PB0326 - Synergistic effects of hypoxia and estrogen on Protein S gene expression

Background: Protein S (PS) is a multifunctional vitamin K-dependent anticoagulant. Protein S deficiency is directly linked to increased risk of thrombosis. Acquired PS deficiency can result from one of several factors, including hypoxia and estrogen, in turn, elevating thrombotic risk

Aims: To measure and establish a synergistic effect of hypoxia and estrogen on PS gene expression

Methods: HepG2 cells were incubated with oxygen concentrations (10%-1%) in a hypoxic chamber ± 25 nM estrogen). In addition, mice were grouped into four categories based on their estrogen treatment and weight as follows: control, control+estrogen, obese, and obese+estrogen. Immunoblotting, qPCR, TGA, aPTT, and ELISA were performed to measure PS protein and mRNA levels, as well as the amounts of PS

Results: Quantification of immunoblot data indicated lower PS protein and mRNA levels in HepG2 cells incubated in 10%-1% O2 without estrogen (Fig. 1 A). However, the amounts of PS protein and mRNA were yet lower in HepG2 cells incubated with the same O2 concentrations plus 25 nM estrogen (Fig. 1B). Similarly, thrombin formation in obese mice treated with estrogen for five weeks was significantly higher compared with obese mice and mice treated with estrogen alone (Fig. 2A). ELISA and immunoblot data in obese +estrogen treated mice showed a reduction in PS level during the five-week period compared to obese or estrogen alone (Fig. 2B and C)

Conclusion(s): Our data show that protein and mRNA levels of PS were significantly lower in HepG2 cells under hypoxia and estrogen treatment. Similarly, PS levels were significantly reduced in obese+estrogen treated mice compared to obese and estrogen treated mice separately. The results demonstrate a synergistic downregulation of PS expression by hypoxia (associated with obesity) and estrogen. Work is underway to assess the hypothesis that hypoxia inducible factor 1 α and the estrogen receptor transcription factors are responsible for PS gene repression