Obesity and Oral Contraceptives Synergistically Increase Thrombotic Risk By Downregulating Protein S

Background:

The increased thrombotic effects of estrogen-based oral contraceptives and obesity have been documented independently. However, obesity and oral contraceptives combined are associated with a far greater thrombotic risk, but we have a poor understanding of the mechanism of this greater effect. Increasingly, women are using oral contraceptives, and the national obesity rate has been skyrocketing. Thus, it is imperative to explain how obesity and oral contraceptives work together to significantly elevate thrombotic risk. We know that hypoxia-inducible factor 1-ɑ (HIF-1ɑ) and the estrogen receptor (Erɑ) bind to the promoter of the Protein S gene; binding occurs at sites within ~450 nucleotides of each other, and the two transcription factors downregulate Protein S expression independently. We hypothesize that the two factors, by binding the promoter simultaneously, synergistically downregulate Protein S to a degree much greater than the downregulation mediated by each factor separately.

Aims:

The goal of this project is determining whether estrogen and obesity-induced hypoxia work synergistically to downregulate Protein S transcription and increase thrombotic risk.

Methods:

We measured the effects of obesity and oral contraceptives on hepatocarcinoma (HEP G2) cells because the liver is the major producer of Protein S. Estrogen of varying concentrations (25-150 μM) was used to mimic the effects of oral contraceptives, and cobalt chloride of varying concentrations (25-150 μM) was used to stimulate hypoxia and HIF-1ɑ expression. Cells were exposed to estrogen only, cobalt chloride only, and estrogen and cobalt chloride together for 24 hours, after which the cells were harvested and subjected to q-PCR and immunoblot blot analyses to measure Protein S transcription and protein expression. Although cobalt chloride is a reliable inducer of hypoxia and HIF-1ɑ expression, we also performed hypoxia experiments by incubating cells in a chamber with varying O ₂ concentrations (20%, 15%, 10%, 5%, 1%).

Results and Conclusions:

Immunoblot analysis of cells treated with either CoCl ₂ and estrogen supplementation revealed a ~20% reduction in Protein S levels compared to control conditions and a more significant reduction in (~60%) Protein S expression in cells treated with estrogen and CoCl ₂ together. These results supported our hypothesis that obesity and estrogen-based contraceptives increase thrombotic risk by downregulating anticoagulant Protein S transcription and subsequently decreasing Protein S level.