The critical role of the G protein‐coupled receptor 30 (GPR30), a novel estrogen receptor, in the formation of lithogenic bile through a non‐transcriptional regulatory mode in 17β‐estradiol (E2)‐treated mice

Abstract

BackgroundA failure of hepatic cholesterol (Ch) homoeostasis, in which the physical‐chemical balance of Ch solubility in bile is disrupted, induces the formation of lithogenic bile. However, few studies were performed to address the metabolic abnormalities underlying the source of excess Ch that causes bile supersaturation induced by E2 through GPR30. Of note is that ~50% of Ch is converted to bile acids (BA) in the liver each day in humans and mice. Because GPR30 is expressed in the endoplasmic reticulum and not in the nucleus of hepatocytes, we hypothesized that the epidermal growth factor receptor (EGFR) is required for GPR30 to produce a non‐transcriptional regulatory role in suppressing hepatic expression of Ch 7α‐hydroxylase (CYP7A1) and the classical pathway of BA synthesis, leading to the availability of excess amounts of Ch for hepatic hypersecretion and the formation of supersaturated bile.MethodsThe effect of GPR30 on the formation of lithogenic bile and Ch crystallization were studied in ovariectomized (OVX) GPR30(−/−)/ERα(−/−) and GPR30(+/+)/ERα(−/−) mice implanted subcutaneously with pellets releasing E2 at 6 μg/day and fed a lithogenic diet for 8 weeks. To study whether the metabolic abnormalities underlying the major source of the excess Ch molecules lead to Ch‐supersaturated bile as induced by GPR30, we examined the role of GPR30 in regulating CYP7A1 and the classical pathway of BA synthesis through EGFR.ResultsE2 induced rapid Ch crystallization mainly through the liquid crystalline pathway and promoted gallstone formation in OVX GPR30(+/+)/ERα(−/−) mice fed the lithogenic diet. By contrast, the prevalence of gallstones was drastically reduced in OVX GPR30(−/−)/ERα(−/−) mice. Hepatic secretion of biliary BA was significantly reduced in mice expressing GPR30, leading to an increased ratio of Ch to BA. After E2 treatment, there was a significant increase in mRNA and protein levels of EGFR and a significant decrease in expression levels of liver receptor homolog‐1 (LRH‐1), coupled with reduced CYP7A1 mRNA and protein levels in OVX GPR30(+/+)/ERα(−/−), but not GPR30(−/−)/ERα(−/−) mice. In vitro studies using cultured primary hepatocytes isolated from GPR30(+/+)/ERα(−/−) mice showed that when EGFR was inhibited by AG1478, a highly potent EGFR kinase inhibitor, the mRNA and protein levels of CYP7A1 were unchanged even though GPR30 was activated by E2.ConclusionsThese results indicate that GPR30 regulates Cyp7a1 expression through the EGFR cascade. In the lithogenic state, reduced hepatic synthesis of BA from the classical pathway significantly inhibits the conversion of Ch to BA due to GPR30 repression of CYP7A1 in E2‐treated mice. As a result, a significant increase in the ratio of Ch to BA leads to the imbalance of biliary lipids for keeping Ch solubilized in bile, contributing to the formation of Ch‐supersaturated bile.Support or Funding InformationThis work was supported in part by research grants DK101793 and DK106249 (to D.Q.‐H.W.), both from the National Institutes of Health (US Public Health Service).