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生长因子调控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创面愈合的影响。我们最近发现CTCF是一种结合重要基因CCCTC序列的表观遗传调控因子，在角膜上皮中同源盒Pax6的调控中起核心作用。毫无疑问，表皮生长因子（EGF）诱导的CTCF激活抑制Pax6的表达，从而促进角膜上皮更新和伤口愈合过程中的增殖/分化。相反，高渗透和紫外线应激诱导的CTCF抑制由于角膜上皮细胞凋亡增加而导致伤口愈合迟缓。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。NF-kB二聚体的亚基特异性激活通过与CTCF基因转录起始位点上游的特定元件5'相互作用，二分法控制CTCF的表达。我们的研究结果有力地支持了这样一种观点，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NF-kB异二聚体和同二聚体的形成，它们分别对CTCF基因转录产生积极和消极的影响。我们假设CTCF受到由不同NF-kB亚基在EGF和应激诱导的角膜上皮细胞中形成的NF-kB二聚体的调控，从而控制了调节角膜上皮细胞增殖和凋亡并影响伤口愈合的重要基因表达。为了验证这一假设，我们将进行三个具体目标，包括：1)表征EGF和应激诱导的NF-kB激活对CTCF调节的影响，2)阐明NF-kB如何调节CTCF活性的分子机制，以及3)研究NF-kB在调节CTCF功能中影响角膜创面愈合的作用。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意义提供第一步。此外，研究结果还揭示了新的调控机制，描述了为什么不同刺激激活NF-kB会导致不同的细胞反应。此外，该研究将为NF-kB活化如何调节CTCF介导EGF和应激诱导的细胞命运的机制提供新的见解。公共卫生相关性。角膜上皮的更新在伤口愈合过程中是必不可少的，因为它使组织作为一个屏障，保护角膜内部免受疾病和有害环境因素的伤害。这个创新的项目是第一个提供证据的研究，表明生长因子和环境应激都激活了炎症信号通路，涉及NF-K:B（核因子- κ B）和CTCF （CCCTC结合因子）的重要基因调节因子。随后，这些基因调节因子进一步控制下游基因的表达水平，这些基因决定角膜上皮细胞的增殖、分化和凋亡（程序性细胞死亡）。

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).