In normal chow fed mice EP deficiency
In normal chow fed mice, EP4 deficiency also decreased the expression of CYP8B1, the downstream target of CYP7A1. Similarly, knockdown of EP4 with small interfering RNA reduced the expression of CYP8B1 in HepG2 cells. Therefore, it was anticipated that there would be an increased expression of CYP8B1 upon activation of EP4 receptors with CAY10580 in high fat fed mice. However, an increased expression of CYP8B1 was not observed. One possible reason for this unforeseen observation is that the high fat diet may be a confounder and directly influence the expression of bile Pertussis Toxin synthetic enzymes. Indeed, CYP8B1 has been reported to be downregulated upon high fat diet challenge in mice [34, 35, 36]. The rate of bile acid synthesis is under control of different molecular mechanisms in which nuclear receptors are involved . In most species, the LXRα is involved in the feedforward regulation of CYP7A1 by cholesterol, whereas the feedback regulation by bile acids is mediated via FXR, RXR and SHP [22, 23, 37]. However, none of these nuclear receptors were altered by EP4 deficiency, indicating they are not the cause of the altered production and metabolism of bile acids in EP4 knockout mice. Instead, as evidenced by the high efficiency of ERK1/2 inhibitors, U0126 and PD98059 to inhibit CYP7A1 expression in HepG2 cells, the present findings suggest that EP4 promotes CYP7A1 expression through the phosphorylation of ERK1/2. In support of this interpretation, phosphorylated ERK1/2 was increased in CAY10580-treated mice while phosphorylated ERK was decreased in EP4 deficient mice as compared to their respective controls. Although prostaglandin E2 and its receptors have long been discovered, their roles have not been linked with bile acid or cholesterol homeostasis. However, it has been reported that activation of EP3 facilitates hepatic bile acid synthesis and cholesterol elimination and prevents high fat diet-induced hypercholesterolemia and atherosclerosis development by suppressing the phosphorylation of HNF4α, leading to decreased CYP7A1 expression . There was no difference in the expression of EP3 or phosphorylated HNF4α between livers of EP4 wild type and knockout mice, supporting that a distinct signaling pathway is responsible for the EP4-mediated CYP7A1 increase. The present experiments indicate that not only EP3 , but also EP4, another subtype of prostaglandin E receptor, influences bile acid synthesis and controls cholesterol homeostasis via a distinct signaling pathway. EP4 does not rely on the phosphorylation of HNF4α to increase CYP7A1 expression, but rather depends on the activation of ERK phosphorylation which causes elevation in CYP7A1 expression leading to a greater cholesterol elimination. It is important to note that on the market there is a wide variety of clinical drugs that influence prostaglandin levels (e.g. cyclooxygenase inhibitors), whether they impose any negative impact on bile acid or cholesterol homeostasis deserve cautious reexamination. In the past, bile acids were simply thought to be molecules that facilitate lipid digestion or a means of cholesterol disposal . However, sparked by the identification of bile acid activated receptors (including FXR, pregnane X receptors, constitutive androstane receptors, vitamin D receptors, and G protein-coupled bile acid receptors) bile acids are now recognized to be important signaling molecules that regulate lipid, glucose and energy metabolism, inflammation, as well as drug metabolism and detoxification processes [37, 39]. By means of controlling bile acid levels, EP4 may regulate an array of cellular and physiological processes. Indeed, in the present study, promoting bile acid synthesis through activation of EP4 receptors exerted diverse actions including slowing weight gain and protection against hepatic steatosis. Of particular interest, exogenous administration of bile acids to high fat fed mice dampens serum triglyceride levels . Bile acids elicit the production of apolipoprotein C-II, a co-activator of lipoprotein lipase, the induction of which lowers serum triglycerides . Mice with EP4 deficiency exhibit reduced hepatic mRNA levels of apolipoprotein C-II and developed hypertriglyceridemia . Whether or not the hypertriglyceridemia in EP4 deficient mice is associated with weakened bile acid production causing diminished apolipoprotein C-II release and consequently less activation of lipoprotein lipase is yet to be determined.