Small heat shock proteins in smooth muscle plasticity

描述（由申请人提供）：我们的长期目标是确定小应激蛋白在平滑肌表型可塑性的分子机制中的作用。microRNA（miRNA）在表型决定中的研究的新进展表明，p38 MAPK/MK2信号传导和miRNA诱导的沉默在HSP 27和tristetraprolin水平上的收敛。我们提出了一个实质性的转移项目的重点，以测试新的想法，即通过p38 MAPK/MK2途径磷酸化HSP 27抑制miRNA沉默机制的功能。假设HSP 27的磷酸化减少促炎基因的沉默，从而促进人气道平滑肌细胞（hASM）的增殖、迁移和分泌状态。我们已经证明p38 MAPK/MK2/HSP 27通路影响hASM细胞中的促炎和基质蛋白表达。其他人已经表明HSP 27定位于应激颗粒并降低具有富含AU的3'非翻译区（战神）的mRNA的稳定性，但机制尚不清楚。最近对Argonaut蛋白的研究表明，miRNA、Ago-2、miR-16和tristetraprolin（p38 MAPK/MK2靶标）定位于应激颗粒和mRNA加工体（P体），在那里它们使mRNA不稳定并引起翻译阻断。这些观察结果导致了一种新的假设，即HSP 27的磷酸化也调节人气道平滑肌中miRNA诱导的沉默。为了验证这个假设，我们将：1。定义靶向平滑肌限制性基因表达的miRNA组。将比较用细胞因子处理的细胞与过表达心肌素的细胞中的miRNA表达。2.确定p38 MAPK/MK2/HSP 27和tristetraprolin对收缩、促迁移和促炎蛋白的miRNA抑制的必要性。显性负性过表达和敲低策略将用于改变培养的hASMC和完整猪气管平滑肌中的p38 MAPK信号传导。将评估改变p38 MAPK信号传导对mRNA稳定性和蛋白质表达的影响。3.确定在改变平滑肌细胞表型的条件下，HSP 27和tristetraprolin磷酸化在应激颗粒、P体和miRISC的形成和功能中的必要性。将在收缩与增殖hASM细胞中比较P体标志物、应激颗粒标志物、HSP 27和三曲脯氨酸的细胞分布。在HSP 27敲低后，将测定信使RNA衰变、RNA切割活性和miRISC蛋白组成。这些结果将确定在建立平滑肌表型中重要的miRNAs，并将确定p38 MAPK和HSP 27如何通过miRNAs诱导的沉默来修饰基因表达。将研究与炎症性肺病中的细胞应激反应、平滑肌可塑性和重塑相关的转录后基因沉默的新机制。 公共卫生相关性：小核糖核酸（microRNA）研究的令人兴奋的新进展改变了我们对器官如何发育以及包括肺部疾病在内的慢性疾病如何治疗的理解。我们提出了一个重要的新作用的生化信号转导途径，已经是一个新的抗炎药物的目标。发现这种途径如何改变microRNA的功能可能会导致新的抗炎药物或药物组合更有效地治疗肺部疾病。

DESCRIPTION (provided by applicant): Our longstanding goal is to define the role of small stress proteins in molecular mechanisms of smooth muscle phenotypic plasticity. New developments in studies of microRNAs (miRNA) in phenotype determination suggest a convergence of p38MAPK/MK2 signaling and miRNA-induced silencing at the level of HSP27 and tristetraprolin. We are proposing a substantial shift in focus of the project to test the novel idea that phosphorylation of HSP27 via the p38 MAPK/MK2 pathway inhibits the function of the miRNA silencing machinery. Phosphorylation of HSP27 is hypothesized to reduce silencing of proinflammatory genes thus promoting proliferative, migratory and secretory states of human airway smooth muscle cells (hASM). We have shown the p38 MAPK/MK2/HSP27 pathway influences proinflammatory and matrix protein expression in hASM cells. Others have shown HSP27 localizes to stress granules and reduces stability of mRNAs with AU-rich 3' untranslated regions (AREs), but the mechanism is undefined. Recent work on Argonaut proteins shows miRNAs, Ago-2, miR-16 and tristetraprolin, a p38MAPK/MK2 target, are localized to stress granules and mRNA processing bodies (P-bodies) where they destabilize mRNAs and cause translational block. These observations have led to the novel hypothesis that phosphorylation of HSP27 also modulates miRNA-induced silencing in human airway smooth muscle. To test this hypothesis we will: 1. Define sets of miRNAs that target smooth muscle-restricted gene expression. miRNA expression will be compared in cells treated with cytokines to cells overexpressing myocardin. 2. Determine the necessity of p38MAPK/MK2/HSP27 and tristetraprolin for miRNA repression of contractile, promigratory and proinflammatory proteins. Dominant negative overexpression and knockdown strategies will be used to alter p38MAPK signaling in cultured hASMC and intact pig tracheal smooth muscle. The effects of altering p38MAPK signaling on mRNA stability and protein expression will be assessed. 3. Define the necessity for phosphorylation of HSP27 and tristetraprolin in the formation and function of stress granules, P-bodies and miRISC under conditions that alter smooth muscle cell phenotype. The cellular distribution of P-body marker, stress granule markers, HSP27 and tristetraprolin will be compared in contractile vs proliferating hASM cells. Messenger RNA decay, RNA cleavage activity and miRISC protein composition will be assayed after knockdown of HSP27. The results will identify miRNAs important in establishing smooth muscle phenotypes and will determine how p38MAPK and HSP27 modifies gene expression via miRNA-induced silencing. A novel mechanism of post-transcriptional gene silencing will be investigated relevant to cellular stress responses, smooth muscle plasticity and remodeling in inflammatory lung diseases. PUBLIC HEALTH RELEVANCE: Exciting new developments in studies of small ribonucleic acids called microRNAs have changed our understanding of how organs develop and how chronic diseases including lung disease might be treated. We are proposing an important novel role for a biochemical signal transduction pathway that is already a target for new anti-inflammatory drugs. Discovering how this pathway can alter the function of microRNAs could lead to new anti-inflammatory drugs or combinations of drugs to treat lung diseases more effectively.