Gastroenterology

Gastroenterology

Volume 156, Issue 6, May 2019, Pages 1788-1804.e13
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

https://doi.org/10.1053/j.gastro.2019.01.010Get rights and content

Background & Aims

Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues.

Methods

Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein–coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry.

Results

Exposure to 17β-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase–protein kinase B–mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish.

Conclusions

In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment.

Transcript Profiling

The accession number in the Gene Expression Omnibus is GSE92544.

Section snippets

Zebrafish Husbandry

Zebrafish were maintained according to Institutional Animal Care and Use Committee guidelines at Harvard Medical School.

Chemical Exposures

Zebrafish larvae were exposed to chemical modifiers, dissolved in 0.1% (volume/volume) dimethyl sulfoxide (DMSO) for 5 hours from 110 to 115 hours postfertilization (hpf), and analyzed at 120 hpf (unless otherwise specified).11 Adults were treated for 5–7 hours per exposure. Chemicals are listed in Supplementary Table 1.

Morpholino Injection

Morpholino oligonucleotides designed against esr1, esr2a,

Estrogen Enhances Liver Growth

To demonstrate the potential impact of endogenous estrogen levels on liver size, adult zebrafish livers were assessed in females and males. Livers of adult female zebrafish were bigger than those of male siblings, as visualized in hepatocyte reporter fish expressing green fluorescent protein (GFP) regulated by the fatty acid binding protein 10a promoter, Tg(fabp10a:GFP) (Figure 1A). Baseline liver-to-body weight ratios in 10 WT males and females at 7 months showed a significant 2.6-fold

Discussion

Liver cancer is the second most common cause of cancer death worldwide, and it predominantly affects men.2 Here, we identified a novel role for GPER1 in hepatocytes that cell-autonomously regulates larval liver growth and contributes to the sex dimorphisms observed in liver cancer incidence and growth. E2 promotes hepatocyte cell cycle progression and proliferation through GPER1 and downstream activation of PI3K-mTOR signaling. Importantly, the GPER1-mTOR pathway remains essential for liver

Acknowledgments

The authors thank Isaac Oderberg from the Goessling lab for creating the visual abstract, John Asara at the Metabolomics Core at the Beth Israel Deaconess Medical Center for assistance with the metabolomics experiments, and staff of Beth Israel Deaconess zebrafish core facility for fish husbandry.

Author contributions: Saireudee Chaturantabut and Wolfram Goessling conceived the study, designed experiments, and analyzed data. Saireudee Chaturantabut conducted all zebrafish experiments. Arkadi

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    Conflicts of interest The authors disclose the following: Wolfram Goessling is a consultant and Leonard I. Zon is a founder of Camp4 Therapeutics which develops treatments for liver diseases.

    Funding This study is supported by National Institutes of Health 5K08CA172288 (to Kimberley J. Evason), R24OD017870 (to Leonard I. Zon and Wolfram Goessling), R37DK048873, R01DK056626 and R01 DK103046 (to David E. Cohen) and R01DK090311 and R01DK105198 (to Wolfram Goessling), and the Claudia Adams Barr Program in Cancer Research (to Wolfram Goessling). Wolfram Goessling is a Pew Scholar in the Biomedical Sciences.

    Author names in bold designate shared co-first authorship.

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