Deliciency of prostaglandin E2 receptor subtype 4 causes dysregulation of cholesterol homeostasis in mice

Abstract

Mice lacking prostaglandin E2 receptor subtype 4 (EP4), one of the four receptors identified for prostaglandin E2 developed spontaneous hypercholesterolemia. The present study investigated the cause of the elevated plasma cholesterol in EP4 knockout mice. 12–15 weeks old normal diet fed EP4 knockout mice show a 48% increase in total cholesterol as compared to their wildtype littermates. The proportion of plasma cholesterol present in very low density lipoprotein (VLDL)/low density lipoprotein (LDL) increased by 84% and that in high density lipoprotein (HDL) increased by 15.6%. The increase in plasma cholesterol was independent of changes in intestinal cholesterol absorption, as there were no changes in fecal neutral sterol excretion and in intestinal ATP-binding cassette (ABC) A1 and G5 protein expression between EP4 wildtype and knockout mice, while hepatic ABCA1 was significantly increased in the latter – suggesting that the deficiency of EP4 increases cholesterol efflux from the liver. The absence of EP4 reduced phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) and in so doing increased HMG-CoA reductase activity, the rate-limiting enzyme for cholesterol synthesis. Furthermore, deficiency of EP4 reduced hepatic mRNA (by 23%) and protein expression (by 36.7%) of low density lipoprotein receptor (LDLR), suggesting that there is impaired cholesterol clearance by the liver. Such conclusion was further reinforced by studies where EP4 knockdown in human hepatoma cell lines reduced cell surface LDLR protein, impaired LDL uptake and reduced intracellular cholesterol. Lastly, EP4 deficiency decreased the catabolism of cholesterol to bile acids. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting step in the classic pathway of bile acid biosynthesis, and its mRNA expression levels were decreased by 57.8% in livers of EP4 deficient mice as compared to those in wildtype mice. While, the expression of sterol 27-hydroxylase (CYP27A1) which initiates the alternate or acidic pathway of bile acid biosynthesis was unchanged. Indeed, EP4 knockout mice exhibited higher plasma but lowered hepatic levels of total bile acids. In summary, the present study shows that the hypercholesterolemia in EP4 deficient mice is mediated by multiple mechanisms including increased hepatic cholesterol biosynthesis, increased cholesterol efflux from the liver, impaired cholesterol clearance by the liver, reduced conversion and/or elimination of cholesterol into bile acids. The lack of EP4 in mice leads to an incapacity to maintain normal plasma cholesterol levels, suggesting a loss of coordinate regulation for cholesterol homeostasis, identifying a new metabolic dimension in the physiological role played by endogenous EP4.