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期间生长因子调节的细胞增殖。我们最近发现，CTCF，一个重要的核蛋白结合CCCTC序列，表皮生长因子（EGF）激活介导的同源框Pax 6基因表达的调控。毫无疑问，EGF诱导的CTCF激活控制Pax 6表达在角膜上皮更新和伤口愈合过程中的增殖/分化调节中起着核心作用。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表达和磷酸化。CTCF通过与Pax 6 P0启动子上游5'侧翼区的特异性元件相互作用，作为阻遏物调控Pax 6基因的表达。我们的研究结果强烈支持这样的观点，即EGF诱导的角膜上皮细胞增殖需要CTCF的激活，而Pax 6活性必须被抑制，因为它已被建议，Pax 6可能会促进角膜上皮细胞的早期分化。我们假设EGF诱导的角膜上皮细胞增殖的有效性依赖于生长因子增加CTCF活性和降低Pax 6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）调查EGF诱导的CTCF激活和Pax 6抑制对细胞增殖的影响，2）描绘Erk和CTCF之间的相互作用，使EGF能够调节CTCF活性，和3）阐明的调节机制，使CTCF能够调节Pax 6基因表达。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，这些结果还将揭示新的调控机制，描述为什么Pax 6保留在成熟的角膜上皮中以及Pax 6表达如何影响角膜上皮细胞的命运。此外，新的见解将获得介导的生长因子控制细胞增殖和分化的机制。

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.