Biochemical and Biophysical Research Communications
NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600
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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