Inflammatory regulation of steroid sulfatase: A novel mechanism to control estrogen homeostasis and inflammation in chronic liver disease - PubMed (original) (raw)

doi: 10.1016/j.jhep.2015.07.022. Epub 2015 Jul 26.

Marcus Klein 2, Ulrich M Zanger 2, Mohammad K Mohammad 3, Matthew C Cave 3, Nilesh W Gaikwad 4, Natasha J Dias 5, Kyle W Selcer 5, Yan Guo 6, Jinhan He 7, Xiuhui Zhang 8, Qiujin Shen 9, Wenxin Qin 9, Jiang Li 1, Song Li 1, Wen Xie 10

Affiliations

Inflammatory regulation of steroid sulfatase: A novel mechanism to control estrogen homeostasis and inflammation in chronic liver disease

Mengxi Jiang et al. J Hepatol. 2016 Jan.

Abstract

Background & aims: Chronic inflammatory liver diseases are associated with estrogen excess and feminization in men, which is thought to be due to compromised liver function to break down estrogens. The goal of this study is to determine whether the inflammatory induction of steroid sulfatase (STS), which converts inactive estrogen sulfates to active estrogens, may have contributed to the estrogen excess in chronic liver disease.

Methods: We performed bioinformatic analysis, real-time PCR, immunohistochemistry, and UPLC/MS-MS to analyze hepatic STS expression and serum estrogen levels in patients with chronic liver diseases. The crosstalk between NF-κB pathway and STS-regulated estrogen signaling was investigated by electrophoretic mobility shift assay, chromatin immunoprecipitation, luciferase assay and gene knockdown experiments in human hepatocytes.

Results: Hepatic STS was induced in patients with chronic inflammatory liver diseases, which was accompanied by increased circulating estrogen levels. The human STS gene, but not the mouse Sts gene, was induced by inflammatory stimuli in hepatic cells. Mechanistically, STS was established as a novel NF-κB target gene, whose induction facilitated the conversion of inactive estrogen sulfates to active estrogens, and consequently attenuated the inflammatory response. In contrast, genetic or pharmacological inhibition of STS or a direct blockade of estrogen signaling sensitized liver cells to the transcriptional activation of NF-κB and inflammatory response, possibly through the inhibition of IκB kinase activation.

Conclusions: Our results suggest a negative feedback loop in chronic inflammatory liver diseases, in which the inflammatory activation of NF-κB induces STS gene expression. The induced STS facilitates the conversion of inactive estrogen sulfates to active estrogens, which in return attenuates the NF-κB-mediated inflammation.

Keywords: Estrogen metabolism; Estrogens; Inflammation; Liver disease; Steroid sulfatase.

Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Frequent induction of STS in chronic inflammatory liver diseases, and increased estrogen levels in patients of alcoholic cirrhosis

(A) Hepatic mRNA expression of STS (left), EST (middle) and IL-8 (right) in patients with normal or elevated CRP

(GSE32504)

as determined by real-time PCR. (B) Hepatic mRNA expression of STS (left), CYP3A4, CYP1A2 and EST (middle left) and IL-8 (middle right), and correlation between the expression of IL-8 and STS (right) in control subjects and patients with alcoholic hepatitis (GSE28619). (C) IHC analysis of STS expression. The top panels are representative cases showing STS expression in control, hepatitis and cirrhosis patients. The bottom panels are corresponding controls without the use of primary anti-STS antibody. (D) Serum level of estrone (left) and estrone sulfate (right) in control subjects and patients with alcoholic cirrhosis from the University of Louisville Hospitals. (E) The mRNA expression of STS in human primary hepatocytes treated with vehicle (VEH), LPS (1 μg/mL), Ethanol (EtOH, 100 μM) or LPS + Ethanol for 6 h. *, P<0.05; **, P<0.01; ***, P<0.001;

NS, statistically not significant.

Figure 2

Figure 2. Treatment of human hepatocytes with NF-κB activators induces the expression and activity of STS

(A) The mRNA expression of STS (left) and CYP3A4, CYP1A2 and EST (middle) in human primary hepatocytes (HPH) treated with VEH or LPS (1 μg/mL) for 24 h, or mouse liver treated with LPS (5 mg/kg) for 24 h. *, P<0.05; ***, P<0.001. (B) Western blotting results. Top panel: the STS protein expression in HPH treated with VEH, LPS (1 μg/ml), or LPS (1 μg/ml) + PDTC (50 μM) for 24 h. Middle panel: the STS protein expression in HPH treated with VEH, PMA (50 ng/mL) or TNFα (40 ng/mL) for 24 h. Bottom panel: the effect of PDTC (50 μM) co-treatment on PMA (50 ng/mL) or TNFα (40 ng/mL) induced STS protein expression in HPH. (C) The STS enzymatic activities in HPH treated with VEH, TNFα (40 ng/mL), PMA (50 ng/mL), or LPS (1 μg/ml). *, P<0.05; **, P<0.01. (D to F) The mRNA expression (D), protein expression (E) and enzymatic activities (F) of STS in Huh7 cells treated with vehicle, PMA (50 ng/mL) or TNFα (40 ng/mL) for 24 h. When applicable, the cells were co-treated with PDTC (50 μM). (G) The protein expression of p65 and STS in PMA (50 ng/mL)-treated Huh7 cells that were transfected with control siRNA or sip65. *, P<0.05; ***, P<0.001.

Figure 3

Figure 3. STS is a transcriptional target of NF-κB

(A) Schematic diagram of the 5′-flanking region of the human STS gene and the three putative NF-κB binding sites. Sequences of the WT and mutant NF-κB binding sites and a consensus NF-κB binding site are shown. (B) The binding of p65 to the three STS NF-κB binding sites was evaluated by EMSA. (C) The recruitment of p65 to the three STS NF-κB binding sites was evaluated by ChIP assay on Huh7 cells treated with VEH or TNFα using an anti-p65 antibody. The recruitment of p65 to a NF-κB binding site on the IL-8 gene promoter was included as a positive control. (D) The transactivation of the WT and mutant STS promoter by p65 was evaluated by transient transfection and luciferase reporter assay. Results are fold induction over the luciferase activity of the empty vector pGL-3 from triplicate assays. *, P<0.05; **, P<0.01.

Figure 4

Figure 4. The inflammatory induction of STS enhances estrogen activity in Huh7 cells

(A to C) The E2 levels in cell culture supernatant from control siRNA or siSTS-transfected Huh7 cells treated with either VEH or PMA (50 ng/mL) for 24 h (A). The knockdown efficiency of siSTS as determined by real-time PCR (B) and Western blotting (C). (D) The level of E2 in cell culture supernatant of Huh7 cells treated with VEH, E2S (10 μM), or E2S (10 μM) + PMA (50 ng/mL) for 24 h. (E) The effects of PDTC (25 μM), STX (10 μM) or ICI (100 nM) on PMA (50 ng/mL)-induced ERE-luciferase reporter activity. (F) TNFα (40 ng/mL)-induced expression of estrogen-responsive genes in Huh7 cells transfected with control siRNA or siSTS.

Figure 5

Figure 5. The inflammation-responsive and STS-mediated induction of estrogen activity attenuates the inflammatory response in Huh7 cells

(A) Treatment of Huh7 cells with E1S (100 nM) or E2 (10 nM) attenuated the PMA (50 ng/mL)-induced NF-κB luciferase reporter activity. (B) The effect of STX (10 μM) or ICI (100 nM) on PMA (50 ng/mL)-induced NF-κB reporter activity. (C to E) The effect of STX (10 μM) (C), siSTS (D), or ICI (100 nM) (E) on TNFα (40 ng/mL)-induced expression of endogenous NF-κB target genes. (F and G) The effects of E2 (F) and ICI and STX (G) on PMA-induced phosphorylation of IKK α and β as shown by Western blotting.

Figure 6

Figure 6. A proposed STS-mediated negative feedback loop in chronic inflammatory liver disease that limits the activity of NF-κB and attenuates inflammation

During chronic inflammation, inflammatory mediators such as LPS and TNFα act on hepatocytes to elicit NF-κB activation. NF-κB then induces the expression of STS, which converts inactive estrogen sulfates to active estrogens and increases estrogen level in the liver and circulation. The estrogens/ER signaling in return suppresses the NF-κB response and inhibits inflammation.

References

    1. Adlercreutz H. Oestrogen metabolism in liver disease. J Endocrinol. 1970;46:129–163. - PubMed
    1. Gavaler JS. Alcohol effects on hormone levels in normal postmenopausal women and in postmenopausal women with alcohol-induced cirrhosis. Recent Dev Alcohol. 1995;12:199–208. - PubMed
    1. Long RS, Simmons EE. The liver and estrogen metabolism; report of cases. AMA Arch Intern Med. 1951;88:762–769. - PubMed
    1. Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis. 1998;19:1–27. - PubMed
    1. Falany CN. Enzymology of human cytosolic sulfotransferases. FASEB J. 1997;11:206–216. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources