Competitive macrophage microRNA-RNA binding protein interactions in wound repair

伤口修复中竞争性巨噬细胞 microRNA-RNA 结合蛋白相互作用

• 批准号: 9439844
• 负责人: JEFFREY R. BENDER
• 金额: $ 32.24万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9439844
• 关键词: 3' Untranslated Regions; Address; Affinity Chromatography; Appearance; Area; Attention; Beds; Binding; Binding Sites; Biological Assay; Biotin; Blood capillaries; Cells; Chimeric Proteins; Chronic; Complex; Consequentialism; Defect; Diabetes Mellitus; Dorsal; Ear; Economic Burden; Elderly; Elements; Excision; Fibroblasts; Gelatinase B; Gene Expression; Generations; Genes; Health; HuR protein; Immune; Incidence; Individual; Inflammation; Inflammatory; Inflammatory Response; Influentials; Injury; Innate Immune Response; Integrins; Knockout Mice; Luciferases; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Multiple Trauma; Mus; Myeloid Cells; Natural Immunity; Natural regeneration; Oligonucleotides; Operative Surgical Procedures; Pathway interactions; Phase; Phenotype; Play; Porifera; Process; Production; Proteins; RNA; RNA-Binding Proteins; Recruitment Activity; Reporter; Ribosomes; Role; Site; Source; Surgical wound; Therapeutic; Thick; Tissues; Transcript; Transcriptional Activation; Transcriptional Regulation; Translating; Translations; Untranslated Regions; Ursidae Family; Vascular Endothelial Growth Factors; Venous Insufficiency; Width; Work; Wound Healing; aging population; capillary; chronic wound; diabetic; economic cost; gene repair; healing; implantation; improved; insight; mRNA Transcript Degradation; macrophage; monocyte; mortality; novel; pre-clinical; protective effect; regenerative; repaired; response; tool; wound

Inadequate wound healing, resulting in chronic wounds, is a major and increasing U.S. health problem, due to the rising incidence of diabetes and our aging population. Innate immune responses to tissue injury are critical to wound repair. Monocyte/macrophages, both recruited and tissue-resident, secrete factors that are critical mediators of the early proliferative and regenerative wound healing phases. Little attention has been paid to posttranscriptional regulatory influences on gene expression in wound localized macrophages. This is a major checkpoint in macrophage-dependent wound repair, since a majority of influential macrophage-derived products are encoded by mRNAs that bear both AU-rich elements (AREs) and numerous microRNA (miRNA) binding sites in their 3'-untranslated region (3'UTR). That is, a transcriptional activation trigger is often insufficient for adequate gene expression. In particular, RNA-binding proteins (RBPs) protect vulnerable 3'UTRs from miRNA binding and consequent translational arrest (and/or mRNA degradation). We have recently demonstrated that macrophage β2 integrin engagement results in dynamic modulation of the RBP HuR, which protects numerous 3'UTR ARE-bearing mRNAs from degradation or translation blocks. We have shown that macrophage HuR gene-deleted mice have repair defects in multiple wound models. In dynamic fashion, HuR has the ability to relieve miRNA-mediated gene expression constraints. Our hypothesis is that expression of a set of wound healing-promoting genes, both overlapping and distinct in recruited vs. tissue- resident macrophages, is driven by HuR-dependent release of miRNA-mediated translation blocks. We have generated unique, macrophage-specific translational profiling tools, and now propose to: (1) document the presence of candidate, and define novel, macrophage mRNAs undergoing active, HuR-dependent translation in early wound healing responses, using the Translating Ribosome Affinity Purification (TRAP) assay, with pulldowns of the ribosomal fusion protein L10a-EGFP, expressed in myeloid cell-specific fashion, in HuR wild- type or gene-deleted mice; (2) define miRNAs that target those TRAP-defined mRNAs, with biotin-3'UTR riboprobe pulldowns of FACS-sorted L10a-EGFP+, wound localized macrophage extracts, confirming their translation-repressing effects in dual luciferase 3'UTR reporter assays; and (3) release the miRNA-mediated translational constraint on wound healing-promoting mRNAs, topically applying miRNA target site blocker oligonucleotides which interfere with binding to specific mRNA 3'UTR target sites, in dorsal full thickness excisional and ear punch hole wound models, assessing wound area, re-epithelialization, revascularization and fibroblast repopulation. These molecular and preclinical approaches, directed at a gene expression regulatory switch in wound-responsive macrophages, will provide mechanistic insight with therapeutic implications.

伤口愈合不充分，导致慢性伤口，是一个主要的和日益严重的美国健康问题，由于 糖尿病发病率上升和人口老龄化。对组织损伤的先天免疫反应至关重要 伤口修复单核细胞/巨噬细胞，无论是招募和组织驻留，分泌的因素是至关重要的 早期增殖和再生伤口愈合阶段的介质。很少关注 创伤局部巨噬细胞基因表达的转录后调节作用。这是一个重大 检查点在巨噬细胞依赖的伤口修复，因为大多数有影响力的巨噬细胞来源的 产物由携带富含AU的元件（战神）和大量microRNA（miRNA）的mRNA编码， 在它们的3 '-非翻译区（3' UTR）中具有结合位点。也就是说，转录激活触发因子通常是 不足以充分表达基因。特别是RNA结合蛋白（RBP）可以保护脆弱的 来自miRNA结合和随后的翻译停滞（和/或mRNA降解）的3 'UTR。我们有 最近证实巨噬细胞β2整合素参与导致RBP的动态调节 HuR，其保护许多携带3 'UTR ARE的mRNA免于降解或翻译阻断。我们有 显示巨噬细胞HuR基因缺失的小鼠在多种创伤模型中具有修复缺陷。在动态 HuR具有缓解miRNA介导的基因表达限制的能力。我们的假设是 一组伤口愈合促进基因的表达，在招募的与组织中重叠和不同， 驻留的巨噬细胞由miRNA介导的翻译阻断的HuR依赖性释放驱动。我们有 产生了独特的，巨噬细胞特异性的翻译分析工具，现在建议：（1）文件， 候选物的存在，并定义新的巨噬细胞mRNA经历活性的HuR依赖性翻译 在早期伤口愈合反应中，使用翻译核糖体亲和纯化（TRAP）测定， 以髓样细胞特异性方式表达的核糖体融合蛋白L10 a-EGFP在HuR野生型中的下拉， 型或基因缺失的小鼠;（2）定义靶向那些TRAP定义的mRNA的miRNA，具有生物素-3 'UTR FACS分选的L10 a-EGFP+、伤口定位的巨噬细胞提取物的核糖探针下拉，证实了它们的 在双荧光素酶3 'UTR报告基因测定中的抑制作用;和（3）释放miRNA介导的 对伤口愈合促进mRNA的翻译限制，局部应用miRNA靶位点阻断剂 干扰与特异性mRNA 3 'UTR靶位点结合的寡核苷酸，背侧全厚度 切除和耳穿孔伤口模型，评估伤口面积，上皮再生，血管再生和 成纤维细胞再生这些分子和临床前方法，针对基因表达调控， 在创伤响应性巨噬细胞中开关，将提供具有治疗意义的机制见解。