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

Meet the First Author, see p 692

The MST (mammalian Ste20-like kinase) 1 and 2 (STK4/3) form the catalytic core of HIPPO, an evolutionally conserved growth-suppressor cassette.^1,2 In adult somatic cells, MST1 and 2 act as growth suppressors and protect against cancer, fibrosis, and the proliferation-driven remodeling of systemic vasculature.^3–5 MST1/2 are activated by cleavage and auto-phosphorylation at T180/183 and inhibit cell proliferation and induce apoptosis via a broad range of mechanisms, the majority of which include phosphorylation of the large tumor suppressors (LATS)1/2 and concomitant inhibition of the transcriptional co-activators YAP (Yes-associated protein)/TAZ (WWTR1),⁶ nuclear retention and activation of forkhead homeobox type O (FOXO) transcription factors,^7–9 and inhibition of Akt-mTOR.^2,10,11

Pulmonary vascular remodeling because of the excessive growth of resident pulmonary vascular cells is an important component of pulmonary arterial hypertension (PAH), a progressive disease with a high mortality rate and poor prognosis,^12–14 characterized by increased pulmonary artery (PA) pressure, elevated right ventricular afterload, and death by heart failure.^14–17 Increased proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMC) and pulmonary arterial adventitial fibroblasts (PAAF) in small PAs from PAH lungs are supported by the constitutive activation of proproliferative/antiapoptotic YAP/TAZ, Akt-mTOR axis (PAVSMC and PAAF), and the deficiency of antiproliferative/proapoptotic FOXO1 (PAVSMC),^14,18–24 for which MST1 and 2 act as positive upstream regulators. Intriguingly, we previously found that MST1 and 2 have little effect on YAP/TAZ in human PAVSMC,¹⁸ and the role of MST1/2 in PAH is currently unknown.

To evaluate the roles of MST1 and 2 in PAH, we utilized tissue specimens and early-passage distal PAVSMC and PAAF from human nondiseased and PAH lungs. Human pulmonary vascular cells retain the diseased phenotype in culture, constituting a unique disease-related model for in vitro mechanistic studies.^{14,18,22,25,26} For in vivo studies, we used mice carrying an inducible smooth muscle-specific Mst1/2 knockout and a SU5416/hypoxia (SuHx) model of pulmonary hypertension (PH), which shares several key features with human PAH including pulmonary vascular remodeling and increased PA pressure.^18,27

Methods

Data Availability

Data available on request from the authors: the data that support the findings of this study are available from the corresponding author upon reasonable request (expanded in the Supplemental Material).

Human Materials

The human lung tissues (Table S1) were provided by the University of Pittsburgh Medical Center Lung Transplant, Pulmonary Division Tissue Donation and the Department of Pathology Autopsy Programs in accordance with Institutional Review Board (IRB) and the Committee for Oversight of Research and Clinical Training Involving Decedents (CORID) policies and by the ethics committee (Ethik Kommission am Fachbereich Humanmedizin der Justus Liebig Universität Giessen) of the University Hospital Giessen (Giessen, Germany) in accordance with national law and with the Good Clinical Practice/International Conference on Harmonisation guidelines. Primary distal PAVSMC and adventitial fibroblasts from nondiseased subjects and patients with PAH were provided by the PH Breakthrough Initiative (PHBI), the University of Pittsburgh Vascular Medicine Institute Cell Processing Core, and by the Biobank platform of the German Center for Lung Research (DZL) under approved protocols. Cells isolation, characterization and maintenance were performed under PHBI protocols as described in.^14,18 All experiments were repeated on primary (3–7 passage) cells from a minimum of 3 subjects. Before experiments, cells were incubated for 24 to 48 hours in basal media with 0.1% BSA if not stated otherwise.

Immunohistochemical, immunocytochemical, immunoblot analyses, transfection, proliferation, apoptosis, and cell count assays were performed as described in study by Goncharov et al,¹⁴ Kudryashova et al,¹⁸ Krymskaya et al,²⁸ and Goncharova et al.²⁹

Animals

All animal procedures were performed under the protocols approved by the University of Pittsburgh Animal Care and Use Committee. The following transgenic founder lines were used in this study: Mst1fl Mst2 fl mice (Mst1/2^fl/fl, Jackson strain: Stk4^tm1.1RjoStk3^tm1.1Rjo/J), smooth muscle (SM)-MHC (myosin heavy chain)-CreER^T2 mice (Jackson strain: Tg(Myh11-cre/ERT2)1Soff/J) and the mTmG reporter mice (Jackson strain: B6,129 (Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}/J). First, male SM-MHC-CreER^T2 mice, which express a tamoxifen-regulated Cre recombinase directed to smooth muscle cells by the Myh 11 (SM-MHC) promoter were crossed with female mTmG reporter mice with Cre-mediated EGFP (enhanced green fluorescent protein) expression to produce male SM-MHC-CreER^T2/R26RmTmG offspring. Then, male SM-MHC-CreERT/R26RmTmG mice were crossed with female Mst1/2^fl/fl mice to produce SM-MHC-CreER^T2-GFP-Mst1/2^f/f mice (Figure S1A and S1B). Treatment of SM-MHC-CreER^T2-GFP-Mst1/2^f/f mice with tamoxifen results in Cre-dependent removal of floxed exons 4-5 of the Mst1 (Stk4) gene and exons 5-6 of the Mst 2 (Stk3) gene in smooth muscle cells and induces EGFP expression^30,31 (Figure S1C). Because the SM-MHC-CreER^T2 cassette is located in the Y chromosome, only male mice (genotype carriers) were used in further experiments. Six- to 8-week-old mice were randomly assigned to 3 groups. Two groups were exposed to hypoxia (10% O₂) and SU5416 (20 mg/kg, sc; Tocris Bioscience, Bristol, United Kingdom). Injections were performed at days 0, 7, and 14.^14,28 At day 17 of the experiment, mice still under hypoxia, were given injections of tamoxifen (20 mg/kg, IP, Sigma, St. Louis, MO; SuHx-Tx group) or vehicle (SuHx-Veh group) for 5 consecutive days. After 2 more weeks of hypoxia exposure, blinded analysis of hemodynamic measurements was performed; animals were sacrificed, and lung tissues were collected for analysis. Negative controls included normoxia-maintained age-matched animals. Hemodynamic and histochemical analyses were performed as described in study by Goncharov et al,¹⁴ Kudryashova et al,¹⁸ and Kelley et al.³² Blinded analysis of small PAs (25–150 μm outer diameter) was performed as described in study by Goncharov et al¹⁴ and Vitali et al.²⁷

Data Analysis

Immunoblots, DNA synthesis and apoptosis assays were analyzed using ImageJ (NIH, Bethesda, MD), StatView (SAS Institute, Cary, NC), STATA (StatCorp, College Station, TX) and GraphPad Prism 9.2 (GraphPad Software, San Diego, CA) software. Immunohistochemical and immunocytochemical analyses were performed using a Keyence BZ-X800 system and software (Keyence Corporation of America, Itasca, IL). Hemodynamic and morphometric data were performed using Indus Instruments (Webster, TX), IOX2 and Emka (Emka Technologies, Falls Church, VA) and Matlab (MathWorks, Natick, MA). Statistical comparisons between the 2 groups were performed by nonparametric Mann-Whitney U test. Statistical comparisons among 3 or more groups were performed using Kruskal-Wallis tests with post hoc Dunn pairwise comparison (all data with sample size n<6/group and skewed data with sample size n≥6/group) and 1-way ANOVA with Dunnett post hoc test for normally distributed data with sample size n≥6/group. Shapiro-Wilk normality test was used.

Results

MST1 and MST2 Support Increased Proliferation and Survival of PAVSMC and PAAF in PAH

MST1 and MST2 act as growth suppressors in adult somatic cells by inhibiting proliferation and inducing cell differentiation or apoptosis. To evaluate the role of MST1 and MST2 in PAH, we first assessed functional roles of MST1 and MST2 in human PAH and nondiseased (control) pulmonary vascular cells using specific siRNAs. In agreement with published studies,^14,18 PAH PAVSMC had significantly higher growth and proliferation rates compared with PAVSMC from nondiseased (control) subjects (Figure S2). In control PAVSMC, siMST1 and siMST2 induced significant proliferation without affecting apoptosis (Figure 1A through 1C). In contrast, both siMST1 and siMST2 significantly reduced proliferation and promoted significant apoptosis in PAH PAVSMC (Figure 1A through 1C). Further, siMST1 and siMST2 significantly reduced proliferation of PAH PAAF, but not control PAAF and induced significant apoptosis in PAH PAAF, but not control cells (Figure 1D through 1F), suggesting a proproliferative/prosurvival role for MST1/2 in PAH pulmonary vascular cells.

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We observed no significant effects of siMST1 on the MST2 protein levels, and no significant effects of siMST2 on the MST1 protein levels in PAH and control PAVSMC and PAAF (Figure S3). To further evaluate the potential interdependence of MST1 and MST2 in supporting increased growth of PAH vascular cells, we co-transfected human PAH PAVSMC and PAAF with siMST1 and siMST2. The combined depletion of MST1 and MST2 reduced PAH PAVSMC growth and proliferation compared with cells transfected with control siRNA but did not exhibit significant additive effects in comparison to separate knockdowns (Figure S4A through S4C). Similarly, combined transfection with siMST1 and si MST2 did not enhance the inhibitory effects of single siMST1 or siMST2 transfection on PAH PAAF proliferation (Figure S4D).

To support our siRNA-based findings, we next inhibited MST1/2 activity using XMU-MP-1, a selective ATP-competitive MST1/2 inhibitor.^33,34 XMU-MP-1 reduced proliferation and induced apoptosis in PAH PAVSMC and PAAF, while having little effect on control cells (Figure 1G and 1H). Intriguingly, in contrast to siMST1 and siMST2 (Figure 1A through 1C), XMU-MP1 did not promote proliferation of control PAVSMC (Figure 1G), suggesting that both kinase activity and preserved protein levels may be required for MST1/2 function. Indeed, XMU-MP1 had little effect on MST1 and MST2 protein levels in control PAVSMC, while reducing MST2 protein levels in PAH PAVSMC (Figure S5), suggesting that the lack of XMU-MP1 effect in control cells may be, at least in part, explained by preserved MST1/2 protein levels. Interestingly, XMU-MP-1 inhibited the proliferation of PAH PA endothelial cells but did not induce apoptosis (data not shown), indicating that antiapoptotic functions of MST1/2 in PAH may be limited to PAVSMC and PAAF cell types. In line with the inhibitor-based findings, transfection of human PAH PAVSMC with kinase-dead K59R MST1 and K56R MST2, but not wild-type (wt) constructs (HA-MST1 and HA-MST2, denoted as wt-MST1 and wt-MST2, respectively), suppressed PAH PAVSMC proliferation compared with empty vector-transfected cells (Figure 1I through 1L), reinforcing our conclusion that active MST1/2 support the proliferation and survival of PAH pulmonary vascular cells.

To determine the mechanism(s) driving the MST1/2 switch to proproliferative molecules in PAH pulmonary vascular cells, we next evaluated expression patterns of MST1 and MST2 in PAH patient lungs by performing a comparative analysis of human lung tissue specimens. Using immunohistochemistry and western blot analyses, we found that MST1 and MST2 are present and overexpressed in isolated PAs from PAH lungs compared with controls; increased accumulation was detected predominantly in the adventitial area (Figure 2A and 2B, Figures S6A and S7). Supporting our observations, while MST1 and MST2 mRNA levels were higher in both PAVSMC and PAAF from PAH lungs, only isolated PAH PAAF (but not PAH PAVSMC) had a significantly higher accumulation of MST1 and MST2 proteins compared to nondiseased cells (Figure 2C through 2E, Figure S6B), demonstrating that MST1 and MST2 are over-accumulated in PAAF in PAH. To test the functional significance of MST1/2 over-accumulation, we transfected control PAAF with mammalian vectors expressing wt or kinase dead MST1 and MST2 and measured proliferation and apoptosis. Importantly, overexpression of wt-MST1 and wt-MST2, but not its kinase-dead mutants, significantly increased proliferation and reduced apoptosis of control PAAF compared with empty vector-transfected cells (Figure 2F and 2G), suggesting that MST1/2 overexpression may be responsible for inducing PAAF proliferation and survival.

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To investigate the mechanism(s) leading to increased MST1/2 expression in PAH PAAF, we next carried out in silico analysis of human MST1 and MST2 promoters to identify putative-binding sites for PAH relevant transcription factors. Promoters of both genes showed the presence of putative-binding sites for the STAT3 (Figure 3A), a well-documented transcription factor acting downstream of several cytokine/chemokine-related^35–37 and growth factor signaling pathways,^38–40 many of which play an important role in PH pathogenesis.^41,42 To dissect the potential upstream regulator of MST1/2, we treated human nondiseased PAAF with various PH-associated cytokines (IL-6, IL-8, IL-13, IL-18, CCL2, TNF-α) and growth factors (PDGF-BB, TGF-β) and evaluated MST1/2 mRNA expression by RT PCR analysis. We found that only IL-6 stimulation led to a significant increase in both MST1 and MST2 mRNA levels compared with controls, IL-8, IL-13, IL-18, CCL2, PDGF-BB, and TGF-β had no effect or affected only MST1 or MST2, and TNF-α reduced MST1 and MST2 mRNA levels compared with vehicle-treated cells (Figure 3B, Figure S8).

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In line with the in silico and RT PCR analyses, stimulation with 20 ng/mL IL-6 significantly increased MST1 and MST2 protein levels in control PAAF (Figure 3C). Further, STAT3 inhibition using a small molecule inhibitor led to a strong decrease in expression of MST1/2 in PAH PAAF while STAT1 inhibition had no significant effect (Figure 3D and 3E). Collectively, our data indicate that IL-6-driven Stat3 signaling might be involved in MST1/2 upregulation in PAH PAAF. Interestingly, IL-6 had no effect on MST1 and MST2 expression in PAVSMC (Figure S8C), suggesting that this mechanism is specific for PAAF.

Taken together, these data demonstrate that, in contrast to canonical growth-suppressor function in nondiseased pulmonary vasculature, MST1 and MST2 play a proproliferative, antiapoptotic role in mesenchymal pulmonary vascular cells from PAH lungs, and that functional switch may, at least in part, be induced by IL-6/STAT3-governed overexpression.

MST1 and MST2 Regulate the Akt-mTOR and FOXO Axes in Pulmonary Vascular Cells in PAH

Canonically, MST1/2 act via inhibition of YAP/TAZ, whereas noncanonical MST1/2 act via suppression of Akt and mTORC1, or nuclear retention of FOXOs in a YAP/TAZ-independent manner.^{1–5,8,9,11,43–45} In agreement with published studies,^{14,18,19,22,26} we observed that increased proliferation of PAVSMC from patients with PAH in culture is associated with constitutive hyper-phosphorylation of Akt and ribosomal protein S6 (the molecular signature of mTORC1 activation)^14,46 and reduced FOXO1 protein content compared with nondiseased controls (Figure S2C and S2D).

MST1 and 2 in human control PAVSMC appeared to signal YAP/TAZ-independently¹⁸ and had unaltered phosphorylation of MST1/2 at T183/180 compared with controls (Figure S6B). Supporting our previous observations, transfection of control cells with siMST1 and siMST2 reduced the levels of proapoptotic protein Bim and increased S473-Akt and S6 phosphorylation rates without a marked effect on FOXO1 protein levels (Figure 4A and 4B), which is in line with the previously reported growth suppressor action of MST1/2 via inhibition of Akt-mTOR.^10,18 In contrast, siRNA-induced MST1 or MST2 depletion in PAH PAVSMC resulted in accumulation of proapoptotic Bim and FOXO1 and reduced phosphorylation rates of Akt and S6 (Figure 4A and 4B). In agreement with the siRNA-based findings, transfection with kinase-dead K59R MST1 and K56R MST2 suppressed Akt and S6 phosphorylation in PAH PAVSMC (Figure S9). Further supporting our observations, treatment of PAH PAVSMC with MST1/2 kinase inhibitor XMU-MP-1 significantly decreased Akt and S6 phosphorylation rates and increased Bim protein content (Figure S10).

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Interestingly, MST1 and MST2 knockdown in PAH PAAF led to an increase in protein expression of Bim and FOXO3 with no significant changes in phosphorylation of S6 and Akt (Figure 4C and 4D), suggesting potentially different mechanisms of MST1/2 action in PAVSMC and PAAF from PAH lungs.

Taken together, these data show that MST1 and 2 act as proproliferative/prosurvival proteins in human PAH pulmonary vascular cells and promote proproliferative/prosurvival Akt and mTORC1 (PAVSMC) while suppressing Bim and FOXO (PAVSMC and PAAF).

Smooth Muscle Mst1/2 Support Pulmonary Vascular Remodeling and PH in Mice

As increased proliferation and survival of pulmonary vascular cells play an important role in PAH pathogenesis, we next tested whether MST1/2 support PA remodeling and PH in vivo. To this end, we developed mice carrying a smooth muscle (SM)-specific tamoxifen-inducible Mst1/2 knockdown cassette, which simultaneously induces cre-mediated Mst1/2 knockdown and GFP expression in smooth muscle cells upon tamoxifen treatment (SM-MHC-CreER^T2GFP-Mst1/2^fl/fl) (Figure S1), and induced PH by combined exposure to SU5416 and hypoxia.^18,27 At days 17 to 21 of the experiment, mice received 5 sequential injections of tamoxifen to deplete Mst1/2 (SuHx Tx group) or vehicle (SuHx Veh group) and were maintained under hypoxia for 2 more weeks. Controls were same-sex littermates maintained under normoxia (Figure 5A). As expected, animals of the SuHx Veh group developed pulmonary vascular remodeling and PH as evidenced by a significant increase in PA medial thickness and elevated systolic right ventricular pressure and contractility (max dP/dT), but not systolic left ventricular pressure compared with controls (Figure 5B through 5F, Figure S11). This was associated with increased phosphorylation of Akt and S6 in SMA (smooth muscle α-actin)-positive cells in small (25–150 µm) PAs (Figure 5G and 5H). In contrast to vehicle-treated SuHx mice and controls, tamoxifen-treated SuHx mice showed GFP expression in SMA-positive areas of small PAs (Figure S1C), confirming successful tamoxifen delivery to the pulmonary vasculature. In agreement with our in vitro findings, tamoxifen-induced SM-specific Mst1/2 depletion (SuHx Tx group) reversed pulmonary vascular remodeling (PA medial thickness) (Figure 5B and 5C), decreased Akt and S6 phosphorylation (Figure 5G and 5H), and attenuated overall PH (systolic right ventricular pressure and max dP/dT) when compared with the vehicle-treated (SuHx Veh) group without affecting systolic left ventricular pressure (Figure 5D through 5F, Figure S11). These data show that smooth muscle Mst1/2 support pulmonary vascular remodeling and experimental PH in vivo.

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MST1/2 in PAH PAVSMC Form Disease-Specific Interaction With the Cell Cycle Protein BUB3

Because PAVSMC from PAH lungs did not demonstrate MST1 or 2 accumulation or changes in T180/183 phosphorylation status (Figure S6), we hypothesized that the observed switch in MST1/2 function is caused by PAH-specific protein-protein interactions. To test this hypothesis, we immuno-precipitated MST1 and MST2 from control and PAH PAVSMC and performed mass spectrometry analysis (Figure 6A). It revealed that MST1 and MST2 form different interactomes in PAH and nondiseased PAVSMC and identified 13 proteins that interact with MST1 exclusively in PAH, but not control PAVSMC (Figure 6B, left). Out of these 13 proteins, only BUB3 and 60S ribosomal protein L22-like 1 (RPL22L1) also interacted with MST2 exclusively in PAH cells (Figure 6B, right). To test functional selectivity of BUB3 and RPL22L1 toward PAH, we depleted those proteins with specific siRNAs and performed a cell growth assay. We found that the depletion of BUB3 reduced growth of PAH, but not control PAVSMC, while the depletion of RPL22L1 decreased growth of both, control and PAH cells (Figure 6C), showing that only BUB3 acts in a PAH-specific manner. Interestingly, we did not observe any significant interaction between BUB3 and MST1/2 in co-IP studies in PAH PAAF (data not shown), suggesting cell-type-specific mechanisms for MST1/2 action.

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MST1/2 Promote BUB3 Accumulation in PAH PAVSMC via ECM and USP10

BUB3 is a cell cycle protein that promotes mitotic entry⁴⁷ and acts as a member of the mitotic checkpoint complex, a component of the spindle assembly checkpoint.⁴⁸ The mechanism(s) promoting BUB3 accumulation in PAH is unknown. Since MST1 is involved in both, regulating accurate kinetochore-microtubule attachment⁴⁹ and mechanosensing, we hypothesized that MST1/2 support pro-proliferative signaling by promoting BUB3 accumulation via modulating the ECM (extracellular matrix). To test this hypothesis, we first treated human PAH PAVSMC with MST1/2 inhibitor XMU-MP-1 and observed a significant ≈2-fold decrease in BUB3 protein levels (Figure 6D). Next, we plated control PAVSMC on the decellularized matrices produced by nondiseased (control) or PAH PAVSMC (Figure 6E) and found that control cells, grown on PAH PAVSMC-produced matrices, had significantly higher BUB3 protein levels (Figure 6G). This was accompanied by elevated cell growth compared with cells grown on the matrices produced by control PAVSMC (Figure 6F). PAH PAVSMC treated with XMU-MP-1 produced reduced amounts of 2 major PAH-specific ECM proteins, FN (fibronectin) and collagen 1 (Col1A) (data not shown), indicating the importance of MST1/2 in ECM production. Importantly, control PAVSMC, plated on the matrices produced by XMU-MP-1-treated PAH PAVSMC, showed reduced cell growth (Figure 6H and 6I) and decreased protein levels of BUB3 (Figure 6J) compared with the cells grown on the matrices produced by vehicle-treated PAH PAVSMC. Further supporting the relevance of our findings to human PAH, we found that BUB3 protein levels are significantly higher in SMA-positive areas of small remodeled PAs in human PAH lungs and isolated early-passage human PAH PAVSMC as compared with nondiseased controls (Figure 7A and 7B). Together, these data demonstrate that MST1/2 control the accumulation of BUB3 in PAH PAVSMC by modifying ECM.

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In addition to the ECM, proliferation of PAVSMC in PAH could be induced by various soluble pro-PAH mitogens, including proinflammatory cytokines and growth factors.^18,22,35 To test whether soluble pro-PAH factors could also induce PAH-specific MST1/2-dependent signaling in human PAVSMC, we treated nondiseased (control) PAVSMC with IL-6, IL-8, IL-13, IL-18, CCL2, TNF-α, PDGF-BB, or TGF-β and examined BUB3 protein levels and Akt phosphorylation status using immunoblot analysis. In agreement with published studies,^50–52 IL-13, PDGF, TGF-β, and TNF-α increased pAkt/Akt ratio in control PAVSMC. However, none of the analyzed agonists induced accumulation of BUB3 similar to what was seen in PAH PAVSMC (Figure S12), suggesting that MST1/2-induced BUB3 accumulation in PAH PAVSMC is likely growth factors- and cytokine-independent.

Beyond BUB3, our screen of the MST1/2 PAH-specific interactome (Figure 6B) produced the deubiquitinating enzyme (DUB) ubiquitin-specific peptidase 10 (USP10) as another interactor of MST1 and MST2 enriched in PAH. Interestingly, we observed increased protein levels of USP10 in PAH PAVSMC and small PAs from PAH lungs compared with controls (Figure 7B, Figure S13), and siRNA-mediated knockdown of USP10 in PAH PAVSMC resulted in decreased BUB3 expression that was induced by MST1 and, to a lesser extent, MST2 overexpression (Figure 7C through 7H). These findings may be interpreted as BUB3 interacting with USP10 in the presence of MST1/2, which, in turn, increases the BUB3 protein level by interfering with its degradation.

BUB3 Drives PAH PAVSMC Proliferation, Survival, and Activation of Akt-mTORC1

BUB3 knockdown is known to abrogate spindle assembly checkpoint, promote apoptosis, and inhibit proliferation of tumor cells.⁵³ We found that siRNA-induced BUB3 depletion significantly reduced proliferation and induced apoptosis in PAH, but not control PAVSMC (Figure 7I), showing that BUB3 is required for increased proliferation and survival of human PAH PAVSMC.

BUB3 is a member of the Akt1 interactome.⁵⁴ Akt1 is a proproliferative/prosurvival protein-kinase that positively regulates mTORC1.^21,55–57 Because Akt and mTORC1 are upregulated in PAH PAVSMC in an MST1/2-dependent manner (Figure 4A and 4B, Figures S9 and S10) and are required for PAVSMC remodeling,^{14,19,58–61} we reasoned that BUB3 may regulate PAH PAVSMC proliferation and survival via Akt-mTORC1. Transfection of PAH PAVSMC with siRNA BUB3 significantly decreased Akt phosphorylation and mTORC1-specific phosphorylation of S6 (Figure 7J), suggesting that BUB3 is required for the activation of the Akt-mTOR axis in PAH PAVSMC. To test whether MST1/2 act via BUB3, we transfected PAH PAVSMC with a vector expressing human BUB3 (pCMV6-BUB3) or an empty pCMV6 plasmid and examined cell proliferation and apoptosis in the presence or absence of the MST1/2 kinase inhibitor XMU-MP-1. We found that overexpression of BUB3 moderately attenuates XMU-MP-1-dependent inhibition of proliferation and protects from XMU-MP-1-induced apoptosis (Figure 7K). Together, these data suggest that MST1/2 promote BUB3 overaccumulation in PAH PAVSMC in an USP10-dependent manner, leading to BUB3-dependent maintenance of increased proliferation, survival, and activation of Akt-mTORC1 in PAH PAVSMC (Figure 7L). Interestingly, BUB3 silencing did not affect FOXO1 protein levels (data not shown), suggesting that the regulation of FOXOs by MST1/2 may be BUB3-independent.

MST1/2 Promote PAAF Proliferation and Survival via PSMC6-Dependent Inhibition of FOXO3

Based on our observation of MST1/2 knockdown-mediated upregulation of FOXO3 in PAH PAAF (Figure 4C and 4D), we hypothesized that increased MST1/2 downregulate FOXO3 in PAH PAAF resulting in hyperproliferation and increased survival. In concurrence, HEK 293 cells transfected with MST1/2 wt plasmids showed an increased nuclear exclusion of FOXO3 compared with an empty vector, as well as their respective kinase dead mutants (Figure 8A, Figure S14A) as measured by nuclear-cytoplasmic fractionation followed by Western blotting. Further, a luciferase assay employing FOXO3 dependent luciferase reporter plasmid, containing 3 copies of FOXO response elements, showed a significant decrease in luciferase activity under MST1/2 wt plasmids; the effect was attenuated under overexpression of kinase dead mutants (Figure 8B). Conversely, knockdown of both MST1 and MST2 resulted in an increased nuclear inclusion of FOXO3 with a concomitant increase in FOXO response element luciferase activity (Figure 8C and 8D, Figure S14B). To analyze how much the pro-proliferative and pro-survival effect of MST1/2 is mediated by selectively inhibiting FOXO3, we carried out a dual knockdown of MST1/2 and FOXO3. We observed that the FOXO3 knockdown reversed the decrease in proliferation seen in PAH PAAF treated with siMST1 and siMST2 (Figure 8E). Additionally, the FOXO3 knockdown was able to substantially reverse the apoptotic effect of MST1/2 knockdown (Figure 8F). These findings signify that the majority of antiproliferative and proapoptotic effects of MST1/2 is mediated via FOXO3. In further support, we observed a decreased expression of FOXO3 in PAH PAAF compared with control cells (Figure S15A), and the FOXO3 knockdown itself increased proliferation and survival of control PAAF (Figure S15B and S15C).

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To investigate the mechanism of FOXO3 regulation by MST1/2, we immunoprecipitated FOXO3-FLAG from MST1/2 wt and kinase dead mutants overexpressing HEK cells and performed mass spectrometric analysis for post translational modifications and interacting partners (Figure S15D). We did not detect significant changes in post translational modifications; however, we obtained differential interacting partners of FOXO3 in the presence of wt MST1/2 or their kinase dead mutants. PSMC6, an essential component of the assembled 19S proteasome subunit, was revealed as the top interacting partner for FOXO3 in the presence of MST1 and among the top 10 in presence of MST2, while it did not show any interaction with FOXO3 in the presence of kinase dead mutants (Figure S15E). We performed co-immunoprecipitation studies with a FOXO3 pulldown in the presence of a MST1/2 knockdown to confirm these findings. We were indeed able to observe an interaction between FOXO3 and PSMC6, which was abrogated on the MST1/2 knockdown (Figure 8G). Further, a siRNA-mediated knockdown of PSMC6 resulted in restoring FOXO3 expression reduced by MST1 overexpression (Figure 8H and 8I). PSMC6 belongs to the AAA (ATPases associated with diverse cellular activities) proteins, which unfold ubiquitinated target proteins for translocation into the proteolytic chamber of the proteasome leading to degradation.⁶² Collectively, our findings strongly suggest that FOXO3 interacts with the proteasome complex in the presence of MST1/2, leading to its degradation, and ultimately contributing to the hyperproliferative and prosurvival phenotype of PAH PAAF (Figure 8J).

Discussion

We here report that MST1 and MST2 act as proproliferative prosurvival proteins in PAH. Our novel findings (Figure S16) are: (1) MST1 and 2 are upregulated in human PAH PAAF; (2) inflammatory cytokine IL-6, involved in the pathogenesis of PAH, promotes MST1 and MST2 expression in PAAF; (3) in contrast to the well-described canonical growth-suppressor function, MST1 and 2 act as proproliferative/prosurvival molecules in human PAH PAVSMC and PAAF; (4) a distinct MST1 and 2 PAH-specific interactomes direct proproliferative/prosurvival signaling in human PAH PAVSMC and PAAF via BUB3 and FOXO3, respectively; and (5) genetic ablation of Mst1/2 in mice suppresses pulmonary vascular remodeling and attenuates SuHx-induced PH in vivo.

MST1 and 2 are core kinases of the HIPPO growth-suppressor cassette. We observed that, under normal conditions, MST1 and MST2 inhibit proliferation of human microvascular PAVSMC. This is in agreement with previous reports of the growth-suppressor function of MST1/2 in the heart, lungs, and cancers.^1,6,63,64 However, in contrast to healthy PAVSMC and PAAF, MST1 and 2 act as proproliferative/prosurvival molecules in diseased (ie, PAH) human pulmonary vascular cells. Both gain and loss of function studies indicated proproliferative and prosurvival effects of MST1 and 2 in PAH PAVSMC and PAAF. Corroborating our in vitro data, the smooth muscle-specific Mst1/2 double knockout in mice attenuated SuHx-induced vascular remodeling and PH, indicating pro-remodeling and pro-PH roles for Mst1/2 in vivo.

This noncanonical, proproliferative role of MST1 and 2 could be explained mechanistically by formation of context-specific MST1- and 2-dependent interactomes. Specifically, we identified that, first, MST1/2 form a PAH-specific interaction with cell cycle protein BUB3 and USP10, promoting the accumulation of BUB3 and consequent upregulation of proproliferative prosurvival Akt and mTORC1 in PAH PAVSMC. Second, we found that MST1/2-driven PAH-specific FOXO3-PSMC6 interaction leads to proteasomal degradation of FOXO3, permitting activation of proproliferative and prosurvival genes in PAH PAAF. Last, distinct pro-PAH stimuli (ie, ECM in PAVSMCs and IL-6 in PAAFs) led to the formation of differential protein-protein interactions (MST-BUB3-USP10 in PAH PAVSMCs and MST-FOXO3-PSMC6 in PAH PAAFs). These lines of evidence indicate that PAH-specific MST1/2 interactomes determine the predominance of proproliferative over the growth-suppressive responses of MST kinases in PAH pulmonary vascular cells; and that distinct PAH-specific stimuli support interaction of MST with different proteins, leading to cell type-specific pro-proliferative signaling in PAH PAVSMCs versus PAH PAAFs.

One of the interesting findings of this study is the interaction between MST and the mitotic checkpoint protein BUB3, and the MST-mediated upregulation of BUB3 exclusively in the PAH setting. Of note, a marked upregulation of BUB3 in PAH human lung tissues and ex vivo cultured PAVSMC was also observed. BUB3 regulates mitotic exit via quality control of microtubular attachment and chromosome segregation.^65–67 In line with our data, increased protein levels of BUB3 in several human cancers are associated with poor prognosis,^68,69 suggesting a potential role of BUB3 in hyper-proliferation. Importantly, BUB3 knockdown in PAH PAVSMC potently reversed the hyperproliferative and apoptosis-resistant phenotype by modulating Akt and mTORC1 signaling pathways, suggesting that BUB3 functions as a novel downstream mediator of MST kinases in regulating pulmonary vascular cell proliferation.

MST1/2 support increased BUB3 protein levels in PAH PAVSMC via ECM, suggesting that MST kinases cooperate with ECM proteins to increase the protein stability of BUB3 and subsequently the mitotic checkpoint signaling. In this study, we also identified USP10 as a novel deubiquitinase that can stabilize BUB3 and block its degradation in PAH. In addition, upregulation of USP10 protein content, concomitant with increased BUB3 in PAH PAVSMC, suggests that a PAH-specific MST1/2 interactome leads to an accumulation of BUB3. Collectively, our study provides new insight into the regulatory mechanism of BUB3 and mitotic checkpoint signaling in PAH and uncovers the MST1/2-USP10-BUB3 axis as a major regulator of pro-proliferative/pro-survival functions of PAH PAVSMC.

Another interesting finding of this study is a distinct mode of the regulation of FOXO transcription factors by the MST family of kinases. FOXO transcription factors are critical integrators of multiple signaling pathways in pulmonary vascular cells and serve as central downstream effectors in driving PAH and fibrogenesis.^22,70 We identified pro-PAH IL-6/STAT3 signaling as an activator of MST1 and MST2 which, in turn, promote the interaction of FOXO3 with the PSMC6 proteasome complex. The MST1- and MST2-induced interaction of PSMC6 with FOXO3 inhibited the translocation of FOXO3 to the nucleus and promoted FOXO3 degradation, thereby inducing PAAF proliferation and survival. These findings were further supported by the decreased expression of FOXO3 in PAH PAAF. To our knowledge, this is the first study that demonstrates a negative regulation of FOXO proteins by MST kinases, which is in contrast to previous research that suggested a positive regulation of FOXO proteins by MST kinases. Lehtinen et al. demonstrated that under conditions of oxidative stress MST1 is activated and, in turn, phosphorylates FOXO3 at serine 207, disrupting FOXO3 interaction with 14-3-3 protein, promoting FOXO3 translocation to the nucleus, and thereby inducing neuronal cell death.^8,71 However, in our experiments, MST failed to phosphorylate the forkhead domain of FOXO proteins (data not shown), suggesting that MST kinases-mediated FOXO regulation is cellular- and pathophysiological context-specific. Taken together, our findings demonstrate an intimate signaling link between MST kinases and FOXO transcription factors that regulate hyper-proliferation.

Notably, we found that kinase-dead mutants as well as a pharmacological inhibitor of MST1 and 2 abrogated PAH-specific MST signaling and subsequent proproliferative/prosurvival effects. However, T183 and T180 autophosphorylation sites, required for canonical MST1/2 signaling (ie, activation of LATS and inactivation of YAP/TAZ), are unaltered in PAH cells compared with controls. Apart from autophosphorylation, MST1/2 can be activated via trans-phosphorylation by protein kinases (ie, AKT and c-Abl) and by protein-protein interactions. For example, MOB3A/B/C adaptor proteins were found to associate with MST1 and negatively regulate MST1-mediated apoptosis, supporting tumorigenesis in glioblastoma multiform and pancreatic cancer.^60,72 Furthermore, interaction with BUB3 (as identified in this study) as well as interaction with mTORC¹⁴ and SARAH domain-binding proteins such as RASSF1⁷³ may regulate the MST kinase activity.

In summary, the present study provides strong evidence that MST1/2 kinases are activated in the medial and adventitial layers of small PAs, form PH-specific protein-protein interactions with BUB3 and FOXO, and function as proproliferative and prosurvival proteins centrally involved in the pathogenesis of PAH. Thus, inhibition of MST1 and 2 kinase activity provides a potential new therapeutic option to reverse the vascular remodeling and overall PH.

Article Information

Acknowledgments

We thank the Biostatistics, Epidemiology, and Research Design program at the Clinical and Translational Science Center, University of California, Davis for their help with statistical analysis.

Author Contributions

E.A. Goncharova and S.S. Pullamsetti participated in conception and design. T.V. Kudryashova, S. Dabral, S. Nayakanti, A. Ray, D.A. Goncharov, T. Avolio, Y. Shen, A. Pena, A. Pena, L. Jiang, D. Lin, J. Baust, T.N. Bachman, J. Graumann, C. Ruppert, A. Guenther, M. Schmoranzer, Y. Grobs, S. Eve Lemay, E. Tremblay, S. Breuils-Bonnet, O. Boucherat, A.L. Mora, H. DeLisser, J. Zhao, Y. Zhao, S. Bonnet, W. Seeger, S.S. Pullamsetti, and E.A. Goncharova performed experimental work, analysis, and interpretation. E.A. Goncharova, S.S. Pullamsetti, T.V. Kudryashova, and S. Dabral performed drafting the article and intellectual content.

Supplemental Material

Expanded methods

Table S1

Figures S1–S17

Footnotes