Small heat shock proteins in smooth muscle plasticity

描述(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。MicroRNAs(MiRNA)在表型研究中的新进展表明，p38MAPK/MK2信号和miRNA诱导的沉默在HSP27和Tristetraprolin水平上趋同。我们建议大幅转移该项目的重点，以测试通过p38MAPK/MK2途径磷酸化HSP27抑制miRNA沉默机制功能的新想法。HSP27的磷酸化被认为可以减少致炎基因的沉默，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p38MAPK/MK2/HSP27通路影响hASM细胞的促炎和基质蛋白的表达。另一些研究表明，HSP27定位于应激颗粒，并降低了富含AU的3‘非翻译区(ARs)的mRNAs的稳定性，但其机制尚不清楚。最近对ArgAert蛋白质的研究表明，miRNAs、AGO-2、miR-16和p38MAPK/MK2靶标Tristetraprolin定位于应激颗粒和mRNA加工体(P体)，在那里它们破坏mRNAs的稳定并导致翻译阻断。这些观察结果导致了一个新的假设，即HSP27的磷酸化也调节了miRNA诱导的人气道平滑肌沉默。为了验证这一假设，我们将：1.定义一组针对平滑肌限制基因表达的miRNAs。在细胞因子处理的细胞中，miRNA的表达将与过度表达肌钙蛋白的细胞进行比较。2.确定p38MAPK/MK2/HSP27和雷公藤红素对收缩、迁移和促炎蛋白的miRNA抑制的必要性。显性负过表达和基因敲除策略将被用来改变培养的hASMC和完整的猪气管平滑肌中的p38MAPK信号转导。我们将评估改变p38MAPK信号对mRNA稳定性和蛋白表达的影响。3.明确在改变平滑肌细胞表型的条件下，HSP27和Tristetraprolin在应激颗粒、P小体和miRISC的形成和功能中的必要性。将比较收缩和增殖的HASM细胞中P体标记物、应激颗粒标记物、HSP27和雷公藤红素的细胞分布。在HSP27被击倒后，将检测信使RNA衰退、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方面重要的miRNAs，并将确定p38MAPK和HSP27如何通过miRNA诱导的沉默来修改基因表达。一种新的转录后基因沉默机制将被研究与炎症性肺疾病的细胞应激反应、平滑肌可塑性和重塑有关。

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.