Tatiana V Kudryashova1,2,3, Swati Dabral4, Soni S Pullamsetti4,5, Elena A Goncharova1,2,3, Sreenath Nayakanti4, Arnab Ray2, Dmitry A Goncharov1,2, Theodore Avolio2, Yuanjun Shen1,2, Analise Rode2, Andressa Pena2, Lifeng Jiang1, Derek Lin1, Jeffrey Baust2, Timothy N Bachman2, Johannes Graumann6, Clemens Ruppert7, Andreas Guenther7, Mario Schmoranzer4, Yann Grobs8, Sarah Eve Lemay8, Eve Tremblay8, Sandra Breuils-Bonnet8, Olivier Boucherat8, Ana L Mora2,3, Horace DeLisser9, Jing Zhao10, Yutong Zhao10, Sébastien Bonnet8, Werner Seeger4,5. 1. Lung Center, Division of Pulmonary, Critical Care and Sleep Medicine, University of California, Davis School of Medicine (T.V.K., D.A.G., Y.S., L.J., D.L., E.A.G.). 2. Pittsburgh Heart, Lung and Blood Vascular Medicine Institute (T.V.K., A.R., D.A.G., T.A., Y.S., A.R., A.P., J.B., T.N.B., A.L.M., E.A.G.). 3. Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh (T.V.K., A.L.M., E.A.G.). 4. Department of Lung Development and Remodeling, Member of the German Center for Lung Research (DZL), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (S.D., S.N., M.S., W.S., S.S.P.). 5. Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Institute for Lung Health (ILH), Cardio-Pulmonary Institute (CPI), Justus Liebig University, Germany (W.S., S.S.P.). 6. Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (J.G.). 7. Department of Internal Medicine, Justus Liebig University, Giessen, Germany (C.R., A.G.). 8. Department of Medicine, Faculty of Medicine, Université Laval, Quebec City, Canada (Y.G., S.E.L., E.T., S.B.-B., O.B., S.B.). 9. Department of Pathology and Laboratory Medicine, Pulmonary Vascular Disease Program, University of Pennsylvania Perelman School of Medicine, Philadelphia (H.D.). 10. The Ohio State University College of Medicine, Columbus (J.Z., Y.Z.).
Abstract
RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.
RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.
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