Distinct modulatory roles for thyroid hormone receptors TRα and TRβ in SREBP1-activated ABCD2 expression

Abstract

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone (T₃) receptor (TR)α and TRβ bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TRβ, but not TRα, represses ABCD2 induction independently of DNA binding. However, activation by TRα and derepression of TRβ are T₃-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T₃ and TRα levels) but downregulated in adults (with low T₃ and TRα but elevated TRβ levels). This temporal repression of Abcd2 is blunted in TRβ-deficient mice, and the response to manipulated T₃ states is abrogated in TRα-deficient mice. These findings show that TRα and TRβ differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.

Introduction

X-linked adrenoleukodystrophy (X-ALD; OMIM 300100) is a severe neurodegenerative disorder that is biochemically characterized by the accumulation of very long-chain fatty acids in tissues and body fluids (Moser et al., 2001). The molecular basis for X-ALD are mutations in the ABCD1 (ALD) gene, which encodes the peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy protein (ALDP), with currently unknown function (Berger and Gartner, 2006; Moser et al., 2001). To date, no satisfying treatment is available for most X-ALD patients. At the molecular level, the adrenoleukodystrophy-related protein (ALDRP), encoded by the ABCD2 gene, shows a high degree of conservation with ALDP, suggesting related or overlapping functions for ALDRP and ALDP. Indeed, upon overexpression, ALDRP can compensate for ALDP deficiency in X-ALD fibroblasts and in Abcd1-deficient mice (Netik et al., 1999; Pujol et al., 2004). This makes pharmacological induction of ABCD2 an attractive therapeutic approach for X-ALD (Kemp et al., 1998), but requires detailed knowledge about regulation of the ABCD2 gene.

ABCD2 transcription is recognized to be exceptionally complex. Next to induction by ligands of the nuclear peroxisome proliferators-activated receptor (PPAR) α and retinoid X receptor (RXR) α (Berger et al., 1999; Netik et al., 1999; Pujol et al., 2000), we have previously demonstrated that cholesterol regulates ABCD2 expression (Weinhofer et al., 2002, Weinhofer et al., 2005). This is mediated by activation of a class of transcription factors known as sterol regulatory element (SRE)-binding proteins (SREBPs), which are synthesized as membrane-bound precursors and cleaved upon induction to generate the active nuclear form. SREBPs are key regulators of cholesterol and lipid metabolism and occur as three different isoforms: SREBP1a and SREBP1c, produced from a single gene and preferentially regulating genes involved in fatty acid synthesis; and SREBP2, encoded by a separate gene and controlling expression of cholesterogenic genes (Horton et al., 2002).

Intriguingly, the SRE located in the ABCD2 promoter overlaps with a direct repeat separated by 4 nucleotides (DR-4), which serves as binding motif for nuclear receptors and suggests cross-talk between different transcription factors (Fig. 1). Indeed, we could demonstrate that the nuclear cholesterol sensor liver X receptor (LXR) α as a heterodimer with RXRα directly binds to the DR-4 sequence and that ligand-activated LXRα interferes with SREBP1-mediated activation of the Abcd2 promoter (Weinhofer et al., 2005). As SREBP1 and LXRα are reciprocal regulators of cholesterol metabolism but share a stimulatory effect on fatty acid synthesis, this setup could provide a mechanism for a more dynamic regulation of ABCD2 expression.

In addition to LXRα/RXRα heterodimers, also 3,5,3′-triiodothyronine (T₃) thyroid hormone receptor (TR) β homodimers or TRβ/RXRα heterodimers can bind the ABCD2 SRE/DR-4 sequence (Fig. 1), and induction of Abcd2 expression by T3 has been shown in cultured cells and in the liver of rodents (Fourcade et al., 2003). T₃ plays a major role in cholesterol and fatty acid metabolism by activating both TRα and TRβ isoforms, which are encoded by separate genes. Targeted mutations in mice have revealed that TRα and TRβ mediate distinct physiological effects. Whereas ablation of TRα resulted in cardiac and thermoregulatory defects, lack of TRβ led to auditory impairment and dysregulation of the pituitary–thyroid axis with increased T₃ levels (Forrest et al., 1996; Macchia et al., 2001). These distinct phenotypes seem to result from differences in expression patterns of TRα and TRβ and in the regulation of T₃ target genes (Cheng, 2005; Flamant and Samarut, 2003; Nishimura et al., 2004).

Next to activation of target genes, TRs also repress gene expression in the absence of T₃ through interaction with co-repressors that function as a platform for recruitment of subcomplexes containing histone deacetylase activity (Perissi and Rosenfeld, 2005). Moreover, also T₃-dependent inhibition of TR-regulated genes has been described, e.g. negative regulation of the hypothalamic thyrotropin-releasing hormone gene (Feng et al., 1994). The mechanism underlying this repression is poorly defined and is thought to occur by transrepression antagonizing the activities of other transcription factors (Nettles and Greene, 2005; Tagami et al., 1997).

In whole-body metabolism, SREBP1, TRα and TRβ are functionally linked through a complex transcriptional network. Many SREBP1c-regulated lipogenic genes are inducible by T₃, and a clear overrepresentation of putative SREs was observed in the promoters of T₃-regulated genes (Stahlberg et al., 2005). A direct interaction between SREBP1 and TRα while binding to adjacent SRE and DR-4 sites, respectively, is required for the T₃ response of acetyl-CoA carboxylase (Acc) gene expression (Yin et al., 2002). Furthermore, activation of the rat S14 gene by T₃ and TRs was found to be dependent on the presence of an SRE in the promoter region, and a functional interaction between SREBP1 and TRs was proposed also in this context (Jump et al., 2001). Based on these results, a possible role for SREBP1 in mediating tissue-specific effects of T₃ on gene transcription was suggested (Jump et al., 2001).

This raised the question whether the T₃ response of the rat Abcd2 gene observed by Fourcade et al. (2003) requires a functional interaction between SREBP1 and TRs. Thus, the aim of the present study was to determine the relevance of the overlapping SRE/DR-4 motif for activation of the human ABCD2 promoter by evaluating (i) how the presence of either unliganded or T₃-activated TRα or TRβ affects SREBP1-stimulated ABCD2 expression in cultured cells and (b) the integrated effect of SREBP1, TRα and TRβ function in vivo through analysis of Abcd2 expression in TRα- and TRβ-deficient mice.