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Abstract
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.
Highlights
X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is caused by mutations or large deletions of one or more exons in the ABCD1 gene located in Xq28, which encodes the peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D member 1 (ABCD1), known as adrenoleukodystrophy protein (ALDP) [1,2]
We found that the 21300 bp, 2800 bp, and 2500 bp promoter fragments showed full transcriptional activity compared to the pGL3 basic group (Fig. 1B, upper panel)
We examined whether mRNA level of ABCD2 gene was increased by ectopic expression of active mutant b-catenin (4A) and wild-type TCF-4 in HepG2 cells. mRNA expression of ABCD2 was compared between control vector-transfected or bcatenin and TCF-4-transfected cells after total RNA extraction
Summary
X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is caused by mutations or large deletions of one or more exons in the ABCD1 gene located in Xq28, which encodes the peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D member 1 (ABCD1), known as adrenoleukodystrophy protein (ALDP) [1,2]. No therapeutic drugs for X-ALD are available, gene correction of autologous hematopoietic stem cell with a wild-type version of the ABCD1 gene by a lentiviral vector has been shown to provide clinical benefit in X-ALD patients [5]. ABCD1 transports very long chain fatty acids (VLCFAs; those with more than 22 carbon atoms) or their CoA derivatives across the peroxisomal membrane for b-oxidation. It was demonstrated that human ABCD1 was able to transport VLCFA-CoA into the peroxisome in a yeast system [6]. We first reported the generation of X-ALD patient-derived induced pluripotent stem (iPS) cell models [8]. Generated X-ALD iPS cells were successfully differentiated into oligodendrocytes, the main cell type affected by the disease, and notably revealed the underlying pathophysiology which had not been observed in patients’ fibroblasts or animal models
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