Distinct roles of CARP target genes in wound healing

描述(申请人提供)：正常的组织修复包括炎症、肉芽组织形成和组织重塑。在慢性伤口中，这些事件被打乱，导致延迟或缺乏修复。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形成。细胞外基质重塑和由此产生的信号级联决定了组织修复是否进展或慢性受损的程度，而重塑依赖于细胞和细胞外基质之间精确控制的相互作用。基质金属蛋白酶(MMPs)家族调控ECM重塑。细胞表面整合素受体家族共同调节细胞与细胞外基质之间的黏附作用。因此，细胞、基质金属蛋白酶、整合素和其他角色之间的相互作用和串扰决定了伤口愈合过程中的重塑和血管形成。目前对调控ECM重塑的关键转录因子的了解有限。我们的实验室发现了核转录辅助因子心脏锚蛋白重复蛋白(CARP)在伤口愈合过程中新血管的形成和丰富方面的新作用。这一应用的中心假设是，在伤口修复过程中，鲤鱼蛋白调控编码基质降解酶、细胞黏附分子和其他与细胞外基质重塑和毛细血管形态形成过程相关的基因的转录。作为支持，CARP基因ankrd1的缺失会导致1个整合素亚基表达降低。有趣的是，其他整合素亚基的表达不会因缺乏鲤鱼而改变。此外，CARP的过度表达会导致MMP-13启动子活性降低，而CARP的缺失会导致体内外基质金属蛋白酶-13的mRNA表达增加。在小鼠损伤后4天，ankrd1基因的敲除导致未损伤皮肤中较高的基质金属蛋白酶-13蛋白水平，并导致小鼠切除创面中较高水平的基质金属蛋白酶-13。这项应用的目标是识别和表征鲤鱼的目标基因，这些基因在伤口修复过程中对细胞外基质重塑非常重要。为了实现这一目标，提出了两个具体目标。目的研究鲤鱼对基质金属蛋白酶-13(MMP13)的调控作用。这一目标的实现将通过识别与鲤鱼相互作用的因子，如核仁素，来调控MMP-13的活性，以及利用凝胶迁移率改变分析(EMSA)和芯片分析来表征鲤鱼如何准确地调控基质金属蛋白酶-13。目的二旨在阐明鲤鱼在11整合素调控中的作用。为此，将使用体外研究和动物伤口愈合模型相结合的方法来研究鲤鱼如何调节整合素1亚单位以及这一调节的后果。这项拟议的研究将研究转录核辅助因子CARP对整合素和基质金属蛋白酶的调节。了解鲤鱼利用其促血管生成活性的机制，可以开发出一种治疗方法，改善慢性伤口的伤口愈合，特别是在伤口修复严重受损的糖尿病患者。

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.