Distinct roles of CARP target genes in wound healing

描述（由申请方提供）：正常组织修复涉及炎症、肉芽组织形成和组织重塑。在慢性伤口中，这些事件被破坏，导致修复延迟或缺乏。有效的伤口修复需要细胞外基质（ECM）重塑，以允许细胞迁移和毛细血管形态发生。ECM重塑和源自该过程的信号级联决定组织修复是否进展或慢性受损或在何种程度上进展或慢性受损，并且重塑取决于细胞与ECM之间精确控制的相互作用。基质金属蛋白酶（MMP）家族调节ECM重塑。一致地，细胞表面受体的整合素家族介导细胞和ECM之间的粘附相互作用。因此，细胞、MMP、整合素和其他参与者之间的相互作用和串扰决定了伤口愈合期间的重塑和血管形成。目前对调节ECM重塑的关键转录因子的理解是有限的。我们的实验室发现了核转录辅因子心脏锚蛋白重复蛋白（CARP）在伤口愈合过程中新血管形成和丰富中的新作用。本申请的中心假设是CARP蛋白调节编码基质降解蛋白酶、细胞粘附分子的基因和与创伤修复过程中ECM重塑和毛细血管形态发生过程相关的其他基因的转录。作为支持，CARP基因ankrd 1的缺失导致1个整合素亚基的表达降低。有趣的是，其他整合素亚基的表达不因缺乏CARP而改变。此外，在体外和体内，CARP的过表达导致MMP- 13启动子活性降低，而CARP的缺失导致MMP-13的mRNA表达增加。敲除ankrd 1导致未受伤皮肤中更高的MMP-13蛋白水平，并导致损伤后4天小鼠切除伤口中更高丰度的MMP-13。本申请的目的是鉴定和表征在伤口修复期间对ECM重塑重要的CARP靶基因。为了实现这一目标，提出了两个具体目标。目的一是研究CARP对基质金属蛋白酶-13（MMP-13）的调节作用。这一目标将通过鉴定与CARP相互作用以调节MMP-13活性的因子（例如核仁素）以及使用电泳迁移率变动测定（EMSA）和ChIP分析来表征CARP如何精确地调节MMP-13来实现。目的二研究CARP对整合素11的调控作用.为此，将结合体外研究和动物伤口愈合模型来研究CARP如何调节整合素1亚基以及这种调节的后果。本研究将探讨转录核辅因子CARP对整合素和基质金属蛋白酶的调控。了解CARP用于其促血管生成活性的机制可以导致开发改善慢性伤口中伤口愈合的疗法，特别是在伤口修复严重受损的糖尿病患者中。 公共卫生相关性：心脏锚蛋白重复蛋白（CARP）过表达和缺失的数据显示，CARP参与新血管形成，这是糖尿病状态下受损的伤口愈合过程的一个关键方面。我建议研究如何CARP影响组织修复，通过临时确定的目标，如整合素和基质金属蛋白酶。这项研究的结果可能会导致划定新的机制，调节伤口血管生成。

DESCRIPTION (provided by applicant): Normal tissue repair involves inflammation, granulation tissue formation, and tissue remodeling. In chronic wounds, these events are disrupted, resulting in delay or lack of repair. Effective wound repair requires extracellular matrx (ECM) remodeling to allow cell migration and capillary morphogenesis. ECM remodeling and signaling cascades derived from this process dictate whether or to what extent tissue repair progresses or is chronically impaired, and remodeling depends on precisely controlled interactions between cells and the ECM. The matrix metalloproteinase (MMP) family regulates ECM remodeling. In concert, the integrin family of cell surface receptors mediates adhesive interactions between cells and the ECM. Thus, interaction and crosstalk between cells, MMPs, integrins, and other players determines remodeling and vascularization during wound healing. Current understanding of key transcription factors regulating ECM remodeling is limited. Our lab has discovered a novel role of the nuclear transcription co-factor, cardiac ankyrin repeat protein (CARP), in the formation and abundance of new blood vessels during wound healing. The central hypothesis of this application is that CARP protein regulates the transcription of genes coding for matrix-degrading proteases, cell adhesion molecules, and other genes associated with the process of ECM remodeling and capillary morphogenesis during the wound repair process. In support, deletion of CARP gene, ankrd1, results in decreased expression of 1 integrin subunit. Interestingly, expression of other integrin subunits does not change by lack of CARP. Furthermore, overexpression of CARP results in decreased MMP- 13 promoter activity, and deletion of CARP results in increased mRNA expression of MMP-13 in vitro and in vivo. Knockout of ankrd1 results in higher MMP-13 protein levels in unwounded skin and leads to a higher abundance of MMP-13 in mouse excisional wounds 4 days after injury. The goal of this application is to identify and characterize CARP target genes that are important for ECM remodeling during wound repair. To address this goal two Specific Aims are proposed. Aim one will determine the involvement of CARP in regulation of matrix metalloproteinase-13 (MMP-13). This aim will be achieved by identifying factors, e.g. nucleolin, that interact with CARP to regulate MMP-13 activity as well as characterizing how exactly CARP regulates MMP-13 using electrophoretic mobility shift assay (EMSA) and ChIP analysis. Aim two is designed to delineate the role of CARP in regulation of 11 integrin. For this aim, a combination of in vitro studies and animal wound healing models will be used to investigate how CARP regulates the integrin 1 subunit and the consequences of this regulation. The proposed research will investigate the regulation of integrins and matrix metalloproteinases by the transcriptional nuclear co-factor, CARP. Understanding the mechanisms that CARP utilizes for its pro-angiogenic activities can lead to the development of a therapy that improves wound healing in chronic wounds, especially in diabetic patients where wound repair is severely compromised. PUBLIC HEALTH RELEVANCE: Data from cardiac ankyrin repeat protein (CARP) overexpression and deletion show CARP involvement in neovascularization, a critical aspect of the wound healing process that is impaired in the diabetic state. I propose to examine how CARP affects tissue repair through provisionally-identified targets such as integrins and matrix metalloproteinases. Results derived from this study can lead to the delineation of new mechanisms that regulate wound angiogenesis.