Function of IRF6 in regulating E-cadherin dependent adherens junctions

项目摘要 美国每年进行超过7000万例手术。虽然许多伤口愈合没有 问题，一半需要术后伤口护理。每年约有700万人的伤口无法愈合 并产生约1000亿美元的治疗费用，这给美国带来了巨大的财政负担 经济增加我们对调节伤口愈合的分子途径的理解， 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。我们先前表明 转录因子干扰素调节因子6（IRF 6）是伤口正常愈合所需的 作为角质形成细胞分化、增殖和集体细胞迁移的主要调节剂。我们最近 初步数据显示，Irf 6缺陷型角质形成细胞具有较弱的细胞间粘附， 粘附连接成分的定位，包括E-钙粘蛋白，为IRF 6- 依赖性角化细胞迁移缺陷。有趣的是，我们的初步数据还显示， 连接蛋白水平没有改变，表明IRF 6在这些过程中的非转录功能。 IRF 6是干扰素调节转录因子家族的一员，含有一个高度保守的N- 末端、DNA结合结构域和较不保守的蛋白质相互作用结构域。大多数IRF 6描述 功能与其转录活性有关，对其功能知之甚少。 蛋白质相互作用域特别是，IRF 6的哪个结构域有助于细胞间粘附， 伤口愈合情况不明。我们的中心假设是，IRF 6通过一个非- 细胞间粘附分子在细胞质膜上的转录调节。我们将测试我们的中央 假设有两个目标的执行。在目标1中，我们将确定IRF 6如何调节E-钙粘蛋白贩运。 在目标2中，我们将确定IRF 6如何促进集体细胞迁移。为了验证我们的假设，使用广泛的 一系列生化和细胞测定，我们将利用多个IRF 6突变株系来确定 该转录因子的哪些结构域是调节细胞粘附所必需的。同样的突变细胞系 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，这将揭示 IRF 6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，这种转录因子 调节细胞粘附组织所必需的囊泡运输，这可以提供一种分子 在IRF 6突变患者中观察到的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.