NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

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Abstract

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

Introduction

NRs comprise a family of transcription factors that have critical functions during development and in adult physiology. NRs include steroid hormone receptors and receptors for other lipophilic ligands such as vitamin D3 and retinoids [1]. Several members of the NR family lack identified ligands and are referred to as orphan receptors [2]. Nurr1 is an orphan receptor, which together with NGFI-B and Nor1, constitutes the NR4A orphan NRs [3]. Nurr1 is expressed within the embryonic central nervous system where it plays a key role for the development of dopamine neurons [4] and continues to be expressed in the adult brain where it may be critical for dopamine neuron survival and other functions [5], [6]. Nurr1, NGFI-B and Nor1 are unique within the NR family in being encoded by immediate early genes that are rapidly induced by various stimuli such as growth factors, ischemia and seizures [7].

The structural features of the Nurr1 and DHR38 (NR4A-homologue in Drosophila) ligand binding domain show that the NR4A family members lack a hydrophobic pocket for ligand binding and thus function as ligand-independent NRs [8], [9]. NR4A proteins bind to DNA either as monomers or homodimers and promote constitutive activation of transcription [10], [11]. In addition, Nurr1 and NGFI-B, but not Nor1, can form heterodimers with the retinoid X receptor (RXR), which have the ability to promote strong transcriptional activation after binding to RXR ligands like 9-cis-retinoic acid [12] or fatty acids such as docosahexaenoic acid [13].

In this study we identify fatty acid binding protein 5 (FABP5) as a Nurr1-regulated gene. Fatty acid binding proteins are cytosolic proteins that bind long-chain fatty acids [14]. FABP5 is expressed in a variety of tissues [15] and has been implicated in regulation of water permeability barrier of the skin, neurite outgrowth [16] and fatty acid transport during neuronal regeneration [17], [18]. Retinoic acid binding to FABP5 leads to its translocation to the nucleus and enhancement of retinoic acid-induced activation of PPARβ/δ, which in turn can promote cellular survival [19]. DHA, a ligand for RXR, has also been shown to bind to FABP5 [20]. Since Nurr1 is indirectly linked to retinoid and DHA-signaling via its ability to form heterodimers with RXR we were interested to further investigate if Nurr1-mediated regulation of FABP5 could influence cross-regulation between Nurr1 and other NR-mediated signaling pathways. Our experiments indicate that Nurr1 can regulate FABP5 expression via direct binding to the FABP5 promoter and we also provide evidence indicating that Nurr1 can modulate RXR signaling via this regulation. Thus, FABP5 could be a regulated target of Nurr1 and the two other NR4A NRs in an immediate early context and thereby influence signaling by ligands binding to RXR.

Section snippets

Materials and methods

Chemicals. Retinoic acid and DHA (cis-4,7,10,13,16,19-docosahexanoic acid, C22:6) were purchased from Sigma–Aldrich. LG268 was kindly provided by Mark Leibowitz at Ligand Pharmaceuticals.

siRNA. A control siRNA and a pool of three siRNAs designed to knock down human FABP5 expression were purchased from Santa Cruz Biotechnology. Cells were transfected in 24-well plates with 20 pM siRNA per well using Lipofectamine 2000.

Plasmids. pCMX-Nurr1, -Nor1, -NGFI-B, -Nurr1R334A and -Nurr1dim have been

Transcriptional regulation of the FABP5 gene in HEK293 cells

To identify potential Nurr1-regulated genes we searched the human promoters included in a mammalian promoter database (MpromDb) [25] for Nurr1-binding sites situated within 1 kb 5′ the transcriptional start sites. One of the hits scored positive for Nurr1-binding was FABP5. By transfection in HEK293 cells FABP5 could be verified as a Nurr1-regulated gene since FABP5 mRNA expression, was up-regulated in cells transfected with a Nurr1 expression vector (Fig. 1A).

Next, we analyzed if the related

Discussion

Our results show that FABP5 mRNA and protein expression are up-regulated in Nurr1-over-expressing HEK293 cells. Nurr1 also regulates the FABP5 promoter (including 1133 bp of the FABP5 upstream sequence) in reporter assays, but the activation of the promoter by Nurr1 is relatively weak since the FABP5 promoter already has a basal activity in HEK293 cells, emphasized by detection of basal FABP5 mRNA and protein levels (Fig. 1C).

We have shown that Nurr1 up-regulates FABP5 expression in vitro and

Acknowledgments

N.V. was supported by a scholarship from the Alexander S Onassis Public Benefit Foundation. The pSG5-PPARβ/δ was a kind gift from Dr. Beatrice Desvergne, Lausanne University, Switzerland.

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