Abstract
Cholesterol 7alpha-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRalpha by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1alpha plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.
Highlights
Cholesterol 7␣-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids
To assess whether all-trans retinoic acid (RA) (atRA) modulates the expression of farnesoid X receptor (FXR) targets involved with bile acid synthesis, metabolism, and transport, as well as lipid catabolism, we treated HepG2 cells with atRA and/or chenodeoxycholic acid (CDCA) for 24 h and measured the expression of several FXR targets in the liver using real-time quantitative PCR
A time-course experiment showed that 5 M atRA decreased cholesterol 7 alpha-hydroxylase (CYP7A1) mRNA expression in association with increased small heterodimer partner (SHP) and fibroblast growth factor 19 (FGF19) mRNA expression in HepG2 cells beginning 2 h after treatment, while CYP7A1 expression remained low throughout the time period (Fig. 1C)
Summary
Cholesterol 7␣-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Elevated FGF19, diminished CYP7A1, and unchanged levels of SHP mRNA expression have been reported in the livers of patients with extrahepatic bile duct obstruction, findings which are reversed by biliary drainage, further supporting this mechanism [5]. Together, these observations suggest that the FXRFGF19 pathway may play a more important role than FXRSHP in regulating CYP7A1 expression in vivo in human liver. The role of FXR in downregulation of CYP7A1 expression is further confirmed in FxrϪ/Ϫ mice, where Cyp7a1 expression is increased [16]
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