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分子机制中的作用。表型测定中microrna （miRNA）研究的新进展表明，p38MAPK/MK2信号和miRNA诱导的沉默在HSP27和tristeprolin水平上趋同。我们提议将项目重点转移到测试HSP27通过p38 MAPK/MK2途径磷酸化抑制miRNA沉默机制功能的新想法。据推测，HSP27的磷酸化可以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。我们发现p38 MAPK/MK2/HSP27通路影响hASM细胞的促炎和基质蛋白表达。其他研究表明，HSP27定位于应激颗粒并降低具有富au 3'非翻译区（AREs）的mrna的稳定性，但其机制尚不明确。最近对Argonaut蛋白的研究表明，mirna、Ago-2、miR-16和tristetrprolin （p38MAPK/MK2靶点）定位于应激颗粒和mRNA加工体（p -体），在那里它们破坏mRNA的稳定性并导致翻译阻滞。这些观察结果导致了一种新的假设，即HSP27的磷酸化也调节了人类气道平滑肌中mirna诱导的沉默。为了验证这个假设，我们将：1。定义一组靶向平滑肌限制性基因表达的mirna。miRNA的表达将在细胞因子处理的细胞和心肌素过表达的细胞中进行比较。2. 确定p38MAPK/MK2/HSP27和tristeprolin在miRNA抑制收缩、促迁移和促炎蛋白中的必要性。显性负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p38MAPK信号。我们将评估改变p38MAPK信号对mRNA稳定性和蛋白表达的影响。3. 在改变平滑肌细胞表型的条件下，确定应激颗粒、p体和miRISC的形成和功能中HSP27和tristeprolin磷酸化的必要性。比较p体标记物、应激颗粒标记物、HSP27和三曲丙氨酸在收缩性和增殖性hASM细胞中的细胞分布。在敲除HSP27后，将检测信使RNA衰变、RNA裂解活性和miRISC蛋白组成。结果将确定在建立平滑肌表型中重要的mirna，并将确定p38MAPK和HSP27如何通过mirna诱导的沉默修饰基因表达。一种与细胞应激反应、平滑肌可塑性和炎症性肺部疾病重塑相关的转录后基因沉默的新机制将被研究。

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.