Farnesoid X receptor directly regulates xenobiotic detoxification genes in the long-lived Little mice

https://doi.org/10.1016/j.mad.2013.08.003Get rights and content

Highlights

  • Expression and activity of FXR is increased in Little mice.

  • Growth hormone treatment reversed alteration of FXR expression in Little mice.

  • Xenobiotic detoxification genes are direct transcriptional targets of FXR.

Abstract

Activation of xenobiotic metabolism pathways has been linked to lifespan extension in different models of aging. However, the mechanisms underlying activation of xenobiotic genes remain largely unknown. Here we showed that although farnesoid X receptor (FXR, Nr1h4) mRNA levels do not change significantly, FXR protein levels are elevated in the livers of the long-lived Little mice, leading to increased DNA binding activity of FXR. Hepatic FXR expression is sex-dependent in wild-type mice but not in Little mice, implying that up-regulation of FXR might be dependent on the reduction of growth hormone in Little mice. Growth hormone treatment decreased hepatic expression of FXR and xenobiotic genes Abcb1a, Fmo3 and Gsta2 in both wild-type and Little mice, suggesting an association between FXR and xenobiotic gene expression. We found that Abcb1a is transactivated by FXR via direct binding of FXR/retinoid X receptor α (RXRα) heterodimer to a response element at the proximal promoter. FXR also positively controls Fmo3 and Gsta2 expression through direct interaction with the response elements in these genes. Our study demonstrates that xenobiotic genes are direct transcriptional targets of FXR and suggests that FXR signaling may play a critical role in the lifespan extension observed in Little mice.

Introduction

Little mice (Ghrhrlit/lit) are a very useful mammalian model for studying aging because of their retarded aging and extended lifespan. These mice have a mutation in the gene Ghrhr (growth hormone-releasing hormone receptor) and correspondingly have very low levels of circulating growth hormone (GH) and insulin-like growth factor1 (IGF1) (Donahue and Beamer, 1993, Godfrey et al., 1993). The GH/IGF1 pathway has been associated with lifespan extension in several species including C. elegans, Drosophila melanogaster, and Mus muscus and is the focus of several studies to understand the beneficial aspects of this pathway on longevity (Berryman et al., 2008). Our previous studies have proposed that alterations in xenobiotic metabolism and increased xenobiotic resistance may contribute to the longevity in Little mice (Amador-Noguez et al., 2004, Amador-Noguez et al., 2007). Genetic studies showed that the up-regulation of xenobiotic detoxification genes is likely to be mediated by the nuclear receptor FXR (Amador-Noguez et al., 2007). Levels of primary bile acids, the endogenous ligands for FXR, are elevated in Little mice and treatment of wild-type mice with cholic acid mimics the up-regulation of xenobiotic detoxification genes observed in Little mice (Amador-Noguez et al., 2007). We further found that knockout of FXR in Little mice reverses or decreases the up-regulation of these genes (Amador-Noguez et al., 2007). However, the mechanism(s) by which FXR regulates these genes remained unclear.

FXR is a member of the nuclear receptor superfamily and is expressed in liver, small intestine, kidney, adrenals, adipose tissue and vascular smooth muscle (Calkin and Tontonoz, 2012, Modica et al., 2010, Wang et al., 2008). FXR has been shown to control expression of various genes in bile acid, lipid, and glucose metabolism (Modica et al., 2010). Upon activation by its natural ligands, such as bile acids and their metabolites, or synthetic agonists including GW4064, FXR regulates the expression of its target genes by binding either as a monomer or as a heterodimer with RXRα to FXR response elements (FXREs) (Calkin and Tontonoz, 2012, Modica et al., 2010, Wang et al., 2008). The typical FXRE is an inverted repeat of the AGGTCA half-site spaced by 1 nucleotide (IR1). Other FXREs include direct repeat (DR), everted repeat (ER) and monomeric binding sites (Modica et al., 2010, Wang et al., 2008). In addition to regulation of target genes via binding to FXREs, FXR represses a group of genes indirectly via the FXR/SHP (small heterodimer partner) pathway (Calkin and Tontonoz, 2012, Goodwin et al., 2000, Li et al., 2005, Lu et al., 2000). Recently, several coactivators of FXR, including PGC-1α, SRC-1, Brg-1, CARM1, PRMT1, GPS2, DRIP205 and TRRAP, have been reported to interact with FXR and enhance FXR-mediated transactivation of different target genes (Ananthanarayanan et al., 2004, Kemper, 2011, Miao et al., 2009, Pineda Torra et al., 2004, Rizzo et al., 2005, Sanyal et al., 2007, Unno et al., 2005, Wang et al., 2006, Zhang et al., 2004), while Ku proteins are identified as FXR corepressors (Ohno et al., 2009).

Our previous study has shown that the loss of FXR, rather than the classic xenobiotic receptors Car (Constitutive Androstane receptor) and Pxr (Pregnane X receptor), had a major influence on the up-regulation of xenobiotic detoxification genes in Little mice (Amador-Noguez et al., 2007). The up-regulation of Abcb1a, Aldh1a1, Cyp2b10, Cyp2c38, Cyp4a10, Fmo3, Gsto2, Gstt2, Papp2s, Por, Sult1d1, Temt, and Ugt1a1 was abolished in the Fxr−/−/Little double deficient mice (Amador-Noguez et al., 2007). Knockout of FXR also reduced the magnitude of the up-regulation of Cyp2b13, Cyp2b9, Cyp4a14 and mOat6 (Amador-Noguez et al., 2007). Interestingly, several genes, including Gsta2, Gstm2, Gstm3, Mt1, and Sult1e1, were more strongly up-regulated in the Fxr−/−/Little mice than in Little mice (Amador-Noguez et al., 2007). The majority of these genes are not known targets of FXR and whether they are directly regulated by FXR is not clear. A recent ChIP-seq study analyzed genome-wide FXR binding in mice and revealed potential FXR binding sites in several of these xenobiotic detoxification genes up-regulated in Little mice (Thomas et al., 2010). Therefore, we hypothesize that these genes might be directly regulated by FXR via binding to these FXREs. In this study, we found that FXR protein levels are elevated in the livers of Little mice of different ages. We identified novel FXREs in three xenobiotic detoxification genes belonging to different phases, Fmo3 (phase 1), Gsta2 (phase 2), and Abcb1a (phase 3), and revealed that FXR activates these genes via direct binding to these regulatory elements as a heterodimer with RXRα. EMSA and ChIP experiments showed that FXR binding is increased in Little mice compared to that in wild-type mice. On the other hand, growth hormone treatment reduced the expression levels of FXR and several xenobiotic detoxification genes in Little mice. This study elucidated the mechanism by which FXR: RXRα heterodimer regulates xenobiotic detoxification genes in Little mice and suggested that regulation of FXR by the GH/IGF1 pathway may play a critical role in the lifespan extension observed in this mouse strain.

Section snippets

Antibodies and reagents

Antibodies to FXR (H-130 and C-20), Lamin A (H-102) and RXRα (D-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal FXR antibody (clone A9033A) was purchased from Perseus Proteomics (Tokyo, Japan). Anti-p-glycoprotein (C219) antibody was purchased from Calbiochem (San Diego, CA). Mouse monoclonal β-actin antibody, chenodeoxycholic acid, GW4064 and LG100268 were purchased from Sigma–Aldrich (St. Louis, MI). Recombinant murine growth hormone was purchased from Dr.

Levels of FXR protein are elevated in the livers of Little mice and growth hormone treatment reduces the expression levels of FXR and xenobiotic detoxification genes

Because crossing Little mice to FXR knockout mice corrected the expression levels of xenobiotic detoxification genes (Amador-Noguez et al., 2007), we have performed a careful analysis of the expression of FXR in Little mice. We found that FXR protein was increased in the livers of both male and female Little mice of different ages (Fig. 1A and Fig. S1A). However, we did not observe a significant change of the levels of FXR mRNA between wild-type and Little mice (Fig. 1B), whereas the mRNA

Discussion

Our previous studies showed a concerted up-regulation of xenobiotic detoxification genes in the long-lived Little mice (Amador-Noguez et al., 2004, Amador-Noguez et al., 2007). The elevation of xenobiotic metabolism pathways has also been observed in calorically restricted mice, C. elegans and the fruit fly with extended lifespan (McElwee et al., 2004, Pletcher et al., 2002, Swindell, 2007). A recent study reported that rapamycin treatment caused transcriptional activation of xenobiotic genes

Acknowledgements

This work was supported by National Institute of Health grants AG028865 (to G.J.D.) and GM055188, CA100070, AG039885, CA159942 (to N.A.T.).

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