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.
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.
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