Role of CTCF in EGF-Induced Corneal Epithelial Growth

描述(申请人提供)：本研究的长期目标是研究在角膜上皮细胞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是一种重要的核蛋白，它与CCCTC序列结合，由表皮生长因子(EGF)激活，介导了同源盒Pax6基因的表达调控。毫无疑问，EGF诱导的控制Pax6表达的CTCF的激活在调节角膜上皮细胞更新和伤口愈合的增殖/分化过程中起着核心作用。我们还发现，在角膜上皮细胞中，EGF通过ERK(MAP Kinase)信号通路调节CTCF的表达和磷酸化。CTCF通过与Pax6 P0启动子上游5‘侧翼区的特定元件相互作用，调控Pax6基因的表达。我们的结果有力地支持了这样的观点，即EGF诱导的角膜上皮细胞增殖需要CTCF的激活，而Pax6的活性必须被抑制，因为已经表明Pax6可能促进角膜上皮细胞的早期分化。我们推测，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Pax6基因表达的能力。为了验证这一假说，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能够调节CTCF的活性；3)阐明使CTCF能够调节Pax6基因表达的调控机制。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学意义提供第一步。此外，这些结果还将揭示新的调控机制，描述为什么Pax6保留在成熟的角膜上皮细胞中，以及Pax6表达如何影响角膜上皮细胞的命运。此外，新的洞察力将在介导生长因子控制细胞增殖和分化的机制中获得。

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.