Estrogen and obesity synergistically suppress protein S via HIF1α, enhancing thrombosis potential
Mohammad A. Mohammad, Narender Kumar, Sonali Ghosh, Ashley Paysse, Claudia Leonardi, Vijaya Pilli, Ma Lorena Duhaylungsod, Eric Lazartigues, Diana C. Polania-Villanueva, Sadaf Nouman, Logan A. Barrios, Rima Chattopadhyay, Rafika Yasmin, Alaina Guilbeau, Manoj Kumar, Tina Nguyen, Jovanny Zabaleta, Li Li, Luis Del Valle, Mallory T. Barbier, Samarpan Majumder, Laurent O. Mosnier, Rinku Majumder
Mohammad A. Mohammad, Narender Kumar, Sonali Ghosh, Ashley Paysse, Claudia Leonardi, Vijaya Pilli, Ma Lorena Duhaylungsod, Eric Lazartigues, Diana C. Polania-Villanueva, Sadaf Nouman, Logan A. Barrios, Rima Chattopadhyay, Rafika Yasmin, Alaina Guilbeau, Manoj Kumar, Tina Nguyen, Jovanny Zabaleta, Li Li, Luis Del Valle, Mallory T. Barbier, Samarpan Majumder, Laurent O. Mosnier, Rinku Majumder
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Research Article Hematology Vascular biology

Estrogen and obesity synergistically suppress protein S via HIF1α, enhancing thrombosis potential

Abstract

Venous thromboembolism (VTE) is a leading cause of morbidity and mortality, with risk heightened in premenopausal women with obesity or use estrogen-based oral contraceptives. When both risk factors are present, the thrombosis risk increases substantially. Protein S (PS), an essential anticoagulant cofactor, is downregulated by both estrogen and obesity, but the molecular basis for this suppression remains poorly defined. We investigated the effect of estrogen and obesity on PS expression using plasma samples from 157 women stratified by BMI and contraceptive use, alongside 40 mice categorized as lean or obese with or without estrogen pellet treatment. The levels of PS were reduced by either estrogen or obesity alone, and the combined effect increased thrombin generation. In HepG2 hepatocytes, hypoxic conditions (1%–10% O2) mimicking obesity, with or without 17 β-estradiol, suppressed PROS1 transcription and promoter activity. ChIP confirmed direct binding of hypoxia-inducible factor 1α (HIF1α) to the PROS1 promoter, repressing gene expression. These findings define a mechanistic pathway through which estrogen and obesity converge to suppress PS synthesis, providing insight into the elevated thrombosis risk observed in women with obesity using estrogen-based contraceptives.

Authors

Mohammad A. Mohammad, Narender Kumar, Sonali Ghosh, Ashley Paysse, Claudia Leonardi, Vijaya Pilli, Ma Lorena Duhaylungsod, Eric Lazartigues, Diana C. Polania-Villanueva, Sadaf Nouman, Logan A. Barrios, Rima Chattopadhyay, Rafika Yasmin, Alaina Guilbeau, Manoj Kumar, Tina Nguyen, Jovanny Zabaleta, Li Li, Luis Del Valle, Mallory T. Barbier, Samarpan Majumder, Laurent O. Mosnier, Rinku Majumder

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Figure 5

Estrogen and hypoxia downregulate PS expression and mRNA levels in HepG2 cells.

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Estrogen and hypoxia downregulate PS expression and mRNA levels in HepG2...
(A and D) Estrogen-treated (5–30 nM) or hypoxia-induced (10%–1%) HepG2 cells were lysed and separated by SDS-PAGE, and the transferred proteins were probed with either PS or GAPDH to determine the effects on PS levels. Quantification of relative PS expression by immunoblotting (B and E) and mRNA levels by RT-qPCR (C and F). The combined effects of estrogen (25 nM) and oxygen concentrations (10%–1%) on the downregulation of PS were quantified (G), and relative PS expression was determined by immunoblotting (H), RT-qPCR (I), and luciferase assay (J). The reverse effect of 30 μM fulvestrant on PS levels was quantified (K), and relative PS expression was determined by immunoblotting (L), RT-qPCR (M), and luciferase assay (N). (O) Schematic diagram illustrating the possible binding sites of HIF1α on the PS promoter at position –631 to –626 in humans and at –628 to –622 in mice, respectively. (P) ChIP assay indicates the HIF1α binding site within the PS promoter. Lane 1: The presence of the PS amplicon indicates that HIF1α interacted with the PS promoter. Lane 2: Input DNA (isotype control). Lane 3: Absence of the PS promoter amplicon in the control antibody immunoprecipitation indicates the specificity of interaction of HIF1α with the PS promoter. Lane 4: DNA ladder. The bands were quantified by ImageJ software (NIH). The values shown are the mean ± SD of at least 3 independent experiments. Statistical significance was performed by 1-way ANOVA with Kruskal-Wallis correction, since data did not follow a normal distribution (*P < 0.05 and **P < 0.01). C, control.