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 (T(3)) receptor (TR)alpha and TRbeta bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TRbeta, but not TRalpha, represses ABCD2 induction independently of DNA binding. However, activation by TRalpha and derepression of TRbeta are T(3)-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 T(3) and TRalpha levels) but downregulated in adults (with low T(3) and TRalpha but elevated TRbeta levels). This temporal repression of Abcd2 is blunted in TRbeta-deficient mice, and the response to manipulated T(3) states is abrogated in TRalpha-deficient mice. These findings show that TRalpha and TRbeta 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.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Genes, erbA/physiology , Sterol Regulatory Element Binding Protein 1/metabolism , Thyroid Hormone Receptors beta/physiology , ATP Binding Cassette Transporter, Subfamily D , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Electrophoretic Mobility Shift Assay , Gene Expression , Humans , Liver/metabolism , Male , Mice , Mice, Knockout , Plasmids , Point Mutation , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulatory Elements, Transcriptional , Reverse Transcriptase Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/genetics , Thyroid Hormones/pharmacology , Transcription, Genetic , TransfectionABSTRACT
The peroxisomal ATP binding cassette (ABC) transporter adrenoleukodystrophy-related protein, encoded by ABCD2, displays functional redundancy with the X-linked adrenoleukodystrophy-associated protein, making ABCD2 up-regulation of therapeutic value. Cholesterol lowering activates human ABCD2 in cultured cells. To investigate in vivo regulation by sterols, we first characterized a sterol regulatory element (SRE) in the murine Abcd2 promoter that is directly bound by SRE-binding proteins (SREBPs). Intriguingly, this element overlaps with a direct repeat 4, which serves as binding site for liver X receptor (LXR)/retinoid X receptor heterodimers, suggesting novel cross-talk between SREBP and LXR/retinoid X receptor in gene regulation. Using fasting-refeeding and cholesterol loading, SREBP accessibility to the SRE/direct repeat 4 was tested. Results suggest that adipose Abcd2 is induced by SREBP1c, whereas hepatic Abcd2 expression is down-regulated by concurrent activation of LXRalpha and SREBP1c. In cell culture, SREBP1c-mediated Abcd2 induction is counteracted by ligand-activated LXRalpha. Finally, hepatic Abcd2 expression in LXRalpha,beta-deficient mice is inducible to levels vastly exceeding wild type. Together, we identify LXRalpha as negative modulator of Abcd2, acting through a novel regulatory mechanism involving overlapping SREBP and LXRalpha binding sites.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , DNA-Binding Proteins/physiology , Gene Expression Regulation , Receptors, Cytoplasmic and Nuclear/physiology , Sterol Regulatory Element Binding Protein 1/metabolism , ATP Binding Cassette Transporter, Subfamily D , Adenosine Triphosphate/chemistry , Adipose Tissue/metabolism , Amino Acid Motifs , Animals , Binding Sites , Blotting, Northern , Blotting, Western , COS Cells , Cell Line, Tumor , Cells, Cultured , Chlorocebus aethiops , Cholesterol/metabolism , Colestipol/pharmacology , DNA-Binding Proteins/metabolism , Dimerization , Down-Regulation , Humans , Ligands , Liver/metabolism , Liver X Receptors , Lovastatin/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Genetic , Monocytes/metabolism , Mutagenesis , Oligonucleotides/chemistry , Orphan Nuclear Receptors , PPAR alpha/metabolism , Plasmids/metabolism , Point Mutation , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/physiology , Sterols/metabolism , Time Factors , Transcription, Genetic , TransfectionABSTRACT
Adrenoleukodystrophy protein (ABCD1), a peroxisomal membrane protein, is mutated in patients affected by X-linked adrenoleukodystrophy (X-ALD). Adrenoleukodystrophy-related protein (ABCD2) is the closest relative of ABCD1. Pharmacological induction of ABCD2 gene expression has been proposed as a novel therapy strategy for X-ALD. Fibrates induce peroxisome proliferation and Abcd2 expression in rodent liver. Here we evaluate the possibility of using peroxisome proliferator-activated receptor alpha (PPARalpha) agonists for pharmacological induction of ABCD2 expression. In the liver of PPARalpha-deficient mice, both the constitutive and the fenofibrate-inducible Abcd2 gene expression was found to be PPARalpha-dependent. In the brain, PPARalpha-deficiency has no effect on Abcd2 expression. In mice orally treated with the novel, highly selective, and potent PPARalpha agonists GW 7647, GW 6867, and tetradecylthioacetic acid, Abcd2 expression was induced in liver and adrenal glands, but not in brain and testis. None of four putative PPREs identified in the 5(')-flanking DNA and in intron 1 of the Abcd2 gene conferred fibrate response in luciferase reporter assays. Thus, although fibrate-mediated Abcd2 induction is PPARalpha-dependent, it appears to be an indirect mechanism. Within the mouse Abcd2 promoter, a putative sterol regulatory element (SRE) similar in sequence and position to the characterized SRE sequence of the human ABCD2 promoter, was identified. A PPARalpha dependent induction of the sterol regulatory-binding protein 2 (SREBP2) and a down-regulation of SREBP1c mRNA levels could be demonstrated after fenofibrate treatment of mice. Our results suggest that the PPARalpha agonist-mediated induction of Abcd2 expression seems to be indirect and possibly mediated by SREBP2.