Isabelle Weinhofer

Liver X Receptor α Interferes with SREBP1c-mediated Abcd2 Expression NOVEL CROSS-TALK IN GENE REGULATION

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 LXRα and SREBP1c. In cell culture, SREBP1c-mediated Abcd2 induction is counteracted by ligand-activated LXRα. Finally, hepatic Abcd2 expression in LXRα,β-deficient mice is inducible to levels vastly exceeding wild type. Together, we identify LXRα as negative modulator of Abcd2, acting through a novel regulatory mechanism involving overlapping SREBP and LXRα binding sites.

X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism

The neurodegenerative disorder X‐linked adrenoleukodystrophy (X‐ALD) is caused by ABCD1 mutations and characterized by very long‐chain fatty acid (VLCFA) accumulation. Cholesterol‐lowering normalized VLCFA in fibroblasts and plasma of X‐ALD patients. We show that in cultured cells, cholesterol‐loading induces ABCD1. In X‐ALD mice, plasma cholesterol is elevated and not further increasable by cholesterol‐feeding, whereas hepatic HMG‐CoA reductase and Abcd2 are downregulated. Upon cholesterol modulation, brain VLCFA increased in X‐ALD mice, but decreased in controls. In murine X‐ALD fibroblasts, cholesterol‐lowering did not normalize VLCFA. Thus, ALDP‐deficiency and VLCFA are linked to cholesterol but species differences complicate evaluating cholesterol‐lowering drugs in X‐ALD mice.

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

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.

Evaluation of Retinoids for Induction of the Redundant Gene ABCD2 as an Alternative Treatment Option in X- Linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is a clinically heterogeneous disease that can manifest as devastating inflammatory cerebral demyelination (CALD) leading to death of affected males. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT is only effective when performed at an early stage because the inflammation may progress for eighteen months after HSCT. Thus, alternative treatment options able to immediately halt the progression are urgently needed. X-ALD is caused by mutations in the ABCD1 gene, encoding the peroxisomal membrane protein ABCD1, resulting in impaired very long-chain fatty acid metabolism. The related ABCD2 protein is able to functionally compensate for ABCD1-deficiency both in vitro and in vivo. Recently, we demonstrated that of the cell types derived from CD34+ stem cells, predominantly monocytes but not lymphocytes are metabolically impaired in X-ALD. As ABCD2 is virtually not expressed in these cells, we hypothesize that a pharmacological up-regulation of ABCD2 should compensate metabolically and halt the inflammation in CALD. Retinoids are anti-inflammatory compounds known to act on ABCD2. Here, we investigated the capacity of selected retinoids for ABCD2 induction in human monocytes/macrophages. In THP-1 cells, 13-cis-retinoic acid reached the highest, fivefold, increase in ABCD2 expression. To test the efficacy of retinoids in vivo, we analyzed ABCD2 mRNA levels in blood cells isolated from acne patients receiving 13-cis-retinoic acid therapy. In treated acne patients, ABCD2 mRNA levels were comparable to pre-treatment levels in monocytes and lymphocytes. Nevertheless, when primary monocytes were in vitro differentiated into macrophages and treated with 13-cis-retinoic acid, we observed a fourfold induction of ABCD2. However, the level of ABCD2 induction obtained by retinoids alone is probably not of therapeutic relevance for X-ALD. In conclusion, our results suggest a change in promoter accessibility during macrophage differentiation allowing induction of ABCD2 by retinoids.

Impaired plasticity of macrophages in X-linked adrenoleukodystrophy

Weinhofer et al. reveal impaired plasticity of macrophages, with intrinsic pro-inflammatory skewing and a decreased ability to establish proper anti-inflammatory responses, in X-linked adrenoleukodystrophy (ALD). This may contribute to the rapidly progressive demyelination seen in cerebral ALD.

Involvement of Human Peroxisomes in Biosynthesis and Signaling of Steroid and Peptide Hormones

Although peroxisomes exert essential biological functions, cell type-specific features of this important organelle are still only superficially characterized. An intriguing new aspect of peroxisomal function was recently uncovered by the observation that the peptide hormones β-lipotropin (β-LPH) and β-endorphin are localized to peroxisomes in various human tissues. This suggests a functional link between peptide hormone metabolism and peroxisomes. In addition, because endocrine manifestations that affect steroid hormones are often found in patients suffering from inherited peroxisomal disorders, the question has been raised whether peroxisomes are also involved in steroidogenesis. With this chapter, we will review several crucial aspects concerning peroxisomes and hormone metabolism.

Cholesterol regulates ABCD2 gene expression: implications for X-linked adrenoleukodstrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder with impaired very long-chain fatty acid (VLCFA) metabolism (Moser et al 2001). The disease-associated ABCD1 (ALD) gene encodes a peroxisomal membrane protein, which belongs to the superfamily of ATP- binding cassette transporters. Currently, no satisfactory therapy is available for X-ALD. The only treatment with some documented benefit for X-ALD patients is bone marrow transplantation when performed at an early stage of the disease (Shapiro et al 2000). Recently, the cholesterol-lowering drug lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitor, was proposed as a new therapeutic agent for X-ALD and a cause-and-effect relationship between lovastatin therapy and plasma VLCFA levels was observed when plasma VLCFA and serum cholesterol levels were compared (Singh et al 1998b, Pai et al 2000). However, statin treatment successfully normalized VLCFA levels in the plasma of X-ALD patients in only two out of three studies (Pai et al 2000, Singh et al 1998a, Verrips et al 2000). This investigation aimed to disclose the molecular mechanism of successful reduction of VLCFA accumulation in order to fill in the gap in the understanding how dietary cholesterol lowering affects levels of VLCFA in patients with X-ALD and to allow more efficacious treatment. Overexpression of ABCD2, the closest relative of ABCD1, restores VLCFA accumulation in cultured ABCD1 -deficient cells. Here we show by real-time PCR that the ABCD2 gene is induced in cultured human fibroblasts and monocytes upon sterol-depletion via a mechanism requiring the activation of sterol regulatory element binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids. This is unexpected and the first report that extends the mechanism of transcriptional regulation by SREBPs to a peroxisomal protein, thus providing a closer link between peroxisomes, cholesterol and fatty acid biosynthesis. Using reporter gene studies, site directed mutagenesis and gel shift assays, we identified a functional sterol regulatory element in the proximal promoter region of ABCD2. Finally, we demonstrate that ABCD2 induction by sterol depletion significantly reduced the accumulation of VLCFA in X-ALD fibroblasts. Thus, lowering cholesterol leads to SREBP maturation, increased ABCD2 expression and reduced VLCFA accumulation.