Function of IRF6 in regulating E-cadherin dependent adherens junctions

项目总结 美国每年进行的手术超过7000万例。尽管许多伤口没有愈合 问题是，一半需要手术后伤口护理。无法愈合的伤口每年影响约700万人 并产生约1000亿美元的治疗费用，这给美国造成了巨大的财政负担 经济舱。增加我们对调控伤口愈合的分子途径的了解将增强 组织修复和降低医疗成本。 我们的长期目标是确定调节组织修复的分子途径。我们之前演示过 转录因子干扰素调节因子6(IRF6)是通过作用正确的伤口愈合所必需的 作为角质形成细胞分化、增殖和集体细胞迁移的主要调节因子。我们最近 初步数据显示，irf6缺乏的角质形成细胞具有较弱的细胞-细胞粘附力，并使细胞膜减少。 粘附素连接元件的定位，包括E-钙粘附素，为IRF6- 依赖性角质形成细胞迁移缺陷。有趣的是，我们的初步数据还显示，道达尔 连接蛋白水平没有改变，表明IRF6在这些过程中具有非转录功能。 IRF6作为干扰素调节性转录因子家族的成员，含有一个高度保守的N- 末端，DNA结合区，以及一个不太保守的蛋白质相互作用域。描述了IRF6的大部分内容 其功能与转录活性有关，对其功能知之甚少。 蛋白质相互作用域。具体地说，IRF6的哪个结构域对细胞间的黏附起作用 伤口愈合，还不得而知。我们的中心假设是，IRF6通过非细胞因子促进细胞的集体迁移 细胞质膜上细胞间黏附分子的转录调控。我们将测试我们的中央 执行两个目标的假设。在目标1中，我们将确定IRF6如何监管E-钙粘素贩运。 在目标2中，我们将确定IRF6如何促进细胞集体迁移。为了验证我们的假设，使用广泛的 一系列生化和细胞分析，我们将利用多个IRF6突变株来确定 该转录因子的哪些区域是调节细胞黏附所必需的。相同的突变细胞系 将用于在2D模型中执行划痕伤口和在3D模型中执行切除伤口，这将有助于了解 IRF6的每个结构域在集体细胞迁移中的重要性。 在这项研究完成后，我们将确定一种新的机制，通过这种转录因子 调节细胞黏附组织所必需的囊泡运输，这可能提供一种分子 观察到IRF6突变患者手术并发症风险增加的机制。

PROJECT SUMMARY Over 70 million surgeries are performed in the United States annually. Although many wounds heal without problem, half require postsurgical wound care. Wounds that do not heal affect about 7 million people annually and generate treatment costs of about $100 billion, which creates a significant financial burden on the US economy. Increasing our understanding of the molecular pathways regulating wound healing would enhance tissue repair and reduce healthcare costs. Our long-term goal is to identify molecular pathways regulating tissue repair. We previously demonstrated that the transcription factor Interferon Regulatory Factor 6 (IRF6) is required for proper wound healing by acting as a master regulator of keratinocyte differentiation, proliferation, and collective cell migration. Our recent preliminary data show that Irf6-deficient keratinocytes have weaker cell-cell adhesion, and reduced membrane localization of adherens junction components, including E-cadherin, providing a potential rationale for the IRF6- dependent keratinocyte migration defect. Interestingly, our preliminary data also revealed that total adherens junction protein levels were not changed, suggesting a non-transcriptional function of IRF6 in these processes. IRF6, as a member of the Interferon regulatory transcription factor family, contains a highly conserved N- terminal, DNA-binding domain, and a less conserved protein interaction domain. Most of IRF6 described functions have been associated with its transcriptional activity, and very little is known about the functions of its protein interaction domain. Particularly, which domain of IRF6 contributes to cell-cell adhesions required for wound healing, is unknown. Our central hypothesis is that IRF6 promotes collective cell migration via a non- transcriptional regulation of cell-cell adhesion molecules at the cytoplasmic membrane. We will test our central hypothesis with the execution of two aims. In Aim 1 we will determine how IRF6 regulates E-cadherin trafficking. In Aim 2 we will determine how IRF6 promotes collective cell migration. To test our hypothesis, using a wide range of biochemical and cellular assays, we will take advantage of multiple IRF6 mutant lines to determine which domains of this transcription factor are required for regulating cell adhesions. The same mutant cell lines will be used to perform scratch wounds in 2D and excisional wounds in 3D models which will shed light on the importance of each domain of IRF6 in collective cellular migration. At the completion of this study, we will have identified a novel mechanism by which this transcription factor regulates vesicular trafficking necessary for cell adhesion organization, which could provide a molecular mechanism for the increased risk of surgical complications observed in patients with IRF6 mutations.