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Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2- Sox17 ( Sox17 EC-/- ) deletion and attenuated by transgenic Tie2- Sox17 overexpression ( Sox17 Tg ). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17 EC-/- and reduced in those from Sox17 Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17 Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations ( n  = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

With severe right ventricular (RV) pressure overload, women demonstrate better clinical outcomes compared with men. The mechanoenergetic mechanisms underlying this protective effect, and their dependence on female endogenous sex hormones, remain unknown. To investigate these mechanisms and their impact on RV systolic and diastolic functional adaptation, we created comparable pressure overload via pulmonary artery banding (PAB) in intact male and female Wistar rats and ovariectomized (OVX) female rats. At 8 wk after surgery, right heart catheterization demonstrated increased RV energy input [indexed pressure-volume area (iPVA)] in all PAB groups, with the greatest increase in intact females. PAB also increased RV energy output [indexed stroke or external work (iEW)] in all groups, again with the greatest increase in intact females. In contrast, PAB only increased RV contractility-indexed end-systolic elastance (i E es )] in females. Despite these sex-dependent differences, no statistically significant effects were observed in the ratio of RV energy output to input (mechanical efficiency) or in mechanoenergetic cost to pump blood with pressure overload. These metrics were similarly unaffected by loss of endogenous sex hormones in females. Also, despite sex-dependent differences in collagen content and organization with pressure overload, decreases in RV compliance and relaxation time constant (tau Weiss) were not determined to be sex dependent. Overall, despite sex-dependent differences in RV contractile and fibrotic responses, RV mechanoenergetics for this degree and duration of pressure overload are comparable between sexes and suggest a homeostatic target. NEW & NOTEWORTHY Sex differences in right ventricular mechanical efficiency and energetic adaptation to increased right ventricular afterload were measured. Despite sex-dependent differences in contractile and fibrotic responses, right ventricular mechanoenergetic adaptation was comparable between the sexes, suggesting a homeostatic target.

Each year the American Thoracic Society (ATS) Conference brings together scientists who conduct basic, translational and clinical research to present on the recent advances in the field of respirology. Due to the Coronavirus Disease of 2019 (COVID-19) pandemic, the ATS2020 Conference was held online in a series of virtual meetings. In this review, we focus on the breakthroughs in pulmonary hypertension research. We have selected 11 of the best basic science abstracts which were presented at the ATS2020 Assembly on Pulmonary Circulation mini-symposium "What's New in Pulmonary Arterial Hypertension (PAH) and Right Ventricular (RV) Signaling: Lessons from the Best Abstracts," reflecting the current state of the art and associated challenges in PH. Particular emphasis is placed on understanding the mechanisms underlying RV failure, the regulation of inflammation, and the novel therapeutic targets that emerged from preclinical research. The pathologic interactions between pulmonary hypertension, right ventricular function and COVID-19 are also discussed., (© The Author(s) 2021.)

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17β-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-β-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH. NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 ( Klk10 ), and Jun Proto-Oncogene ( Jun ) were identified as potential mediators in ERα-regulated pathways in RV pressure overload.

Left heart failure (LHF) is the most common cause of pulmonary hypertension, which confers an increase in morbidity and mortality in this context. Pulmonary vascular resistance has prognostic value in LHF, but otherwise the mechanical consequences of LHF for the pulmonary vasculature and right ventricle (RV) remain unknown. We sought to investigate mechanical mechanisms of pulmonary vascular and RV dysfunction in a rodent model of LHF to address the knowledge gaps in understanding disease pathophysiology. LHF was created using a left anterior descending artery ligation to cause myocardial infarction (MI) in mice. Sham animals underwent thoracotomy alone. Echocardiography demonstrated increased left ventricle (LV) volumes and decreased ejection fraction at 4 wk post-MI that did not normalize by 12 wk post-MI. Elevation of LV diastolic pressure and RV systolic pressure at 12 wk post-MI demonstrated pulmonary hypertension (PH) due to LHF. There was increased pulmonary arterial elastance and pulmonary vascular resistance associated with perivascular fibrosis without other remodeling. There was also RV contractile dysfunction with a 35% decrease in RV end-systolic elastance and 66% decrease in ventricular-vascular coupling. In this model of PH due to LHF with reduced ejection fraction, pulmonary fibrosis contributes to increased RV afterload, and loss of RV contractility contributes to RV dysfunction. These are key pathologic features of human PH secondary to LHF. In the future, novel therapeutic strategies aimed at preventing pulmonary vascular mechanical changes and RV dysfunction in the context of LHF can be tested using this model. NEW & NOTEWORTHY In this study, we investigate the mechanical consequences of left heart failure with reduced ejection fraction for the pulmonary vasculature and right ventricle. Using comprehensive functional analyses of the cardiopulmonary system in vivo and ex vivo, we demonstrate that pulmonary fibrosis contributes to increased RV afterload and loss of RV contractility contributes to RV dysfunction. Thus this model recapitulates key pathologic features of human pulmonary hypertension-left heart failure and offers a robust platform for future investigations.

17β-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERβ expression. Sprague-Dawley rats and ERα or ERβ knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERβ, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERβ mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERβ, whereas HIF-1α knockdown decreased ERβ abundance in hypoxic PAECs. In turn, ERβ knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERβ knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERβ activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERβ regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERβ knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERβ expression and the E2-ERβ axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development.

Right ventricular (RV) function is the primary prognostic factor for both morbidity and mortality in pulmonary hypertension (PH). RV hypertrophy is initially an adaptive physiological response to increased overload; however, with persistent and/or progressive afterload increase, this response frequently transitions to more pathological maladaptive remodeling. The mechanisms and disease processes underlying this transition are mostly unknown. Angiogenesis has recently emerged as a major modifier of RV adaptation in the setting of pressure overload. A novel paradigm has emerged that suggests that angiogenesis and angiogenic signaling are required for RV adaptation to afterload increases and that impaired and/or insufficient angiogenesis is a major driver of RV decompensation. Here, we summarize our current understanding of the concepts of maladaptive and adaptive RV remodeling, discuss the current literature on angiogenesis in the adapted and failing RV, and identify potential therapeutic approaches targeting angiogenesis in RV failure.

Although extensively used for the study of left ventricular function, limited experience exists with the isolated heart model in the evaluation of right ventricular (RV) function. In particular, no published experience exists with this tool in sugen/hypoxia-induced pulmonary hypertension (SuHx-PH), a frequently used model of severe and progressive PH We sought to characterize markers of RV contractile and diastolic function in SuHx-PH and to establish their relationship with markers of maladaptive RV remodeling. Hearts were excised from anesthetized Sprague Dawley rats with or without SuHx-PH and perfused via the aorta using a Langendorff preparation. We explored the Frank-Starling relationship of RV function (RV developed pressure, d P /d t max , and d P /d t min ; all normalized to RV mass) by increasing RV end-diastolic pressure (RVEDP) from 0 to 40 mmHg. Functional studies were complemented by quantification of RV pro-apoptotic signaling (bcl2/bax), procontractile signaling (apelin), and stress response signaling (p38MAPK activation). Pearson's correlation analysis was performed for functional and biochemical parameters. SuHx-RVs exhibited severe RV dysfunction with marked hypertrophy and decreased echocardiographic cardiac output. For any given RVEDP, SuHx-RVs demonstrated less developed pressure and lower d P /d t max , as well as less pronounced d P /d t min , suggestive of decreased contractile and diastolic function. SuHx-RVs exhibited decreased bcl2/bax ratios, apelin expression, and p38MAPK activation. Bcl2/bax and apelin RNA abundance correlated positively with RV developed pressure and d P /d t max and negatively with d P /d t min p38MAPK activation correlated positively with RV developed pressure. We conclude that SuHx-RVs exhibit severe contractile and diastolic dysfunction. Increased pro-apoptotic signaling and attenuated procontractile and stress response signaling may contribute to these functional alterations., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

17β-estradiol (E2) exerts complex and context-dependent effects in pulmonary hypertension. In hypoxia-induced pulmonary hypertension (HPH), E2 attenuates lung vascular remodeling through estrogen receptor (ER)-dependent effects; however, ER target genes in the hypoxic lung remain unknown. In order to identify the genome regulated by the E2-ER axis in the hypoxic lung, we performed a microarray analysis in lungs from HPH rats treated with E2 (75 mcg/kg/day) ± ER-antagonist ICI182,780 (3 mg/kg/day). Untreated HPH rats and normoxic rats served as controls. Using a false discovery rate of 10%, we identified a significantly differentially regulated genome in E2-treated versus untreated hypoxia rats. Genes most upregulated by E2 encoded matrix metalloproteinase 8, S100 calcium binding protein A8, and IgA Fc receptor; genes most downregulated by E2 encoded olfactory receptor 63, secreted frizzled-related protein 2, and thrombospondin 2. Several genes affected by E2 changed in the opposite direction after ICI182,780 co-treatment, indicating an ER-regulated genome in HPH lungs. The bone morphogenetic protein antagonist Grem1 (gremlin 1) was upregulated by hypoxia, but found to be among the most downregulated genes after E2 treatment. Gremlin 1 protein was reduced in E2-treated versus untreated hypoxic animals, and ER-blockade abolished the inhibitory effect of E2 on Grem1 mRNA and protein. In conclusion, E2 ER-dependently regulates several genes involved in proliferative and inflammatory processes during hypoxia. Gremlin 1 is a novel target of the E2-ER axis in HPH. Understanding the mechanisms of E2 gene regulation in HPH may allow for selectively harnessing beneficial transcriptional activities of E2 for therapeutic purposes.

More than 350 mutations in the type-2 BMP (bone morphogenetic protein) receptor, BMPR2 , have been identified in patients with heritable pulmonary arterial hypertension (HPAH). However, only 30% of BMPR2 mutation carriers develop PAH, and we cannot predict which of these carriers will develop clinical disease. One possibility is that the nature of the BMPR2 mutation affects disease severity. This hypothesis has been difficult to test clinically, given the rarity of HPAH and the complexity of the confounding genetic and environmental risk factors. To test this hypothesis, therefore, we evaluated the susceptibility to experimental pulmonary hypertension (PH) of mice carrying different HPAH-associated Bmpr2 mutations on otherwise identical genetic backgrounds. Mice with Bmpr2 ΔEx4-5 mutations ( Bmpr2 +/- ), in which the mutant protein is not expressed, develop less severe PH in response to hypoxia or hypoxia with vascular endothelial growth factor receptor inhibition than mice with an extracellular-domain Bmpr2 ΔEx2 mutation ( Bmpr2 ΔEx2/+ ), in which the mutant protein is expressed. This was associated with a marked decrease in stabilizing phosphorylation of threonine 495 endothelial nitric oxide synthase (pThr495 eNOS) in Bmpr2 ΔEx2/+ compared to wild-type and Bmpr2 +/- mouse lungs. These findings provide the first experimental evidence that BMPR2 mutation types influence the severity of HPAH and suggest that patients with BMPR2 mutations who express mutant BMPR2 proteins by escaping non-sense-mediated messenger RNA decay (NMD- mutations) will develop more severe disease than HPAH patients with NMD+ mutations who do not express BMPR2 mutant proteins. Since decreased levels of pThr495 eNOS are associated with increased eNOS uncoupling, our data also suggest that this effect may result from defects in eNOS function.

17β-Estradiol (E2) exerts protective effects on right ventricular (RV) function in pulmonary arterial hypertension (PAH). Since acute exercise-induced increases in afterload may lead to RV dysfunction in PAH, we sought to determine whether E2 allows for superior RV adaptation after an acute exercise challenge. We studied echocardiographic, hemodynamic, structural, and biochemical markers of RV function in male and female rats with sugen/hypoxia (SuHx)-induced pulmonary hypertension, as well as in ovariectomized (OVX) SuHx females, with or without concomitant E2 repletion (75 μg·kg(-1)·day(-1)) immediately after 45 min of treadmill running at 75% of individually determined maximal aerobic capacity (75% aerobic capacity reserve). Compared with males, intact female rats exhibited higher stroke volume and cardiac indexes, a strong trend for better RV compliance, and less pronounced increases in indexed total pulmonary resistance. OVX abrogated favorable RV adaptations, whereas E2 repletion after OVX markedly improved RV function. E2's effects on pulmonary vascular remodeling were complex and less robust than its RV effects. Postexercise hemodynamics in females with endogenous or exogenous E2 were similar to hemodynamics in nonexercised controls, whereas OVX rats exhibited more severely altered postexercise hemodynamics. E2 mediated inhibitory effects on RV fibrosis and attenuated increases in RV collagen I/III ratio. Proapoptotic signaling, endothelial nitric oxide synthase phosphorylation, and autophagic flux markers were affected by E2 depletion and/or repletion. Markers of impaired autophagic flux correlated with endpoints of RV structure and function. Endogenous and exogenous E2 exerts protective effects on RV function measured immediately after an acute exercise challenge. Harnessing E2's mechanisms may lead to novel RV-directed therapies.

Estrogens are disease modifiers in PAH. Even though female patients exhibit better right ventricular (RV) function than men, estrogen effects on RV function (a major determinant of survival in PAH) are incompletely characterized. We sought to determine whether sex differences exist in RV function in the SuHx model of PAH, whether hormone depletion in females worsens RV function, and whether E2 repletion improves RV adaptation. Furthermore, we studied the contribution of ERs in mediating E2's RV effects. SuHx-induced pulmonary hypertension (SuHx-PH) was induced in male and female Sprague-Dawley rats as well as OVX females with or without concomitant E2 repletion (75 μg·kg(-1)·day(-1)). Female SuHx rats exhibited superior CI than SuHx males. OVX worsened SuHx-induced decreases in CI and SuHx-induced increases in RVH and inflammation (MCP-1 and IL-6). E2 repletion in OVX rats attenuated SuHx-induced increases in RV systolic pressure (RVSP), RVH, and pulmonary artery remodeling and improved CI and exercise capacity (V̇o2max). Furthermore, E2 repletion ameliorated SuHx-induced alterations in RV glutathione activation, proapoptotic signaling, cytoplasmic glycolysis, and proinflammatory cytokine expression. Expression of ERα in RV was decreased in SuHx-OVX but was restored upon E2 repletion. RV ERα expression was inversely correlated with RVSP and RVH and positively correlated with CO and apelin RNA levels. RV-protective E2 effects observed in females were recapitulated in male SuHx rats treated with E2 or with pharmacological ERα or ERβ agonists. Our data suggest significant RV-protective ER-mediated effects of E2 in a model of severe PH., (Copyright © 2015 the American Physiological Society.)

Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

Unlabelled: Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/β-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of β-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/β-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/β-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents., Significance: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT–SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans., (©2013 AACR.)

Bone morphogenetic protein (BMP) signaling has been linked to the development of pulmonary hypertension (PH). Inhibitors of differentiation (ID) proteins (ID1-4) are a family of basic helix-loop-helix transcription factors that are downstream targets of the BMP signaling pathway, but the role that ID proteins play in the development of PH is unknown. To address this, we evaluated pulmonary expression of ID proteins in a mouse model of hypoxia-induced PH. There is selective induction of ID1 and ID3 expression in hypoxic pulmonary vascular smooth muscle cells (VSMCs) in vivo, and ID1 and ID3 expression are increased by hypoxia in cultured pulmonary VSMCs in a BMP-dependent fashion. ID4 protein is barely detectable in the mouse lung, and while ID2 is induced in hypoxic peripheral VSMCs in vivo, it is not increased by hypoxia or BMP signaling in cultured pulmonary VSMCs. In addition, the PH response to chronic hypoxia is indistinguishable between wild type and Id1 null mice. This is associated with a compensatory increase in ID3 but not ID2 expression in pulmonary VSMCs of Id1 null mice. These findings indicate that ID1 is dispensable for mounting a normal pulmonary vascular response to hypoxia, but suggest that ID3 may compensate for loss of ID1 expression in pulmonary VSMCs. Taken together, these findings indicate that ID1 and ID3 expression are regulated in a BMP-dependent fashion in hypoxic pulmonary VSMCs, and that ID1 and ID3 may play a cooperative role in regulating BMP-dependent VSMC responses to chronic hypoxia.

The SYT proto-oncoprotein (also known as SS18) is a gene expression regulator conserved across species. Although its biological function is still unknown, the importance of SYT as a housekeeping protein is illustrated by the lethal phenotype of SYT-null embryos. Notably, SYT is a component of the synovial sarcoma-associated translocation product, the SYT-SSX oncogene. SYT was previously reported as a mediator of cell adhesion. In the present study we show that SYT possesses distinct domains that control MDCK cyst formation in three-dimensional collagen cultures. While the carboxy-half of SYT, the QPGY domain, is required for cyst growth, the amino-terminal region appears to exert on this process a regulatory effect. Further analysis suggested that the purinergic G protein-coupled P2Y receptor signaling is involved in SYT-induced cystogenesis. Activation of this cascade is due to facilitation of ATP release in the extracellular space of polarized MDCK cells by SYT. These studies allow us to begin to understand the vital role of SYT in controlling epithelial morphogenesis and might explain the lethality of its loss in the developing embryo.

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.