Abstract
The bile acid (BA) nuclear receptor, farnesoid X receptor (FXR/NR1H4), maintains metabolic homeostasis by transcriptional control of numerous genes, including an intestinal hormone, fibroblast growth factor-19 (FGF19; FGF15 in mice). Besides activation by BAs, the gene-regulatory function of FXR is also modulated by hormone or nutrient signaling-induced post-translational modifications. Recently, phosphorylation at Tyr-67 by the FGF15/19 signaling-activated nonreceptor tyrosine kinase Src was shown to be important for FXR function in BA homeostasis. Here, we examined the role of this FXR phosphorylation in cholesterol regulation. In both hepatic FXR-knockout and FXR-knockdown mice, reconstitution of FXR expression up-regulated cholesterol transport genes for its biliary excretion, including scavenger receptor class B member 1 (Scarb1) and ABC subfamily G member 8 (Abcg5/8), decreased hepatic and plasma cholesterol levels, and increased biliary and fecal cholesterol levels. Of note, these sterol-lowering effects were blunted by substitution of Phe for Tyr-67 in FXR. Moreover, consistent with Src's role in phosphorylating FXR, Src knockdown impaired cholesterol regulation in mice. In hypercholesterolemic apolipoprotein E-deficient mice, expression of FXR, but not Y67F-FXR, ameliorated atherosclerosis, whereas Src down-regulation exacerbated it. Feeding or treatment with an FXR agonist induced Abcg5/8 and Scarb1 expression in WT, but not FGF15-knockout, mice. Furthermore, FGF19 treatment increased occupancy of FXR at Abcg5/8 and Scarb1, expression of these genes, and cholesterol efflux from hepatocytes. These FGF19-mediated effects were blunted by the Y67F-FXR substitution or Src down-regulation or inhibition. We conclude that phosphorylation of hepatic FXR by FGF15/19-induced Src maintains cholesterol homeostasis and protects against atherosclerosis.
Highlights
The bile acid (BA) nuclear receptor, farnesoid X receptor (FXR/NR1H4), maintains metabolic homeostasis by transcriptional control of numerous genes, including an intestinal hormone, fibroblast growth factor-19 (FGF19; FGF15 in mice)
To examine the effect of the FXR phosphorylation on expression of FXR target genes, we examined the changes in gene expression after rescue of FXR expression with liver-specific expression of FXR-WT or phosphorylation-defective (p-defective) Y67F-FXR in mice in which FXR was downregulated in the liver by expression of hepatocyte-targeting AAV-TBG-Cre [27] in FXR-floxed mice [26] (Fig. 1, A and B)
Analysis of previously published RNA-Seq data [16] from these mice revealed that expression of hepatic genes involved in the uptake and transport of cholesterol for hepatobiliary secretion, including an high-density lipoprotein (HDL) uptake transporter, scavenger receptor class B member 1 (Scarb1) (8 –11); key transporters for biliary cholesterol excretion, including Abcg5 and Abcg8 [8, 9]; and BA transporters, Abcb11 (Bsep) and Atp8b1, are up-regulated by hepatic expression of WT-FXR compared with Y67F-FXR (Fig. 1A)
Summary
To examine the effect of the FXR phosphorylation on expression of FXR target genes, we examined the changes in gene expression after rescue of FXR expression with liver-specific expression of FXR-WT or phosphorylation-defective (p-defective) Y67F-FXR in mice in which FXR was downregulated in the liver by expression of hepatocyte-targeting AAV-TBG-Cre [27] in FXR-floxed mice [26] (Fig. 1, A and B). Expression of WTFXR resulted in decreased plasma/liver cholesterol levels and increased biliary/fecal cholesterol levels, and each of these changes was blocked by the Y67F mutation (Fig. 4D) These results indicate that phosphorylation of hepatic FXR at Tyr-67 is important for its beneficial effects in atherosclerosis-prone mice. Consistent with the changes in hepatic sterol transport gene expression and cholesterol levels, down-regulation of Src increased atherosclerotic plaques, necrotic areas, and macrophage infiltration in the aortic sinus (Fig. 5E) and brachiocephalic artery (Fig. 5F) and substantially increased collagen content of the plaques in the brachiocephalic artery These results demonstrate that atherosclerosis in hypercholesterolemic ApoE-KO mice is exacerbated by down-regulation of Src. Endogenous FGF15 signaling is important for induction of Scarb and Abcg5/8, after feeding or FXR activation. These results from hepatocytes and mice, together, strongly suggest that Src is required for FGF19-mediated induction of direct FXR targets, Abcg5/8 and Scarb, and increased cholesterol efflux transport for hepatobiliary excretion
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