侧区特定元件相互作用，调控Pax6基因的表达。我们的研究结果强烈支持egf诱导的角膜上皮细胞增殖需要CTCF的激活，而Pax6的活性必须被抑制，因为已有研究表明Pax6可能促进角膜上皮细胞的早期分化。我们假设egf诱导角膜上皮细胞增殖的有效性取决于生长因子增加CTCF活性和降低Pax6基因表达的能力。为了验证这一假设，我们将进行三个具体的目标，包括：1)研究EGF诱导的CTCF激活和Pax6抑制对细胞增殖的影响，2)描述Erk和CTCF之间的相互作用，使EGF调节CTCF活性，以及3)阐明CTCF调节Pax6基因表达的调节机制。这些研究将为测试CTCF在介导生长因子诱导的角膜上皮细胞增殖中的生理意义提供第一步。此外，研究结果还将揭示Pax6在成熟角膜上皮中保留的新调控机制，以及Pax6的表达如何影响角膜上皮细胞的命运。此外，将对介导生长因子控制细胞增殖和分化的机制获得新的见解。

DESCRIPTION (provided by applicant): 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.