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

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.

Isoform-specific characterization of class I histone deacetylases and their therapeutic modulation in pulmonary hypertension.

Pharmacological modulation of class I histone deacetylases (HDAC) has been evaluated as a therapeutic strategy for pulmonary hypertension (PH) in experimental models of PH. However, information of their expression, regulation and transcriptional targets in human PH and the therapeutic potential of isoform-selective enzyme modulation are lacking. Comprehensive analysis of expression and regulation of class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8) was performed in cardiopulmonary tissues and adventitial fibroblasts isolated from pulmonary arteries (PAAF) of idiopathic pulmonary arterial hypertension (IPAH) patients and healthy donors. Cellular functions and transcriptional targets of HDAC enzymes were investigated. Therapeutic effects of pan-HDAC (Vorinostat), class-selective (VPA) and isoform-selective (CAY10398, Romidepsin, PCI34051) HDAC inhibitors were evaluated ex vivo (IPAH-PAAF, IPAH-PASMC) and in vivo (rat chronic hypoxia-induced PH and zebrafish angiogenesis). Our screening identifies dysregulation of class I HDAC isoforms in IPAH. Particularly, HDAC1 and HDAC8 were consistently increased in IPAH-PAs and IPAH-PAAFs, whereas HDAC2 and HDAC8 showed predominant localization with ACTA2-expressing cells in extensively remodeled IPAH-PAs. Hypoxia not only significantly modulated protein levels of deacetylase (HDAC8), but also significantly caused dynamic changes in the global histone lysine acetylation levels (H3K4ac, H3K9/K14ac and H3K27ac). Importantly, isoform-specific RNA-interference revealed that HDAC isoforms regulate distinct subset of transcriptome in IPAH-PAAFs. Reduced transcript levels of KLF2 in IPAH-PAAFs was augmented by HDAC8 siRNA and HDAC inhibitors, which also attenuated IPAH-associated hyperproliferation and apoptosis-resistance ex vivo, and mitigated chronic hypoxia-induced established PH in vivo, at variable degree. Class I HDAC isoforms are significantly dysregulated in human PAH. Isoform-selective HDAC inhibition is a viable approach to circumvent off-target effects.

A RASSF1A-HIF1α loop drives Warburg effect in cancer and pulmonary hypertension.

Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 α (HIF-1α) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1α, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1α forms a feedforward loop driving hypoxia signaling in PH and cancer.