Ocular Surface Functions of KLF4 and KLF5

项目摘要 角膜上皮（CE）是一种自我再生的分层鳞状组织，通过以下方式保护眼睛的其余部分： 作为最前面的障碍。1，2我们的长期目标是确定管理 CE分层和稳态，有助于更好地了解视力威胁的分子基础 角膜疾病之前，我们证明了Krüppel样因子Klf 4和Klf 5在非细胞性肿瘤中起着至关重要的作用。 在眼表的成熟和维持中起着多余的作用。2 -17在本提案中，我们试图确定 它们在调节CE分层和稳态的不同细胞和分子途径中的作用，以及 阐明这些途径的缺陷如何影响CE分裂平面和遗传稳定性， 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测试新的中心假设，即Klf 4和Klf 5 通过协调CE细胞的分裂平面和遗传平衡来协调CE细胞分层和稳态 以TGF β 1-、BMP 6-和Pard 3-依赖方式稳定。这一假设是根据我们的 发表的工作，2-16和令人兴奋的初步结果。我们将通过以下三个具体步骤来验证这一假设： 目标。在目的1中，我们将测试KLF 4和KLF 5是否在调节CE细胞形状、顶-底极性和细胞周期中起关键作用。 (ABP)和分裂平面，从而阐明这些转录因子与细胞凋亡之间的联系。 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Klf 4和Klf 5之间，以及涉及TGF β和BMP 6途径的信号网络， 从而阐明调节CE分层和稳态的分子机制。在目标3中， 将测试Klf 4是否是基因组稳定性的关键调节因子，保护CE免受辐射诱导的DNA损伤 通过上调ABP基因Pard 3（也称为Par 3）的表达而抑制肿瘤的发生。通过描绘 角膜上皮分化、分层和癌变的调节途径，并阐明 眼表疾病如翼状胬肉和眼表鳞状细胞癌的分子基础 肿瘤（OSSN），这项建议直接解决了NIH的使命“寻求基本知识， 生命系统的性质和行为，并提供了转化为眼表疾病的可能性 这在世界上造成了相当大的医疗负担。这项研究具有重要意义，因为 预期的结果将确定对正常生长和功能重要的分子因子和途径 CE细胞，以及它们的缺陷，导致视力威胁角膜疾病。

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.