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A7396 - Non-Canonical Vascular Smooth Muscle HIPPO-MST1/2 Signaling in Pulmonary Arterial Hypertension
Author Block: A. Ray, T. Kudryashova, A. Rode, D. Goncharov, E. A. Goncharova; Department of Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
RATIONALE: Pulmonary vascular remodeling is a key pathological feature of pulmonary arterial hypertension (PAH). Increased proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMC) are important components of pulmonary vascular remodeling, the mechanisms of which are not completely understood. We and others recently reported the roles for HIPPO and mechanistic target of rapamycin (mTOR) signaling networks as a negative and positive regulator, respectively, of proliferative, apoptosis-resistant PAVSMC phenotype in PAH. HIPPO is a growth suppressor cassette formed by two sets of catalytic subunits, mammalian STE20-like protein kinases 1/2 (MST1/2) and large tumor suppressors 1/2 (LATS1/2). We found that MST1/2-independent dysfunction of LATS1 in PAVSMC promotes hyper-activation of transcriptional co-activators Yap/Taz, pulmonary vascular remodeling and PH. The role of MST1/2 in PAVSMC proliferation, survival and mTOR up-regulation in PAH is unknown.
METHODS AND RESULTS: Immunoblot analysis of early-passage distal PAVSMC from subjects with pulmonary arterial hypertension (PAH) and non-diseased (control) donor lungs (n=5 subjects/group) demonstrated that human PAH PAVSMC had marked reduction of cleaved phospho-Thr183 MST1 and phospho-Thr180 MST2 compared to controls, suggestive of deficiency of active (cleaved and phosphorylated) MST1/2. siRNA-induced depletion of either MST1 or MST2 in control human PAVSMC significantly up-regulated mTOR complex 1 (mTORC1) and mTORC2 signaling pathways (assessed by mTORC2-specific S473Akt and mTORC1-specific ribosomal protein S6 phosphorylation, respectively). Further, siRNA MST1 and siRNA MST2-transfected control PAVSMC showed increased cell growth (detected by cell count assays) and reduced levels of pro-apoptotic protein Bim without significant effect on apoptosis, demonstrating canonical anti-proliferative/pro-apoptotic role for MST1/2 in non-diseased human PAVSMC. Surprisingly, in human PAH PAVSMC, siRNA-induced depletion of MST1 or MST2 resulted in down-regulation of both, mTORC1-Akt and mTORC2-S6 pathways, suppression of cell growth, marked accumulation of pro-apoptotic Bim and significant induction of apoptosis compared to cells transfected with control siRNA, suggesting that MST1 and MST2 are required for pathological up-regulation of mTORC2-Alt and mTORC1-S6 pathways, increased proliferation and reduced apoptosis of human PAH PAVSMC.
CONCLUSIONS: To the best of our knowledge, this is the first report of the pathological switch of MST1/2 function from anti-proliferative/pro-apoptotic to pro-proliferative/pro-survival protein. We also demonstrate that this MST1/2 function is specific for PAVSMC from PAH lungs and is required for up-regulation of mTORC1/2 axis and maintenance of disease-specific proliferative/pro-survival PAVSMC phenotype. This data suggests the potential attractiveness of MST1/2 as a molecular targets to selectively inhibit proliferation and promote apoptosis in diseased PAVSMC and reduce pulmonary vascular remodeling.