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
基本信息
- 批准号:10001549
- 负责人:
- 金额:$ 32.24万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:3&apos Untranslated RegionsAddressAffinity ChromatographyAppearanceAreaAttentionBindingBinding SitesBiological AssayBiotinBlood capillariesCellsChimeric ProteinsChronicComplexConsequentialismDefectDiabetes MellitusDorsalEarEconomic BurdenElderlyElementsEpithelialEpitheliumExcisionFibroblastsGelatinase BGene ExpressionGenerationsGenesHealthHuR proteinImmuneIncidenceIndividualInflammationInflammatoryInflammatory ResponseInfluentialsInnate Immune ResponseIntegrinsKnockout MiceLuciferasesMediatingMediator of activation proteinMessenger RNAMicroRNAsMolecularMorbidity - disease rateMultiple WoundsMusMyeloid CellsNatural ImmunityNatural regenerationOligonucleotidesOperative Surgical ProceduresPathway interactionsPhasePhenotypePlayPoriferaProcessProductionProteinsRNARNA-Binding ProteinsReporterRibosomesRoleSiteSourceSurgical woundTherapeuticThickTissuesTopical applicationTranscriptTranscriptional ActivationTranscriptional RegulationTranslatingTranslationsUrsidae FamilyVascular Endothelial Growth FactorsVenous InsufficiencyWidthWorkWound modelsaging populationchronic wounddiabeticeconomic costgene repairhealingimplantationimprovedinsightmRNA Transcript Degradationmacrophagemonocytemortalitynovelpre-clinicalprotective effectrecruitregenerativerepairedresponsesevere injurytissue injurytoolwoundwound bedwound healing
项目摘要
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.
伤口愈合不足,导致慢性伤口,是美国一个主要且日益严重的健康问题,原因是
糖尿病发病率的上升和我们的人口老龄化。对组织损伤的先天免疫反应至关重要
伤口修复。单核/巨噬细胞,无论是招募的还是组织驻留的,都会分泌至关重要的因子
创面早期增殖期和再生期的中介物。很少有人注意到
转录后调控对创伤局部巨噬细胞基因表达的影响。这是一名少校
巨噬细胞依赖的伤口修复中的检查点,因为大多数有影响力的巨噬细胞来源
产物由同时含有富含AU的元素(ARs)和大量的microRNA(MiRNA)的mRNA编码。
其3‘-非翻译区的结合位点(3’-UTR)。也就是说,转录激活触发器通常
不足以充分表达基因。特别是,RNA结合蛋白(RBPs)保护脆弱的
3‘UTRs来自miRNA结合和随之而来的翻译停滞(和/或mRNA降解)。我们有
最近发现巨噬细胞β-2整合素参与导致RBP的动态调节
HUR,它保护许多3‘非编码区携带的mRNAs免受降解或翻译块的影响。我们有
显示巨噬细胞HUR基因缺失小鼠在多创面模型中存在修复缺陷。在动态中
时尚,HUR有能力解除miRNA介导的基因表达限制。我们的假设是
一组促进伤口愈合的基因在招募的和组织中重叠和不同的表达-
驻留的巨噬细胞,是由依赖Hur的miRNA介导的翻译块的释放驱动的。我们有
生成了独特的、巨噬细胞特定的翻译简档工具,现在建议:(1)记录
存在正在进行HUR依赖的主动翻译的候选和定义新的巨噬细胞mRNAs
在早期伤口愈合反应中,使用翻译核糖体亲和纯化(TRAP)试验,
以髓系细胞特异性方式表达的核糖体融合蛋白L10a-EGFP在呼和浩特-野生型
类型或基因缺失的小鼠;(2)定义针对这些陷阱定义的mRNAs的miRNAs,带有生物素-3‘非编码区
FACS分选的L10a-EGFP+的核探针下拉,伤口定位的巨噬细胞提取物,证实了他们的
双荧光素酶3‘非编码区报告分析中的翻译抑制效应;以及(3)释放miRNA介导的
局部应用miRNA靶点阻滞剂对促进伤口愈合的mRNAs的翻译限制
干扰与背侧全厚的特定mRNA3‘非编码区靶点结合的寡核苷酸
切除和耳穿孔伤口模型,评估伤口面积,再上皮化,再血运重建和
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
切换创伤反应巨噬细胞,将提供具有治疗意义的机械洞察力。
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages.
- DOI:10.1016/j.isci.2023.107985
- 发表时间:2023-12-15
- 期刊:
- 影响因子:5.8
- 作者:Wilcox, Nicholas S.;Yarovinsky, Timur O.;Pandya, Prakruti;Ramgolam, Vinod S.;Moro, Albertomaria;Wu, Yinyu;Nicoli, Stefania;Hirschi, Karen K.;Bender, Jeffrey R.
- 通讯作者:Bender, Jeffrey R.
T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.
T 细胞 LFA-1 诱导的促炎 mRNA 稳定是由 p38 通路激酶 MK2 介导的,该过程受 hnRNP C、H1 和 K 调节。
- DOI:10.1371/journal.pone.0201103
- 发表时间:2018
- 期刊:
- 影响因子:3.7
- 作者:Rao,GauthamK;Wong,Albert;Collinge,Mark;Sarhan,Joseph;Yarovinsky,TimurO;Ramgolam,VinodS;Gaestel,Matthias;Pardi,Ruggero;Bender,JeffreyR
- 通讯作者:Bender,JeffreyR
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