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了组织修复是否进展或程度如何或长期受损，而重塑取决于细胞和 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。细胞表面受体的整合素家族一致介导细胞和 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过程中的重塑和血管化。目前对调节 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度中的新作用。该应用的中心假设是，CARP 蛋白调节基质降解蛋白酶、细胞粘附分子的编码基因的转录，以及与伤口修复过程中 ECM 重塑和毛细血管形态发生相关的其他基因的转录。删除 CARP 基因 ankrd1 会导致 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会因 CARP 的缺乏而改变。此外，CARP 的过表达导致 MMP-13 启动子活性降低，CARP 的缺失导致体外和体内 MMP-13 mRNA 表达增加。敲除 ankrd1 会导致未受伤皮肤中 MMP-13 蛋白水平升高，并导致受伤 4 天后小鼠切除伤口中 MMP-13 丰度更高。该应用的目标是识别和表征对伤口修复过程中 ECM 重塑非常重要的 CARP 靶基因。为了实现这一目标，提出了两个具体目标。目标一将确定 CARP 在基质金属蛋白酶-13 (MMP-13) 调节中的参与。这一目标将通过确定因素来实现，例如核仁素，与 CARP 相互作用调节 MMP-13 活性，并使用电泳迁移率变动分析 (EMSA) 和 ChIP 分析表征 CARP 如何准确调节 MMP-13。目标二旨在描述 CARP 在 11 整合素调节中的作用。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。拟议的研究将调查转录核辅助因子 CARP 对整合素和基质金属蛋白酶的调节。了解 CARP 促血管生成活性的机制可以促进开发一种改善慢性伤口愈合的疗法，尤其是伤口修复严重受损的糖尿病患者。 公共健康相关性：心脏锚蛋白重复蛋白 (CARP) 过度表达和缺失的数据显示，CARP 参与新生血管形成，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 CARP 如何通过暂时确定的靶标（例如整合素和基质金属蛋白酶）影响组织修复。这项研究的结果可以帮助描述调节伤口血管生成的新机制。