Distinct roles of CARP target genes in wound healing

CARP 靶基因在伤口愈合中的独特作用

• 批准号: 8320448
• 负责人: Karinna Almodovar
• 金额: $ 3.58万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320448
• 关键词: Address; Adhesives; Affect; Animals; Ankyrin Repeat; Basement membrane; Binding; Blood Vessels; Blood capillaries; Cardiac; Cell Adhesion Molecules; Cell Surface Receptors; Cell Survival; Cell-Matrix Junction; Cells; Chronic; Code; Collagen; Complex; Data; Deposition; Development; Electrophoretic Mobility Shift Assay; Event; Extracellular Matrix; Extracellular Matrix Proteins; Family; Gene Proteins; Gene Targeting; Genes; Genetic Transcription; Goals; Granulation Tissue; Immune; In Vitro; Inflammation; Injury; Integrins; Knock-out; Lead; Leucocytic infiltrate; Matrix Metalloproteinases; Mediating; Modeling; Molecular; Morphogenesis; Mus; Natural regeneration; Normal tissue morphology; Nuclear; Peptide Hydrolases; Process; Promoter Regions; Protein Overexpression; Proteins; Regulation; Research; Role; Series; Signal Pathway; Signal Transduction; Skin; Testing; Tissues; Vascular remodeling; Vascularization; Wound Healing; angiogenesis; capillary; cell motility; collagenase 3; design; diabetic; diabetic patient; extracellular; improved; in vivo; mRNA Expression; member; migration; neovascularization; novel; nucleolin; overexpression; prevent; promoter; protein expression; repaired; therapeutic target; tissue repair; transcription factor; vasculogenesis; wound

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.

描述（由申请人提供）：正常的组织修复涉及炎症，肉芽组织形成和组织重塑。在慢性伤口中，这些事件被破坏，导致延迟或缺乏修复。有效的伤口修复需要细胞外MATRX（ECM）重塑，以允许细胞迁移和毛细管形态发生。从该过程得出的ECM重塑和信号传导级联反应决定组织修复的进展或长期受损，而重塑取决于细胞与ECM之间的精确控制的相互作用。基质金属蛋白酶（MMP）家族调节ECM重塑。在协同的过程中，整合素的细胞表面受体家族介导细胞与ECM之间的粘附相互作用。因此，细胞，MMP，整联蛋白和其他玩家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化。当前对调节ECM重塑的关键转录因子的理解是有限的。我们的实验室在伤口愈合过程中发现了核转录辅助因子，心氨基酸重复蛋白（CARP）的新作用。该应用的中心假设是，腕蛋白调节编码基因降解蛋白酶，细胞粘附分子以及与ECM重塑过程和毛细血管形态发生过程中的基因的转录。为了支持，鲤鱼基因的缺失ANKRD1导致1个整联蛋白亚基的表达降低。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，鲤鱼的过表达导致MMP-13启动子活性降低，鲤鱼的缺失导致体外和体内MMP-13的mRNA表达增加。 ANKRD1的敲除导致未能的皮肤中的MMP-13蛋白水平较高，并在损伤后4天导致小鼠伸缩伤口中MMP-13的丰度更高。该应用的目的是识别和表征对伤口修复过程中ECM重塑很重要的鲤鱼靶基因。为了解决这个目标，提出了两个具体目标。 AIM ONE将确定鲤鱼参与基质金属蛋白酶13（MMP-13）的调节。这个目标将通过确定因素，例如核苷与鲤鱼相互作用以调节MMP-13活性，并表征鲤鱼如何使用电泳迁移率转移测定法（EMSA）和CHIP分析来准确调节MMP-13。 AIM二的设计旨在描述鲤鱼在调节11个整合素中的作用。为此，将使用体外研究和动物伤口愈合模型的组合来研究鲤鱼如何调节整联蛋白1亚基及其调节的后果。拟议的研究将通过转录核副因素Carp调节整合素和基质金属蛋白酶的调节。了解鲤鱼用于其促血管生成活性的机制可能会导致一种疗法的发展，从而改善慢性伤口的伤口愈合，尤其是在严重损害伤口修复的糖尿病患者中。 公共卫生相关性：来自心脏踝重复蛋白（CARP）过表达和缺失的数据显示鲤鱼参与新血管形成，这是伤口愈合过程的关键方面，在糖尿病状态下受损。我建议检查鲤鱼如何通过临时识别的靶标（例如整联蛋白和基质金属蛋白酶）影响组织修复。这项研究得出的结果可能导致描述调节伤口血管生成的新机制。