Hepatic steroid sulfatase critically determines estrogenic activities of conjugated equine estrogens in human cells in vitro and in mice

Ye Feng,Yang Xie,Meishu Xu,Linhao Li,Kyle W Selcer,Patrick J Oberly,Samuel M Poloyac,Hongbing Wang,Chengjiang Li,Fengqin Dong,Chaohui Yu,Wen Xie

doi:10.1074/jbc.ra119.009181

Abstract

Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women. Extracted from pregnant mare urine, CEEs are composed of nearly a dozen estrogens existing in an inactive sulfated form. To determine whether the hepatic steroid sulfatase (STS) is a key contributor to the efficacy of CEEs in HRT, we performed estrogen-responsive element (ERE) reporter gene assay, real-time PCR, and UPLC-MS/MS to assess the STS-dependent and inflammation-responsive estrogenic activity of CEEs in HepG2 cells and human primary hepatocytes. Using liver-specific STS-expressing transgenic mice, we also evaluated the effect of STS on the estrogenic activity of CEEs in vivo We observed that CEEs induce activity of the ERE reporter gene in an STS-dependent manner and that genetic or pharmacological inhibition of STS attenuates CEE estrogenic activity. In hepatocytes, inflammation enhanced CEE estrogenic activity by inducing STS gene expression. The inflammation-responsive estrogenic activity of CEEs, in turn, attenuated inflammation through the anti-inflammatory activity of the active estrogens. In vivo, transgenic mice with liver-specific STS expression exhibited markedly increased sensitivity to CEE-induced estrogenic activity in the uterus resulting from increased levels of liver-derived and circulating estrogens. Our results reveal a critical role of hepatic STS in mediating the hormone-replacing activity of CEEs. We propose that caution needs to be applied when Premarin is used in patients with chronic inflammatory liver diseases because such patients may have heightened sensitivity to CEEs due to the inflammatory induction of STS activity.

Highlights

Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women
The expression and activity of STS are required for the estrogenic activity of CEEs in a reporter gene assay
To evaluate the estrogenic activity of CEEs and its dependence on STS in vitro, 293T cells were transfected with estrogen receptor (ER)␣ expression vector and the estrogen-responsive luciferase reporter gene estrogen-responsive element (ERE)-Luc in the absence or presence of the cotransfection of STS expression vector

Summary

Steroid sulfatase activates conjugated equine estrogens

Steroid sulfatase (STS) is the enzyme responsible for the hydrolysis of steroid sulfates to their unconjugated forms [7]. STS mediated desulfation and sulfotransferase (SULT) mediated sulfation are critical metabolic mechanisms to regulate the chemical and functional homeostasis of endogenous chemicals, including the estrogens. Compared with the parent estrogens, estrogen sulfates have higher concentrations and prolonged half-life in the circulation. The desulfation of steroid sulfate is catalyzed by STS, which is believed to be the only enzyme responsible for the activation of conjugated estrogens. It is known that CEEs are a natural mixture of sulfated estrogens, the specific role of STS in the hormone-replacement effect of CEEs has yet to be established.

Results

Discussion

Mouse and human primary hepatocyte isolation

Statistical analysis