Estrogen-mediated SOX17 Regulation in the Development of Pulmonary Arterial Hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a sexually dimorphic disease with female predominance. This gender bias is attributed, in part, to estrogen signaling and the estrogen metabolite, 16α-hydroxyestrone (16αOHE) is considered a major contributor to the pathogenesis of PAH. Recent studies have also highlighted both, common and rare genetic variants, in the SOX17 gene, an endothelial cell (EC)-specific transcription factor, in patients with PAH, suggesting that SOX17 deficiency contributes to PAH development. To date, however, functional studies of SOX17 in PAH are absent and the relationship between SOX17 and sexual dimorphism in PAH remains unknown. We hypothesized that 16αOHE-mediated estrogen signaling downregulates SOX17 during the development of PAH. Sox17 mRNA and protein expression were reduced in the lungs of monocrotaline (MCT) and sugen hypoxia (SuHx) rodent models of PH as well as in human pulmonary artery endothelial cells (HPAECs) isolated from patients with PAH compared to controls. Staining of human lung samples from patients with PAH reveals decreased expression of SOX17 in the endothelium. Inducible conditional EC-specific Sox17 knockout (Sox17EC-/-) mice exhibited increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), and PA wall thickness (PAWT) after exposure to chronic hypoxia (4 weeks) compared to controls. While not evident across Sox17EC-/- murine sex, lungs from male rats at baseline, however displayed increased Sox17 expression compared to those from age-matched female rats. To further study these latter expression differences, in silico analysis revealed the presence of five estrogen response element (ERE) sequences on the SOX17 promoter, suggesting estrogen-mediated transcriptional regulation of the SOX17 gene. Treatment of HPAECs with 16αOHE reduced mRNA and protein levels of SOX17 in a dose-dependent manner. HPAECs transfected with the SOX17 promoter and treated with 16αOHE showed reduced activity of SOX17 promoter when measured by dual-luciferase reporter assay. Leveraging ERE mutated constructs for promoter luciferase studies, 16αOHE transcriptionally repressed SOX17 promoter activity at predicted ERE sites and via ERα in HPAECs. Supporting these in vitro observations, ERα loss-of-function (ERα-/-) mutant rats generated via CRISPR-Cas9 editing revealed reduced Sox17 protein expression in vehicle and MCT-exposed lungs compared to cage controls. To validate these data in vivo, we tested whether Sox17 rescues 16αOHE-mediated hypoxic PH in mice. Consistent with prior reports, 16αOHE induced increases in RVSP, RVH, and PAWT in control mice under normoxic and hypoxic conditions. Inducible conditional EC-specific Sox17 transgenic over-expressing (Sox17Tg) mice attenuated chronic hypoxia-induced PH with reduced RVSP, RVH, and PAWT. Hypoxic mice with Sox17Tg exhibited reduced RVSP, RVH, and PAWT after 16αOHE exposure. Inflammatory markers such as NLR family pyrin domain containing 3 (NLRP3), Caspase-1, Interleukin (IL)-1, and IL-18 were reduced in lungs of Sox17Tg mice compared to WT controls despite exposure to chronic hypoxia and 16αOHE. These results suggest that Sox17 mediates PH protection, in part, via regulation of lung inflammation. To translate these experimental data to patients, we identified a functional common variant residing in the DNA binding domain of the ESR1 gene (encoding ERα), rs746432, which was previously shown to reduce transcriptional activation of ERα protein on target ERE reporter genes. This polymorphism was associated with reduced pulmonary vascular resistance (PVR) in patients with PAH (n=702) in fully adjusted analyses. In summary, validating recent genetic studies, deficiency of Sox17 augments preclinical PAH. Moreover, 16αOHE mediates the development of PAH, in part, via downregulation of SOX17, linking sexual dimorphism in PAH with SOX17-based genetic underpinnings.