Prostaglandin E receptor subtype EP
Prostaglandin E receptor subtype 4 (EP4) is a transmembrane G-coupled protein receptor activated by prostaglandin E2 (PGE2). EP4 activation exerts anti-inflammatory effects in adipose tissue by dampening the levels of inflammatory chemokines . In the mouse, EP4 deficiency aggravates fragmentation of lipid droplets and promotes mitochondrial biogenesis in white adipose tissue, elevating whole-body metabolic rate . The pivotal study that illustrated a role of EP4 in lipid homeostasis showed that EP4 deficiency compromised the activation of lipoprotein lipase, an enzyme responsible for trafficking plasma triglycerides into peripheral tissues, and attenuated triglyceride clearance resulting in hypertriglyceridemia in mice . In that same study, EP4 deficient mice were reported to be hypercholesterolemic, shedding light that EP4 also acts as a regulator of cholesterol homeostasis . However, the detailed mechanism(s) by which EP4 affects cholesterol levels remains unexplored. Hence, the present study was designed to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol.
Materials and methods
Discussion The novel finding of this study is that EP4 deficiency resulted in impaired bile diltiazem hydrochloride synthesis, decreased fecal bile acid excretion and contributed to elevated plasma cholesterol levels in mice. The conversion of cholesterol into bile acids and their subsequent fecal excretion is quantitatively the most important way for elimination of this lipid from the body. Two pathways determine bile acid synthesis in the liver, the classical and alternative pathways, mediated by the rate-limiting enzymes cholesterol 7α hydroxylase and cholesterol 27α hydroxylase, which are encoded by CYP7A1 and CYP27A1 genes, respectively [9, 10]. The mRNA transcript and protein levels of the bile synthetic enzyme CYP7A1 but not CYP27A1 were downregulated in EP4 deficient mice, indicating that EP4 explicitly modulate the classical route of bile acid synthesis. Previous studies support an association between CYP7A1 expression and plasma cholesterol levels [30, 31]. Transient adenoviral overexpression of CYP7A1 protected mice against high fat feeding-induced hypercholesterolemia and elevated fecal bile acid elimination . In humans, a genetic variant that is resistant to diet-induced hypercholesterolemia increased CYP7A1 expression and fecal bile acid excretion . The phenotype of EP4 knockout mice closely resembles that of the genetically engineered CYP7A1 knockout mice [30, 32]. EP4 knockouts have decreased hepatic bile acid synthesis, bile acid excretion and display hypercholesterolemia, effects similar to those observed in CYP7A1 knockout mice [30, 32]. The present findings identify EP4 as a critical regulator of bile acid synthesis and thereby of cholesterol homeostasis. The hypercholesterolemic phenotype of EP4 knockout mice can be attributed to the low expression of CYP7A1 in the liver which decreased bile acid synthesis and impaired the ability to dispose cholesterol in this form. EP4 deficiency compromised cholesterol-to-bile acid conversion and contributed to hypercholesterolemia. This conclusion was further supported by treating mice with CAY10580, the pharmacological EP4 activator which effectively restored plasma cholesterol levels in high fat fed mice by increasing CYP7A1-mediated elimination of cholesterol. Mice receiving the EP4 agonist not only exhibited resistance to diet-induced hypercholesterolemia, but were protected against diet-induced hepatic steatosis and obesity. Promoting bile acid elimination is an effective way to reduce plasma cholesterol, as evidenced by the use of bile acid sequestrants, such as cholestyramine or colestimide, which bind to bile acids in the intestine and prevent them from being reabsorbed into the bloodstream and as a result boost their excretion . The accelerated loss of bile acids will activate further conversion of cholesterol into bile acids through the induction of CYP7A1 in the liver. The decrease in hepatic cholesterol levels induces the expression of LDL receptors, in order to promote the delivery of more cholesterol into the liver resulting in a drop in plasma cholesterol levels . In line with this notion, knockdown of EP4 with small interfering RNA decreased LDL receptor expression and resulted in defective cholesterol clearance in HepG2 cells. Thus the present experiments identified EP4 as a potential target to modulate bile acid synthesis for the treatment of hypercholesterolemia. Taken in conjunction, the present findings indicate that the primary mechanism by which EP4 activation lowers cholesterol is by increasing the conversion of cholesterol into bile acids in the liver following stimulation of CYP7A1. Development of novel cholesterol-lowering agents with mechanism of actions distinct from currents approved drugs may help to achieve appropriate cholesterol levels in patients. The present findings hence permit to hope that enhancing endogenous bile acid synthesis and its subsequent removal through the activation of EP4 signaling may be translated into an effective stand alone or adjunct therapy to lower cholesterol levels, ameliorating problems associated with hypercholesterolemia.