Small heat shock proteins in smooth muscle plasticity

平滑肌可塑性中的小热休克蛋白

• 批准号: 8220912
• 负责人: William T Gerthoffer
• 金额: $ 33.08万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220912
• 关键词: 3' Untranslated Regions; Adenovirus Vector; Anti-Inflammatory Agents; Anti-inflammatory; Biochemical; Biological Assay; Biology; Cells; Cellular Stress Response; Chronic Disease; Contractile Proteins; Cultured Cells; Cytoplasmic Granules; Data; Development; Dominant-Negative Mutation; Drug Combinations; Enzyme-Linked Immunosorbent Assay; Family suidae; Gene Expression; Genes; Goals; HSPB1 gene; Heat shock proteins; Human; Inflammation; Inflammatory; Lead; Lentivirus Vector; Lung diseases; MAP Kinase Gene; MAPK14 gene; Measures; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Molecular; Molecular Profiling; Muscle Contraction; Northern Blotting; Obstructive Lung Diseases; Organ; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Process; Proliferating; Proteins; Publishing; RNA; RNA Decay; RNA Interference; Repression; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Stress; TIS11 protein; Testing; Tissues; Western Blotting; Work; airway remodeling; cell motility; cytokine; inhibitor/antagonist; knock-down; locked nucleic acid; mRNA Decay; mRNA Stability; migration; mutant; myocardin; novel; overexpression; protein expression; public health relevance; respiratory smooth muscle; stress protein

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.

描述（由申请人提供）：我们的长期目标是定义小压力蛋白在平滑肌表型可塑性的分子机制中的作用。 MicroRNA（miRNA）在表型测定中的新发展表明，p38MAPK/MK2信号传导和miRNA诱导的HSP27和Tristetraprolin水平上的沉默。我们提出了该项目重点的重大转变，以测试通过p38 MAPK/MK2途径磷酸化HSP27的新思想，抑制了miRNA沉默机制的功能。假设Hsp27的磷酸化以减少促炎基因的沉默，从而促进人类气道平滑肌细胞的增殖，迁移和分泌状态（HASM）。我们已经显示了p38 MAPK/MK2/HSP27途径影响HASM细胞中的促炎和基质蛋白表达。其他人则显示HSP27本地化以应对颗粒的应力并降低了MRNA的稳定性，而Au富3'未翻译区域（ARES），但该机制不确定。关于Argonaut蛋白的最新研究表明，P38MAPK/MK2靶标MiRNA，AGO-2，miR-16和Tristetraprolin，被定位为应力颗粒和mRNA加工体（P-Bodies（P-Bodies），它们使mRNA破坏了mRNA并引起翻译块。这些观察结果导致了一个新的假设，即Hsp27的磷酸化还调节了miRNA诱导的人类气道平滑肌的沉默。为了检验这一假设，我们将：1。定义靶向平滑肌限制基因表达的miRNA集。在用细胞因子治疗的细胞中，将比较miRNA的表达与过表达心肌素的细胞。 2。确定p38MAPK/MK2/HSP27和Tristetraprolin的必要性，以抑制收缩，降低和促炎蛋白的miRNA抑制。主要的负过表达和敲低策略将用于改变培养的HASMC和完整的猪气管平滑肌中的p38mapk信号传导。将评估改变p38mapk信号对mRNA稳定性和蛋白质表达的影响。 3。在改变平滑肌细胞表型的条件下，定义了Hsp27和Tristetraprolin磷酸化的必要性。 P体标记，应力颗粒标记，Hsp27和Tristetraprolin的细胞分布将在收缩与增殖的HASM细胞中进行比较。 Messenger RNA衰减，RNA裂解活性和Mirisc蛋白组成将在HSP27敲低后测定。结果将确定对建立平滑肌表型重要的miRNA，并确定p38mapk和Hsp27如何通过miRNA诱导的沉默修饰基因表达。转录后基因沉默的一种新型机制将与细胞应激反应，平滑肌可塑性和炎症性肺部疾病中的重塑有关。 公共卫生相关性：在称为microRNA的小核糖核酸研究中令人兴奋的新发展改变了我们对器官如何发展的理解以及如何治疗包括肺部疾病在内的慢性疾病。我们提出了生化信号转导途径的重要新作用，该途径已经是新型抗炎药的目标。发现该途径如何改变microRNA的功能可能会导致新的抗炎药或药物组合以更有效地治疗肺部疾病。