Regulation of bile acid receptor activity☆.

Many receptors can be activated by bile acids (BAs) and their derivatives. These include nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR), as well as membrane receptors Takeda G protein receptor 5 (TGR5), sphingosine-1-phosphate receptor 2 (S1PR2), and cholinergic receptor muscarinic 2 (CHRM2). All of them are implicated in the development of metabolic and immunological diseases in response to endobiotic and xenobiotic exposure. Because epigenetic regulation is critical for organisms to adapt to constant environmental changes, this review article summarizes epigenetic regulation as well as post-transcriptional modification of bile acid receptors. In addition, the focus of this review is on the liver and digestive tract although these receptors may have effects on other organs. Those regulatory mechanisms are implicated in the disease process and critically important in uncovering innovative strategy for prevention and treatment of metabolic and immunological diseases.

Introduction

Upon catalysis by hepatic and bacterial enzymes, cholesterol converts into bile acids (BAs).[1,2] In addition, BAs have bacteriostatic effects. Thus, BAs are the intrinsic links that explain how foods, through gut microbiota, affect host metabolism and immunity. Hepatic enzymes generate free primary BAs such as chenodeoxycholic acid (CDCA) and cholic acid (CA). Hepatic conjugation of BAs increases the hydrophilicity of BAs and changes their binding affinity to their receptors. In the gut, bacterial enzyme, i.e., bile salt hydrolase deconjugates BAs. Moreover, bacterial enzyme 7α- dehydroxylase that can be found in Firmicutes converts primary BAs into secondary BAs such as deoxycholic acid (DCA) and lithocholic acid (LCA).[3,4] Therefore, host and bacteria jointly produce various BAs, and eubiosis is essential for maintaining BA homeostasis. In contrast, dysregulated BA synthesis accompanied by dysbiosis is implicated in the development of metabolic diseases including obesity, steatosis, steatohepatitis, as well as liver and colon cancer.[3,5-10]

Free and conjugated primary as well as secondary BAs have differential binding affinities to various receptors including nuclear farnesoid X receptor (FXR) as well as membrane Takeda G protein receptor 5 (TGR5), and sphingosine-1-phosphate receptor 2 (S1PR2). Additionally, pregnane X receptor (PXR), vitamin D receptor (VDR), constitutive androstane receptor (CAR), and cholin-ergic receptor muscarinic 2 (CHRM2) can be activated by BAs or their precursors and metabolites (Table 1). Thus, BA receptors are essentially endobiotic and xenobiotic sensors. For an organism to adapt to constant environmental change, epigenetic mechanism is used to regulate host response. Epigenetic effects such as acetylation and methylation are ways to switch genes on and off without changing deoxyribonucleic acid (DNA) sequence. Thus, as nutrient and chemical sensors, epigenetic mechanisms should be important for regulating the expression and activity of BA receptors. This review article summarizes epigenetic regulation and post-transcriptional modification of BA receptors. The information is critically important to understand how these receptors are activated or silenced, thereby leading to metabolic or detoxification function or dysfunction. We focus on FXR, TGR5, and S1PR2 since the information available for other receptors in this area is limited. The search was done using combinations of following keywords: FXR, G protein-coupled bile acid receptor, TGR5, S1PR2, acetylation, methylation, glycosylation, epigenetics, and bile acid in the PubMed.

Table 1

Bile acids and their derivatives as agonists for the listed receptors.

Receptors	Ligands	References
FXR	CDCA > DCA > LCA > CA > UDCA	[11,12]
	Bile alcohols, 6α-ethyl-CDCA	[13,14]
	5β-cholanoic acid, 5β-norcholanoic acid, 5α-cholanoic acid	[15]
TGR5	LCA > DCA > CDCA > CA > UDCA	[16]
	TLCA	[17]
S1PR2	Conjugated BAs (GCA, TCA, GDCA, TDCA, TUDCA)	[18]
PXR	3-keto-LCA, LCA, CDCA, DCA, CA	[19,20]
	7α-hydroxy-4-cholesten-3-one	[21]
VDR	LCA, 3-keto-LCA	[22]
CAR	CA, 6-keto-LCA, 12-keto-LCA	[23,24]
CHRM2	TCA	[25]

Note: Humans mainly make glycine conjugates of bile acids while mice make taurine conjugates.

Abbreviations: FXR, farnesoid X receptor; TGR5, Takeda G protein receptor 5; S1PR2, sphingosine-1-phosphate receptor 2; PXR, pregnane X receptor; VDR, vitamin D receptor; CAR, constitutive androstane receptor; CHRM2, cholinergic receptor muscarinic 2; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; CA, cholic acid; TLCA, taurolithocholic acid; UDCA, ursodeoxycholic acid; GCA, glycocholic acid; TCA, taurocholic acid; GDCA, glycodeoxycholic acid; TDCA, taurodeoxycholic acid; TUDCA, tauroursodeoxycholic acid.

FXR

FXR introduction

BAs regulate glucose and lipid metabolism as well as the inflammatory process. This paradigm shift was spurred by identification of the BA receptor FXR. The function of FXR has been extensively reviewed by recent articles.[1,26-34] We only provide a general introduction here. FXR activation plays a key role in regulating BA homeostasis in the liver and intestine.[3,5,35-39] The activation of FXR leads to the regulation of genes whose function is to decrease the concentrations of BAs. FXR increases the expression of hepatic small heterodimer partner (SHP) and intestinal fibroblast growth factor 15 (FGF15), which in turn inhibits hepatic cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1), reducing BA synthesis. In addition, FXR activation increases the expression of canalicular transporters, such as the bile-salt export pump (BSEP), providing a pathway for excreting cholesterol and BAs. These regulatory pathways are important in part because accumulation of hydrophobic BAs leads to inflammation, injury, cirrhosis, and carcinogenesis.[40,41] In contrast to the dysregulated BA synthesis found in metabolic disease patients, activation of FXR increases metabolism and insulin sensitivity, and FXR agonists are used to treat non-alcoholic steatohepatitis.[42] In consistency, whole-body FXR knockout mice, which have dysregulated BA synthesis and dysbiosis, spontaneously develop non-alcoholic steatohepatitis and liver cancer.[43-47] Moreover, reduced FXR is found in patients who have cirrhosis and colon or liver cancer as well as ulcerative colitis.[48-51] Thus, it is important to understand the mechanism by which FXR is regulated. Taken together, FXR, which is mainly expressed in the liver and intestine, has a pivotal role in regulating BAs homeostasis leading to metabolic and anti-inflammatory beneficial outcomes (Fig. 1).

Fig. 1

Schematic overview of the functions of bile acid receptors.

The key functions of bile acid receptors are summarized in the figure. Abbreviations: FXR, farnesoid X receptor; PXR, pregnane X receptor; VDR, vitamin D receptor; CAR, constitutive androstane receptor; TGR5, Takeda G protein receptor 5; S1PR2, sphingosine-1-phosphate receptor 2; CHRM2, cholinergic receptor muscarinic 2.

Methylation regulates FXR expression and activity

Methylation is a mechanism that affects FXR activity. Silencing the FXR gene through CpG methylation is found in mouse models of colon cancer in adenomatous polyposis coli mutant mice, human colon cancer cells, and human colon cancers.[51-53] By direct-sequence analyses of bisulfonated genomic DNA, there are 13 CpG methylation sites located in the region flanking the transcription start site on exon-3 of the FXR.[52] In addition, methylation of the FXR is implicated in pregnancy related diseases. The intrahepatic cholestasis of pregnancy is a liver disorder that involves the inter-play between dysregulated BA synthesis, sex hormones, genetic susceptibility, as well as environmental factors. There is a clear relationship between the status of methylation in the FXR promoter and the profile of BAs in intrahepatic cholestasis of pregnancy patients compared with healthy pregnant women.[54] Specifically, reduced methylation of the FXR promoter is found in intrahepatic cholestasis of pregnancy cases when compared with healthy pregnancy controls. In addition, increased methylation level at the distal promoter (–1890) is positively correlated with elevated conjugated BAs; whereas methylation level at the proximal promoter (–358) is negatively correlated with serum CA and DCA concentration.[54] Methylation of the FXR gene is also implicated in another pregnancy related disease, i.e., preeclampsia. Altered methylation pattern of the FXR as well as liver X receptor (LXR) is found in early onset of preeclampsia based on genome-wide methylation study using cord blood DNAs.[55] These findings implicate the potential role of BAs and FXR in immunological disorders.

At the histone level, methylation of H3 and H4 occurs on lysine or arginine and is catalyzed by histone methyltransferases that use S-adenosylmethionine as a methyl donor. It has been shown that methylation by Set7/9, a lysine methyltransferase, increases FXR binding to its target gene leading to increased transcriptional activity.[56] In addition, FXR activity incorporates histone methyl-transferase activity within the BSEP gene locus.[57] This methyltransferase activity is directed specifically to arginine 17 of H3. By interacting with arginine methyl-transferase type I, the transcriptional activity of FXR is activated, thereby leading to increased expression of SHP and BSEP and decreased CYP7A1.[58] Moreover, 5′-deoxy-5′-methylthioadenosine, a methylation inhibitor, reduces the expression of BSEP.[58] In consistency, reduced recruitment of H3K4me3 to the BSEP and multidrug resistance-associated protein 2 (Mrp2) promoter of the FXR-binding elements was found in mouse livers after bile duct ligation.[59] Thus, histone 3 lysine 4 trimethylation (H3K4me3) is essential to increase the transcription of the BSEP, sodium-taurocholate cotransporting polypeptide (NTCP), and Mrp2 genes that are controlled by FXR.

Acetylation and FXR activity

The transcriptional activity of FXR can be modulated by sirtuin1 (SIRT1), a protein deacetylase. SIRT1 activity is dependent on nicotinamide adenine dinucleotide (NAD+) levels. Hepatic over-expression of microRNA (miR)-34a, which reduces nicotinamide phosphoribosyltransferase and NAD+ levels, decreases SIRT1 leading to reduced transcriptional activity of FXR.[60] It has been shown that the FXR acetylation site targeted by SIRT1 deacetylase and p300 acetylase is at lysine 217.[61] Acetylated FXR has increased stability, but reduced capability to dimerize with retinoid X receptor α (RXRα), thereby leading to decreased transcriptional activity. In mouse models of metabolic disease, FXR acetylation level is elevated. Therefore, potentially, inhibiting FXR acetylation by increasing SIRT1 or reducing p300 can be used to treat metabolic disorders.[61] Moreover, FXR acetylation increased pro-inflammatory gene expression, macrophage infiltration, and hepatic cytokine and triglyceride levels. Mechanistically, acetylated FXR prevented small ubiquitin-like modifier 2 (SUMO2) modification. SUMOylation of activated FXR increased its interaction with nuclear factor k B (NF-kB), but reduced the dimerization with RXRα.[62] Taken together, SIRT1 modulates the FXR signaling pathway by directly deacetylating FXR. Another mechanism by which SIRT1 regulates FXR transcriptional activity is through hepatocyte nuclear factor 1a (HNF1α). Knockout hepatic SIRT1 reduces FXR activity is mainly due to reduced occupancy of HNF1α in the FXR promoter leading to decreased FXR expression.[63]

The role of SIRT1 in regulating proliferation mediated by FXR is also revealed using a partial hepatectomy model. SIRT1 transgenic mice have increased mortality, impairs hepatocyte proliferation, and BA accumulation after partial hepatectomy. This is in part due to persistent deacetylation and reduced FXR expression. In contrast, 24-nor-ursodeoxycholic acid increases miR-34a and reduces SIRT protein, resulting in increased acetylation of FXR and neighboring histones. Thus, 24-nor-ursodeoxycholic acid is able to establish BA homeostasis and restores liver regeneration capability in SIRT1 transgenic mice.[64] Moreover, inversed expression of SIRT1 and FXR is also found in liver cancer; human hepatocellular carcinoma has increased SIRT1 and reduced FXR compared with normal liver.[64]

At the histone level, the occupancy of FXR and co-activator-associated arginine methyltransferase 1 on the human BSEP locus is associated with increased Arg-17 methylation and Lys-9 acetylation of H3 of the BSEP.[57] Moreover, by acetylating histones at the promoter and FXR itself, p300 acetylase is a coactivator of FXR to increase the expression of SHP.[65] Taken together, acetylation and deacetylation of FXR should be a dynamic process to maintain FXR activity. Sustained FXR activation and deactivation lead to metabolic imbalance impaired liver regeneration, and potentially carcinogenesis.

Other mechanisms affecting FXR activity

O-GlcNAc transferase, responsible for O-GlcNAcylation, is a nutrient sensor that links glucose and the hexosamine biosynthetic pathway to the regulation of transcriptional factors that regulate energy homeostasis. By interacting FXR, hepatic carbohydrate response element-binding protein (ChREBP) can regulate glycolytic and lipogenic gene expression. It is interesting to note that FXR as well as ChREBP are both O-GlcNAcylated in response to glucose. High glucose increases FXR O-GlcNAcylation and enhances its stability as well as transcriptional activity. Moreover, in vivo fasting and refeeding experiments show that FXR undergoes O-GlcNAcylation in the fed condition, which is associated with increased expression of FXR target gene.[66]

MiRNA regulation of protein deacetylases may indirectly affect FXR activity. MiR-34a is one example mentioned above. It is interesting to note that FXR-activation induced miR-22 can also silence SIRT1, which in turn affects FXR stability or transcriptional activation. Such pathway potentially forms a self-regulatory loop.[67,68]

TGR5

Function of TGR5

In contrast to nuclear receptor FXR, TGR5 is a membrane receptor ubiquitously expressed in adipocytes, endocrine glands, muscles, as well as immune organs.[69] It is also known as G protein-coupled bile acid receptor 1 (GPBAR1) or G-protein coupled receptor 19 (GPCR19). TGR5 is also expressed in the gut, liver, and gallbladder, where BAs are produced and stored.[70,71] Because TGR5 is expressed in cells of the hematopoietic system, such as monocytes and macrophages, it confers a potent anti-inflammatory property at the systemic level.[17,72-76] Our recent publication revealed the potential role of TGR5 in neuro-inflammation as well as neuroplasticity.[77] Activation of TGR5 also increases intracellular cyclic adenosine monophosphate (cAMP), thereby activating cAMP response element binding protein. One of the downstream effects of cAMP production is to induce the expression of thyroid hormone deiodinase 2, which generates thyroxine, a key player in basal metabolism.[78] In addition to metabolism and inflammation, TGR5 also regulates proliferation, muscle relaxation, and itchiness among many others, which have been reviewed in recent articles (Fig. 1).[69,79-82]

In the liver, although TGR5 is not expressed in hepatocytes, it is found in Kupffer cells and endothelial and biliary epithelial cells, which are involved in regulating immune, inflammatory signaling, and circulation.[83] In the intestine, TGR5 activation induces the expression of the preproglucagon gene (Gcg) and glucagon-like peptide-1 (GLP-1) secretion in the intestinal enteroendocrine L-cells.[84,85] GLP-1 is an incretin that potentiates postprandial insulin secretion.[86] Activation of TGR5 also releases neuropeptide hormone peptide tyrosine tyrosine (PYY), which regulates immune signaling and intestinal mobility.[87]

Regarding the ligands, unconjugated BAs such as CDCA, DCA, and ursodeoxycholic acid (UDCA) induce a large cAMP response in neonatal mouse cardiomyocytes.[88] Secondary BAs such as LCA and taurine-conjugated LCA are also endogenous ligands for TGR5 (Table 1).[16,17] In addition, TGR5 potentially can be activated by many other chemicals. Those include allogregnanolone, betulinic acid, linolenic acid, etc.[89-91] We recently showed that supplementation of Western diet-fed mice with epigallocatechin-3-gallate activates TGR5 signaling pathways leading to a lean phenotype.[10] Whether the effect is mediated via epigenetic regulation of TGR5 remains to be investigated.

Regulation of TGR5

FXR induces the expression of the TGR5 gene in mouse intestine. An inverted repeat with one-nucleotide spacing (IR1) that can be occupied by FXR/RXRα has been uncovered in the proximal promoter of the human TGR5 gene.[92] FXR and TGR5 are co-expressed in the enteroendocrine L cells, and activation of FXR induces TGR5 to stimulate the secretion of GLP-1.[93] Because the expression of the TGR5 gene is transcriptionally regulated by FXR, methylation and acetylation likely influence TGR5 activity, which remains to be proved.

It is interesting to note that INT-777-mediated TGR5 activation induces renal expression of SIRT1 and SIRT3. Increased SIRT3 activity induces acetylation of mitochondrial superoxide dismutase 2 and isocitrate dehydrogenase 2 found in db/db mice.[94] Since acetylation has a known role in regulating FXR expression and activity, it is possible TGR5-induced SIRT expression may have an impact on FXR activity as well. The hypothesis that TGR5 and FXR may mutually regulate each other warrants further investigation.

Methylation has a role in TGR5 expression. Methylation status of the TGR5 promoter has been studied in peripheral mononuclear cells in patients with acute-on-chronic hepatitis B liver failure. The frequency of TGR5 promoter methylation is significantly higher in liver failure patients than chronic hepatitis patients. In addition, hyper-methylation is accompanied by reduced TGR5 mRNA level.[95] This study concludes that aberrant TGR5 promoter methylation is a potential prognostic marker for acute-on-chronic hepatitis B liver failure. Hyper-methylation of the TGR5 promoter is also found in hepatocellular carcinoma patients by studying their circulating cell-free DNA. Moreover, the methylation rate of TGR5 is age-related, much higher in patients older than 60 than in those younger than 60 years old. It has been suggested that a combination of serum TGR5 promoter methylation level along with the value of a-fetoprotein may increase the sensitivity for hepatocellular carcinoma diagnosis.[96]

S1PR2

S1PR2 or S1P2 is a G protein-coupled receptor for sphingosine-1-phosphate (S1P). S1PR2 was also found to be the receptor for conjugated BAs such as taurocholic acid (TCA) and taurodeoxycholic acid (TDCA) (Table 1).[18,97] S1P is a bioactive lipid mediator that regulates proliferation, immunity, cell trafficking, inflammation, etc.[98,99] TCA-activated S1PR2 induces the expression and activity of sphingosine kinase (SphK2) to increase the conversion of sphingosine into S1P and leading to increased lipid and sterol metabolism in the liver.[18] Thus, conjugated BAs have a pivotal role in S1P singling via SphK2 induction as well as S1PR2. Consistent with these findings, both SphK2 and S1PR2 knockout mice are susceptible to diet-induced fatty liver.[97,100]

It is interesting to note that nuclear S1P, produced by either induction of SphK2 or inhibition of S1Plyase, binds to histone deacetylases (HDAC) 1 and 2, thereby increasing histone acetylation and up-regulating the expression of metabolic genes.[100] Through such HDAC inhibitory mechanism, sphingosine has a role in regulating apoptosis and metabolism. Furthermore, glycochenodeoxycholate (GCDC) via S1PR2 as well as cell entrance have an apoptotic effect in human liver cancer Huh7 cells.[101] These results suggested that S1PR2 activation has a pro-apoptotic effect in GCDC-treated liver cancer cells, but the effect is not simply due to just binding between the GCDC and S1PR2.

Conjugated BAs via S1PR2 also activate ERK1/2- and AKT- signaling pathways leading to the growth and invasion of cholangiocarcinoma cells.[102] The role of conjugated BAs via S1PR2 to regulate apoptosis or cancer progression remains to be dissected. Nevertheless, there is no doubt that the composition of free and conjugated BAs has an impact on regulating BA receptor activity.

Conclusions and perspectives

BA receptors can be found in many types of cells within and outside the digestive tract. By activating G protein-coupled membrane receptors, i.e., TGR5, S1PR2, and muscarinic receptor, BAs exert their effects without crossing the cell membrane. Similarly, those receptors are readily accessible to enzymes that regulate methylation, acetylation, glycosylation, etc. Thus, in addition to transcriptional regulation, it is important to study post–transcriptional modification of those receptors. It is likely that due to transcriptional and post-transcriptional modification, those receptors exert various biological effects ranging across metabolism, energy homeostasis in skeletal muscle and adipose tissue, inflammatory signaling in macrophages, muscle relaxation, hormonal secretion, as well as cell proliferation and apoptosis, etc. The current knowledge limits to acetylation and methylation of a few receptors. More research should be done to understand the mechanism that influences their expression, modification, and biological effects.