NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600

doi:10.1016/j.bbrc.2012.01.102

Biochemical and Biophysical Research Communications

Volume 418, Issue 4, 24 February 2012, Pages 780-785

https://doi.org/10.1016/j.bbrc.2012.01.102 Get rights and content

Abstract

Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and can be activated by 9-cis retinoic acid (9CRA). RXRs form homodimers and heterodimers with other nuclear receptors such as the retinoic acid receptor and NR4 subfamily nuclear receptors, Nur77 and NURR1. Potential physiological roles of the Nur77-RXR and NURR1-RXR heterodimers have not been elucidated. In this study, we identified a gene regulated by these heterodimers utilizing HX600, a selective RXR agonist for Nur77-RXR and NURR1-RXR. While 9CRA induced many genes, including RAR-target genes, HX600 effectively induced only carnitine palmitoyltransferase 1A (CPT1A) in human teratocarcinoma NT2/D1 cells, which express RXRα, Nur77 and NURR1. HX600 also increased CPT1A expression in human embryonic kidney (HEK) 293 cells and hepatocyte-derived HepG2 cells. Although HX600 induced CPT1A less effectively than 9CRA, overexpression of Nur77 or NURR1 increased the HX600 response to levels similar to 9CRA in NT2/D1 and HEK293 cells. A dominant-negative form of Nur77 or NURR1 repressed the induction of CPT1A by HX600. A protein synthesis inhibitor did not alter HX600-dependent CPT1A induction. Thus, the rexinoid HX600 directly induces expression of CPT1A through a Nur77 or NURR1-mediated mechanism. CPT1A, a gene involved in fatty acid β-oxidation, could be a target of RXR-NR4 receptor heterodimers.

Highlights

► The function of RXR heterodimers with NR4 receptors remains unknown. ► The RXR ligand HX600 induces expression of carnitine palmitoyltransferase 1A (CPT1A). ► HX600-induced CPT1A expression is mediated by the NR4 receptors, Nur77 and NURR1. ► CPT1A induction by HX600 is not mediated by de novo protein synthesis. ► CPT1A could be a target of the Nur77-RXR and NURR1-RXR heterodimers.

Introduction

Retinoid X receptor α (RXRα; NR2B1), RXRβ (NR2B2), and RXRγ (NR2B3) are receptors for 9-cis retinoic acid (9CRA) and regulate many biological processes by forming heterodimers with other members of the nuclear receptor superfamily, including retinoic acid receptor (RAR; NR1B), vitamin D receptor (NR1I1), thyroid hormone receptor (NR1A), peroxisome proliferator-activated receptor (PPAR; NR1C), liver X receptor (LXR; NR1H2/3), and farnesoid X receptor (NR1H4) [1], [2]. Heterodimers composed of RXR and permissive partners, such as PPAR, LXR and farnesoid X receptor, can be activated by agonists for both RXR and the partner receptor [2], [3]. In contrast, RXR heterodimers with nonpermissive partners, such as vitamin D receptor and thyroid hormone receptor, can be activated only by the partner receptor’s agonist but not by an RXR agonist. Although several lines of evidence indicate that RXR plays an important role in multiple nuclear receptor functions, an understanding of RXR ligand signaling in RXR heterodimers is only recently emerging [2], [3].

The orphan nuclear receptors growth factor-inducible immediate early gene nur/77-like receptor (Nur77; NR4A1, also known as NGFI-B and TR3) and Nur-related protein 1 (NURR1; NR4A2) belong to the NR4A nuclear receptor subfamily, which also includes neuron-derived orphan receptor 1 (NOR1; NR4A3) and the Drosophila ortholog hormone receptor-like 38 (NR4A4) [4], [5]. Structural studies reveal that the NR4A subfamily receptors lack a cavity for ligand binding due to the presence of bulky hydrophobic amino acid residue side chains [6], [7], [8]. NR4A receptors bind to a specific sequence as monomers or homodimers and promote constitutive activation of transactivation [4]. The activity of NR4A receptors is determined by their expression level, subcellular localization, and posttranslational modifications [4], [9]. Nur77 and NURR1, but not NOR1, can heterodimerize with RXR. RXR ligands can induce transactivation of Nur77-RXR and NURR1-RXR heterodimers [3], [10], indicating that Nur77 and NURR1 are permissive heterodimerization partners for RXR signaling. In addition to transcriptional regulation, Nur77 plays a role in apoptosis by interacting with Bcl-2 in mitochondria [11]. While unliganded RXR is required for export of Nur77 from nuclei to mitochondria, RXR ligand suppresses nuclear export of the Nur77-RXR heterodimer and inhibits apoptosis [12]. These findings suggest that RXR ligand regulates the transactivation of Nur77- and NURR1-containing RXR heterodimers on specific target genes. Although NR4A response elements have been identified in several genes, including proopiomelanocortin and tyrosine hydroxylase, which are targets for monomers, homodimers, or heterodimers of NR4A receptors [13], endogenous target genes of the Nur77-RXR and NURR1-RXR heterodimers have not been established.

We previously reported that the RXR heterodimers of Nur77 and NURR1 are selectively activated by the dibenzodiazepine RXR ligand HX600 [14]. In contrast to 9CRA, which can activate permissive RXR heterodimers, HX600 induces activation of Nur77-RXR and NURR1-RXR as well as RXR homodimers, but not RXR heterodimers with LXR, farnesoid X receptor or PPARγ [14]. In this study, we identified carnitine palmitoyltransferase 1A (CPT1A) as an HX600-inducible gene. Induction of CPT1A by HX600 was enhanced by overexpression of Nur77 or NURR1 and suppressed by cotransfection of a dominant-negative form of Nur77 or NURR1. Thus, CPT1A could be a target of RXR-NR4A receptor heterodimers.

Section snippets

Chemicals

HX600 (Fig. 1A) was synthesized as reported previously [15]. All-trans retinoic acid (ATRA) and 9CRA were purchased from Wako Pure Chemical Industries (Osaka, Japan), and cycloheximide was from Nacalai Tesque (Kyoto, Japan).

Cell lines and cell cultures

Human embryonic kidney (HEK) 293 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. Human

Induction of mRNA expression of CPT1A by the rexinoids 9CRA and HX600

Nur77 was originally identified as an immediate-early gene that is rapidly induced by serum and growth factors such as nerve growth factor in neuron-derived cell lines [18]. NURR1 is necessary for the development of dopaminergic neurons [19]. Nur77 and NURR1 are highly expressed in the adult brain [20] as well as in neural cell lines, including NTera2-N cells [21]. We detected mRNA expression of Nur77 and NURR1 as well as RXRα and NOR1 in NT2/D1 cells, a cell line derived from NTera2 cells (

Discussion

While 9CRA induced expression of RARβ, CRABP2, CPT1A, LEFTY1, LEFTY2, DHRS3 and ABCA1 in NT2/D1 cells, CPT1A was the sole HX600-responsive gene (Fig. 1, Fig. 2). A RAR-binding element has been identified in the promoter of the mouse Lefty1 gene [28], and ATRA treatment increases expression of LEFTY1 and LEFTY2 mRNA in mouse embryos [29]. The RAR-RXR heterodimer binds to a direct-repeat-type element in the mouse Dhrs3 gene [30]. ATRA and 9CRA have been suggested to induce expression of LEFTY1,

Acknowledgments

The authors thank members of the Makishima lab for technical assistance and helpful comments and Dr. Andrew I. Shulman for editorial assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant-in-Aid for Scientific Research on Priority Areas (No. 18077995)), and Research Foundation Istuu Laboratory.

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NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600

Abstract

Highlights

Introduction

Section snippets

Chemicals

Cell lines and cell cultures

Induction of mRNA expression of CPT1A by the rexinoids 9CRA and HX600

Discussion

Acknowledgments

Cell

Structure

Cell

J. Biol. Chem.

Cell

Biochem. Pharmacol.

J. Lipid Res.

Biochem. Pharmacol.

Neuron

Eur. J. Cancer

Mol. Aspects Med.

J. Biol. Chem.

Dev. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Retinoid x receptor heterodimers in the metabolic syndrome

N. Engl. J. Med.

The retinoid X receptors and their ligands

Biochim. Biophys. Acta

Minireview: nuclear hormone receptor 4A signaling: implications for metabolic disease

Mol. Endocrinol.

Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors

Nature