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生长因子调节和应激诱导的细胞增殖、分化和凋亡对角膜上皮更新过程和伤口愈合的影响。我们最近发现，CTCF是一种表观遗传调节因子，它与重要基因的CCCTC序列结合，在调节角膜上皮细胞同源盒Pax6的过程中发挥着核心作用。毫无疑问，在角膜上皮细胞更新和伤口愈合过程中，表皮生长因子(EGF)诱导的CTCF的激活抑制了Pax6的表达，从而促进了细胞的增殖和分化。相反，高渗和紫外线应激诱导的CTCF抑制会由于增加角膜上皮细胞凋亡而导致伤口愈合的延迟。我们还发现，EGF和其他应激通过激活炎症相关的NF-kB信号通路来调节CTCF的表达。亚基特异性激活的核因子-kB二聚体通过与CTCF基因转录起始点上游的特定元件5‘-相互作用来二分地控制CTCF的表达。我们的结果有力地支持了这样的观点，即EGF和应激诱导的NF-kB激活对角膜上皮细胞命运的双重影响是由于形成了分别对CTCF基因转录产生正向和负向影响的核因子-kB异源二聚体和同源二聚体。我们假设CTCF是由EGF和应激诱导的角膜上皮细胞中不同的核因子-kB亚基形成的核因子-kB二聚体调节的，从而控制了调控角膜上皮细胞增殖和凋亡的重要基因表达，并影响伤口愈合。为了验证这一假说，我们将承担三个具体目标：1)表征表皮生长因子和应激诱导的NF-kB激活对CTCF调节的影响；2)阐明NF-kB调节CTCF活性的分子机制；3)研究NF-kB在调节CTCF功能从而影响角膜伤口愈合中的作用(S)。这些研究将为测试CTCF在介导生长因子和应激诱导的角膜上皮细胞增殖、分化和凋亡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，该机制描述了为什么不同刺激激活的NF-kB会导致不同的细胞反应。此外，这项研究将为核因子-kB激活调节CTCF如何介导EGF和应激诱导的细胞脂肪的机制提供新的见解。角膜上皮再生由生长因子促进，但因环境压力而延迟，在伤口愈合过程中是必不可少的，因为它使组织起到屏障的作用，保护角膜内部免受疾病和有害环境因素的伤害。这一创新项目是第一个提供证据表明生长因子和环境应激都激活了涉及重要基因调节因子-核因子-kappaB(核因子-kappa B)和CTCF(CCCTC结合因子)的炎症信号通路的研究。随后，这些基因调节器控制决定角膜上皮细胞增殖、分化和凋亡(程序性细胞死亡)的进一步下游基因的表达水平。

DESCRIPTION (provided by applicant): The long-term goal of the present study is to investigate the effects of growth factor-regulated and stress-induced cell proliferation, differentiation and apoptosis on the corneal epithelial renewal process and wound healing. We recently found that CTCF, an epigenetic regulator that binds to the CCCTC sequence of important genes, plays a central role in the regulation of homeobox Pax6 in corneal epithelia. Undoubtedly, epidermal growth factor (EGF)-elicited activation of CTCF inhibits Pax6 expression to promote proliferation/differentiation during corneal epithelial renewal and wound healing. In contrast, hyper-osmotic and UV stress-induced suppression of CTCF results in retardation of wound healing due to increased corneal epithelial apoptosis. We also found that EGF and other stresses regulate CTCF expression through activation of the inflammation- related NF-kB signaling pathway. Subunit-specific activation of NF-kB dimers dichotomously controls CTCF expression by interacting with a specific element 5'-upstream of the transcription initiation site of the CTCF gene. Our results strongly support the notion that the dichotomous effects of EGF- and stress-induced NF-kB activation on corneal epithelial cell fates are due to the formations of NF-kB heterodimers and homodimers that exert positive and negative effects on CTCF gene transcription, respectively. We hypothesize that CTCF is regulated by NF-kB dimers formed by different NF-kB subunits in EGF- and stress-induced corneal epithelial cells, resulting in controls of important gene expressions that regulate corneal epithelial proliferation and apoptosis and affect wound healing. To test the hypothesis, we will undertake three specific aims including: 1) to characterize the effects of EGF- and stress-induced NF-kB activation on the regulation of CTCF, 2) to elucidate the molecular mechanisms of how NF-kB regulates CTCF activity, and 3) to investigate the role(s) of NF-kB in regulating CTCF function to affect corneal wound healing. Such studies will provide the first step towards testing the physiological significance of CTCF in mediating growth factor- and stress-induced corneal epithelial cell proliferation, differentiation and apoptosis. In addition, the results will also reveal novel regulatory mechanisms that describe why activation of NF-kB by different stimuli results in different cellular responses. Furthermore, the study will provide new insight into the mechanisms of how regulation of CTCF by NF-kB activation mediates EGF- and stress-induced cell fates.PUBLIC HEALTH RELEVANCE. Corneal epithelial renewal, which is promoted by growth factors and delayed by environmental stresses, is essential in the wound healing process because it enables the tissue to act as a barrier that protects the corneal interior from becoming injured by diseases and noxious environmental agents. This innovative project is the first study to provide evidence showing that both growth factors and environmental stresses activate an inflammatory signaling pathway involving important gene regulators of NF-K:B (nuclear factor-kappa B) and CTCF (CCCTC binding factor). Subsequently, these gene regulators control the expression levels of further downstream genes that determine corneal epithelial proliferation, differentiation and apoptosis (programmed cell death).