We demonstrate in these studies that acetylation of FXR normally dynamically modulates its activity and that abnormally elevated levels of acetylated FXR are present in mouse models of metabolic disease. The level of FXR acetylation is reciprocally regulated by the acetylase p300 and the deacetylase SIRT1. The major site of FXR acetylated by p300 is K217 within the hinge domain and acetylation was also detected at K157 within the DNA binding domain. Mutation of K157 and K217 resulted in decreased stability of FXR but increased heterodimerization with RXRα, binding to DNA, and transactivation activity indicating that acetylation of FXR increases its stability but inhibits its DNA binding and transactivation activity. Down-regulation of endogenous SIRT1 in mouse liver increased acetylation levels of endogenous FXR demonstrating that SIRT1 affected FXR acetylation in vivo. Interestingly, down-regulation of SIRT1 was associated with deleterious gene expression patterns and metabolic outcomes. Consistent with this observation, FXR acetylation levels were highly elevated in ob/ob mice and high fat western-style diet mice. Furthermore, activation of SIRT1 by treatment with resveratrol, which has been shown to improve metabolic outcomes (Baur et al., 2006
; Lagouge et al., 2006
), demonstrated reduced the elevated FXR acetylation in these metabolic disease mice. Our results suggest an intriguing connecting between elevated FXR acetylation and decreased FXR activity in animals with deleterious metabolic profiles.
Based on results from the present and published studies (Fang et al., 2008
), acetylation is likely to play a complex role in gene regulation by FXR (). Upon activation, FXR recruits p300 to target gene promoters and SIRT1 is dissociated, resulting in increased acetylation of FXR and histones. Acetylation of histones by p300 is associated with gene activation and is probably the major factor in the activation of the FXR target gene as we previously demonstrated (Fang et al., 2008
). At the same time, however, acetylation of FXR itself inhibits the activity of FXR. This seemingly paradoxical effect may be important to limit or terminate the response to a stimulus response, which is essential in a dynamically regulated system. Once the acetylated FXR is released from the promoter, FXR is deacetylated by SIRT1, which then either interacts with RXRα and rebinds to the DNA as a heterodimer, or is degraded via the ubiquitin-proteasomal degradation pathway. In the absence of further stimulation, SIRT1 is recruited to the target genes by the unliganded FXR and histones are deacetylated so that gene expression remains at a low basal level. Importantly, acetylation and deacetylation of FXR appear to be a dynamic process under normal physiological conditions, so that the activity of FXR is tightly balanced by the opposing actions of p300 and SIRT1.
A proposed model of FXR acetylation in normal and disease states
In contrast to normal mice, in mice with abnormal metabolic profiles such as ob/ob mice, western diet mice, or mice expressing siSIRT1, the FXR acetylation levels are constitutively and highly elevated. The increased acetylation of FXR may be caused by low activity of SIRT1 since down-regulation of SIRT1 in normal mice led to deleterious metabolic outcomes. In metabolic disease states, continuously acetylated FXR would show impaired interaction with RXRα and DNA binding of FXR/RXRα, and subsequently decreased FXR transactivation of its metabolic target genes. This result suggests that the dynamic acetylation and deacetylation of FXR in normal mice may be required for activation of the genes, while continuously elevated acetylation in the diseased states blocks activation. Therefore, our studies provide a potential intriguing correlation between elevated FXR acetylation, decreased FXR activities, and deleterious metabolic effects in metabolic disease states.
The correlation of elevated FXR acetylation and metabolic disease suggests that activation of SIRT1 should have beneficial effects. Indeed, activation of SIRT1 by resveratrol has already been demonstrated to have beneficial metabolic effects in mouse models of metabolic disease by enhancing mitochondria function through activation of PGC-1α (Baur et al., 2006
; Lagouge et al., 2006
). Our observation that activation of resveratrol or overexpression of SIRT1 results in decreased acetylation of FXR in disease model mice suggests that effects of resveratrol on FXR, in addition to PGC-1α, may also contribute to improved metabolic outcomes. These results are consistent with our previous studies showing that down-regulation of p300, which should reduce acetylation of FXR, altered expression of metabolic target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected (Fang et al., 2008
). In addition to the direct effects of SIRT1 on FXR, SIRT1 may also indirectly increase FXR activity by activating PGC-1α (Baur et al., 2006
; Rodgers et al., 2008
). PGC-1α has been shown to coactivate FXR transactivation and increase FXR expression (Zhang et al., 2004
). It is possible that SIRT1-mediated PGC-1α coactivation and FXR deacetylation synergistically regulate hepatic FXR activity under physiological conditions.
It has been demonstrated that bile acids, not only activate FXR signaling by binding to the ligand binding domain of FXR, but also activate upstream cellular kinase signaling pathways, such as protein kinase B (PKB, Akt), PKC, JNK, and ERK kinases, in hepatocytes (Dent et al., 2005
; Gineste et al., 2008
; Hylemon et al., 2009
; Miao et al., 2009
). We recently demonstrated that bile acids increase the stability of SHP, a well known FXR target, by inhibiting ubiqutin-proteasomal degradation in an ERK-dependent manner (Miao et al., 2009
). A recent study showed that inhibition of PKC impaired ligand-mediated regulation of FXR activity of its target genes by blocking FXR interaction with PGC-1α (Gineste et al., 2008
). Interestingly, one of the two reported PKC sites in FXR, S154, is located near K157, an acetylation site in FXR. It will be, therefore, interesting to know whether phosphorylation of FXR by PKB or PKC affects acetylation of FXR and whether impaired signaling pathways are associated with elevated FXR acetylation in metabolic disease states.
Although K217 was identified as the major acetylation site for FXR, acetylation at K157 was also observed. Recent studies demonstrated that acetylation of p53 at different lysine residues affected different biological processes, for example, acetylation of p53 at K120 in the DNA binding domain by Tip60 acetylase is crucial for apoptosis but is dispensable for cell cycle arrest (Tang et al., 2006
). Likewise, acetylation at K157 and K217 in FXR may have different functional outcomes and may selectively play a role in regulation of subsets of target genes involved in different metabolic pathways, such as cholesterol/bile acids, fatty acid, and glucose. It will be, therefore, important to determine whether acetylation in the disease models is generally increased or if acetylation at specific sites is increased and whether FXR acetylated at different sites is recruited in a gene-selective manner.
In this study, we demonstrate for the first time that FXR acetylation is tightly and reciprocally regulated by p300 and SIRT1 and is critical for activation of FXR target genes in response to FXR signaling under normal conditions and further, this dynamic regulation of FXR acetylation is disrupted in metabolic disease model mice, resulting in constitutively elevated FXR acetylation. We propose that small molecules that inhibit FXR acetylation by increasing SIRT1 or decreasing p300 activity may be promising therapeutic agents for treatment of metabolic disorders, such as, fatty liver, diabetes, and obesity.