Noncanonical HIPPO/MST Signaling via BUB3 and FOXO Drives Pulmonary Vascular Cell Growth and Survival

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 artery 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 artery 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 fibroblast, 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 cell, 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 cell and pulmonary arterial adventitial fibroblast, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.

The Hippo signalling pathway and its implications in human health and diseases

“…306,307 Interestingly, recent research revealed that MST1/2, a Hippo component that usually plays an antiproliferative role, supports the abnormal proliferation of PASMCs in pulmonary arterial hypertension via forehead homeobox type O and BUB3. 308 Second, the Hippo pathway could regulate the inflammatory response in the lung. In a bacterial pneumonia model, the relative levels of p-YAP and p-TAZ were decreased in alveolar epithelial type II cells (AECIIs).…”

Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension

“…Cell proliferation was assessed using Ki-67 immunostaining as described previously ( 10 , 32 , 34 ). Briefly, cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized using Triton X-100/PBS solution.…”

“…Cell growth analysis was performed as described previously ( 10 , 32 , 34 ). Briefly, 300,000 cells per well were plated in a six well plate in cell culture media supplemented with 5% FBS.…”

“…Immunoblot analysis was performed as described previously ( 10 , 32 , 34 ). Antibodies for ACLY (#4332), P-S79-ACC (#11818), ACC (#3676), FASN (#3180), P-S473-Akt (#4060), P-T450-Akt (#12178), Akt (#9272), PThr183/Tyr185-JNK (#4668), JNK (#9252), α/β-Tubulin (#2148), CPT1A (#97361), hexokinase II (HKII) (#2867), phosphofructokinase (PFKP) (#8164) were purchased from Cell Signaling (Danvers, MA, United States).…”

Cardiovascular complications in insulin resistance and endocrine diseases

“…FoxO3a promotes VSMC growth arrest and apoptosis after vascular injury [57,58], suggesting that Aktdependent regulation of FoxOs activity also plays an important role in VSMC biology. Moreover, in VSMCs of human pulmonary arteries, degradation of FOXO3 is promoted by interleukin-6-induced mammalian Ste20like kinases 1/2 expression, which enhances proliferation and inhibits apoptosis in VSMCs to contribute to the pathogenesis of pulmonary arterial hypertension [59].…”