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 ?2 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.
Diabetics and the elderly typically heal wounds slowly, sometimes inadequately, with consequential morbidity and even mortality. Inflammatory responses to wounds, by immune cell macrophages, are important early components of proper wound healing. This work addresses molecular controls on production of wound healing- promoting factors, with aims at generating a novel class of topical therapeutics to enhance wound repair.
