Ocular Surface Functions of KLF4 and KLF5

项目总结 角膜上皮(CE)是一种自我再生的复层鳞状组织，通过以下方式保护眼睛的其余部分 作为最前面的屏障1，2我们的长期目标是确定管理 CE分层和动态平衡，有助于更好地理解视力威胁的分子基础 角膜疾病。在此之前，我们证明了类Krüppel因子Klf4和Klf5在 在眼表的成熟和维持中的多余角色。2-17在这项建议中，我们试图确定 它们在调节CE层积和内稳态的不同细胞和分子途径中的作用，以及 阐明这些通路中的缺陷是如何影响CE分裂平面和遗传稳定性的，从而导致眼睛 表面疾病。我们将采用互补和创新的细胞培养和成像系统，转基因 小鼠模型和下一代测序方法来检验新的中心假设：KLF4和KLF5 通过协调CE细胞平面的分裂和遗传来协调CE细胞分层和动态平衡 在转化生长因子、BMP6和PARD3依赖的方式下的稳定性。这一假设是基于我们的 发表的作品，2-16个令人振奋的初步结果。我们将通过追求三个具体的假设来检验这一假设 目标。在目标1中，我们将测试KLF4和KLF5是否在调节CE细胞形状、心尖-基底极性方面发挥关键作用 (ABP)和分裂平面，从而阐明了这些转录因子和细胞之间的联系 调节CE分层的过程。在目标2中，我们将测试CE分层是否由串扰驱动 在转录因子KLF4和KLF5之间，以及涉及转化生长因子和BMP6通路的信号网络之间， 从而阐明了调控CE层积和动态平衡的分子机制。在目标3中，我们 将测试KLF4是否是保护CE免受辐射诱导的DNA损伤的基因组稳定性的关键调节因子 并通过上调ABP基因PARD3(也称为Par3)的表达来促进肿瘤的发展。通过勾勒出 角膜上皮细胞分化、层化和癌变的调控途径及阐明 翼状胬肉和眼表鳞状上皮等眼表疾病的分子基础 肿瘤(OSSN)，这项建议直接涉及美国国立卫生研究院的使命，即寻求关于 生命系统的性质和行为，为转化为眼表疾病提供了可能性 这在世界上造成了相当大的医疗负担。这项拟议的研究具有重要意义，因为它 预期的结果将确定对正常生长和功能至关重要的分子因素和途径。 CE细胞及其缺陷导致了威胁视力的角膜疾病。

PROJECT SUMMARY Corneal epithelium (CE) is a self-regenerating stratified squamous tissue that protects the rest of the eye by serving as the anterior-most barrier.1, 2 Our long-term goal is to identify the regulatory networks that govern the CE stratification and homeostasis, facilitating better understanding of the molecular basis for sight-threatening corneal disorders. Previously, we demonstrated that the Krüppel-like factors Klf4 and Klf5 play crucial non- redundant roles in maturation and maintenance of the ocular surface.2-17 In this proposal we seek to determine their role in regulating distinct cellular and molecular pathways of CE stratification and homeostasis, and elucidate how defects in these pathways affect CE plane of division and genetic stability, causing ocular surface diseases. We will employ complementary and innovative cell culture and imaging systems, transgenic mouse models and Next-Gen sequencing approaches to test the novel central hypothesis that `Klf4 and Klf5 orchestrate CE cell stratification and homeostasis by coordinating the CE cell plane of division and genetic stability in a TGF-, BMP6-, and Pard3-dependent manner'. This hypothesis was formulated based on our published work, 2-16 and exciting preliminary results. We will test this hypothesis by pursuing three Specific Aims. In Aim 1, we will test if KLF4 and KLF5 play key roles in regulating CE cell shape, apical-basal polarity (ABP) and plane of division, thereby elucidating the link between these transcription factors and the cellular processes that regulate CE stratification. In Aim 2, we will test if CE stratification is driven by a crosstalk between transcription factors Klf4 and Klf5, and signaling network involving TGF and BMP6 pathways, thereby elucidating the molecular mechanisms that regulate CE stratification and homeostasis. In Aim 3, we will test if Klf4 is a key regulator of genome stability that protects the CE from radiation-induced DNA damage and tumor development by upregulating the ABP gene Pard3 (also called Par3) expression. By delineating the regulatory pathways of corneal epithelial differentiation, stratification and carcinogenesis, and elucidating the molecular underpinnings of ocular surface disorders such as pterygium and ocular surface squamous neoplasia (OSSN), this proposal directly addresses the NIH mission of `seeking fundamental knowledge about the nature and behavior of living systems' and offers the potential for translation to ocular surface disorders which account for considerable healthcare burden in the world. The proposed research is significant as its anticipated outcomes will identify the molecular factors and pathways important for normal growth and function of CE cells, and their deficiencies that lead to sight-threatening corneal disorders.