Susanne Heimerl

The Zinc Finger Protein 202 (ZNF202) Is a Transcriptional Repressor of ATP Binding Cassette Transporter A1 (ABCA1) and ABCG1 Gene Expression and a Modulator of Cellular Lipid Efflux

The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the effect of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the promotors of ABCA1 (−229/−210) and ABCG1 (−572/−552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the promotor activities of ABCA1 and ABCG1. This transcriptional effect required the presence of the SCAN domain in ZNF202 and the functional integrity of a TATA box at position −24 of ABCA1, whereas the presence of GnT binding motifs was nonessential. The state of ZNF202 SCAN domain oligomerization affected the ability of the adjacent ZNF202 Krüppel-associated box domain to recruit the transcriptional corepressor KAP1. Overexpression of ZNF202m1 in RAW264.7 macrophages prevented the induction of ABCA1 gene expression by 20(S)OH-cholesterol and 9-cis-retinoic acid, further substantiating the interference of ZNF202 in critical elements of transcriptional activation. Finally, HDL and apoAImediated lipid efflux was significantly reduced in RAW264.7 cells stably expressing ZNF202m1. In conclusion, we have identified ABCA1 and ABCG1 as target genes for ZNF202-mediated repression and thus, provide evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia.

Mapping ATP-binding cassette transporter gene expression profiles in melanocytes and melanoma cells.

ATP-binding cassette (ABC) transporters regulate the transport of a variety of physiologic substrates. Moreover, several human ABC proteins are responsible for drug exclusion in compound-treated tumor cells, providing cellular mechanisms for the development of multidrug resistance and, therefore, playing an important role in malignant transformation. As only limited information exists on the role of ABC transporters in melanoma, the aim of the study was to generate a complete expression profile of ABC transporters in this tumor entity. Using a TaqMan low-density array for 47 human ABC transporters, mRNA expression analysis was performed from normal human epidermal melanocytes (NHEM P2 and NHEM P3), nine different cell lines originating from primary melanoma (Mel Ei, Mel Juso, Mel Ho and Mel Wei), and metastases of malignant melanoma (Mel Im, Mel Ju, SK Mel 28, HTZ 19 and HMB2). Cell line-specific expression levels were compared with gene expression in pooled RNA from a variety of other human tissues. High expression levels were detected in pooled tissue RNA as well as in cells of melanocytic origin for ABCA5, ABCB2, ABCB6, ABCD3, ABCD4, ABCF1, ABCF2 and ABCF3, whereas ABCB5 revealed a melanocyte-specific high transcript level. In relation to normal melanocytes, ABCB3, ABCB6, ABCC2, ABCC4, ABCE1 and ABCF2 were significantly increased in melanoma cell lines, whereas ABCA7, ABCA12, ABCB2, ABCB4, ABCB5 and ABCD1 showed lower expression levels. In summary, we present here for the first time an ABC-transporter mRNA expression profile in melanoma in comparison to normal melanocytes. The differentially regulated ABC transporters detected by our approach may be candidate genes involved in melanoma tumorigenesis, progression and therapy resistance and could therefore be of great importance to identify novel options for melanoma therapy.

Identification of Sterol-independent Regulatory Elements in the Human ATP-binding Cassette Transporter A1 Promoter ROLE OF Sp1/3, E-BOX BINDING FACTORS, AND AN ONCOSTATIN M-RESPONSIVE ELEMENT

The ATP-binding cassette transporter A1 (ABCA1) shows a differentiation-, cAMP-, and sterol-dependent up-regulation in human monocytes. As part of an ongoing study, we investigated the proximal promoter regions that are highly conserved between the human and murine ABCA1 genes. Using reporter gene assays, we show here that a TATA box 24 bp upstream of the transcription initiation site is essential for promoter activity in RAW 264.7 and HepG2 cells, whereas further enhancement of transcriptional activity is mediated by the −175 bp promoter region. Gel shift assays revealed in vitro binding of Sp1 to a −91 GnC motif as well as binding of Sp1 and Sp3 to a −157 GnC promoter region. In co-transfection experiments using Drosophila S2 cells, we demonstrate that Sp3 competes with Sp1 for binding to the −157 GnC motif and acts as a repressor. On the other hand, overexpression of Sp1 increased ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. We also show here that the conserved E-box at position −140 binds upstream stimulatory factors 1 and 2 and hepatic nuclear factor 1α and that mutagenesis of the E-box enhanced constitutive ABCA1 expression in RAW 264.7 cells, implying a role for this element in silencing ABCA1 expression. Besides the functional importance for basal gene expression, we have identified that the core promoter region (−175 to +224) is also responsible for the induction of ABCA1 by the cytokine oncostatin M, resulting in a rapid increase in ABCA1 mRNA levels in HepG2 cells. Interestingly, this oncostatin M-induced expression is not dependent on the currently known sequence motifs in the ABCA1 promoter. In conclusion, a functional complex of cis-elements within the proximal human ABCA1 promoter associated with the transcription factors Sp1/3, upstream stimulatory factors 1 and 2, and hepatic nuclear factor 1α has been characterized, which allows a subtle tissue-specific regulation of ABCA1 gene expression.

Glycerophospholipid and Sphingolipid Species and Mortality: The Ludwigshafen Risk and Cardiovascular Health (LURIC) Study

Vascular and metabolic diseases cause half of total mortality in Europe. New prognostic markers would provide a valuable tool to improve outcome. First evidence supports the usefulness of plasma lipid species as easily accessible markers for certain diseases. Here we analyzed association of plasma lipid species with mortality in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. Plasma lipid species were quantified by electrospray ionization tandem mass spectrometry and Cox proportional hazards regression was applied to assess their association with total and cardiovascular mortality. Overall no differences were detected between total and cardiovascular mortality. Highly polyunsaturated phosphatidylcholine species together with lysophosphatidylcholine species and long chain saturated sphingomyelin and ceramide species seem to be associated with a protective effect. The predominantly circulating phosphatidylcholine-based as well as phosphatidylethanolamine-based ether species and phosphatidylethanolamine species were positively associated with total and cardiovascular mortality. Saturated and monounsaturated phosphatidylcholine species, especially phosphatidylcholine 32∶0 (most probably dipalmitoyl-phosphatidylcholine) and palmitate containing sphingomyelin and ceramide species showed together with 24∶1 containing sphingomyelin and ceramide species strongest positive association with mortality. A quotient of the sums of the six most protective species and the six species with the strongest positive mortality association indicated an almost 3-fold increased risk of mortality, which was higher than the hazard ratio for known risk factors in our cohort. Plasma lipid species levels and especially ratios of certain species may be valuable prognostic marker for cardiovascular and total mortality.

Alterations of plasma lysophosphatidylcholine species in obesity and weight loss.

Background Obesity and related diseases of the metabolic syndrome contribute to the major health problems in industrialized countries. Alterations in the metabolism of lipid classes and lipid species may significantly be involved in these metabolic overload diseases. However, little is known about specific lipid species in this syndrome and existing data are contradictive. Methods In this study, we quantified plasma lipid species by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in obese subjects before and after 3 month weight loss as well as in a control group. Results The comparison of obese subjects with control subjects before weight loss revealed significantly lower lysophosphatidylcholine (LPC) concentrations in obesity. LPC concentrations did not significantly increase during the observed period in the weight loss group. Analysis of LPC species revealed a decrease of most species in obesity and negative correlations with C-reactive protein (CRP) and body mass index (BMI). Correlating BMI ratio before and after weight loss with the ratio of total LPC and individual LPC species revealed significant negative relationships of LPC ratios with BMI ratio. Conclusions Our findings contribute to the contradictive discussion of the role of LPC in obesity and related chronic inflammation strongly supporting pre-existing data in the literature that show a decrease of LPC species in plasma of obese and a potentially anti-inflammatory role in these subjects.

ABCA10, a novel cholesterol-regulated ABCA6-like ABC transporter

We recently identified several novel members of the A subclass of ABC transporters. In this study, we report the cloning of an additional ABC A subfamily transporter, denoted ABCA10, from macrophages. The coding sequence of ABCA10 is of 4.6 kb size and codes for a 1543-amino acid protein that bears the structural features of a full-size ABC transporter. Intriguingly, ABCA10 contains a PEST sequence downstream of the N-terminal transmembrane domain which may be potentially involved in the control of its turnover rate by proteasomal degradation. Several distinct ABCA10 transcripts are expressed in human macrophages that predict the existence of various truncated forms of the novel transporter. Moreover, we identified seven single nucleotide polymorphisms in ABCA10 transcripts. ABCA10 displays high amino acid sequence homology with ABCA6 (63%), ABCA8 (62%), and ABCA9 (63%), respectively, known members of the subgroup of ABCA6-like transporters. Like other transporters of this subfamily, ABCA10 mRNA is ubiquitously expressed and highest gene expression levels are detectable in heart, brain, and the gastrointestinal tract. Analysis of the gene structure demonstrated that the ABCA10 gene consists of 40 exons that extend across a genomic region of approximately 97kb size (Chr. 17q24.3). ABCA10 mRNA is expressed in similar quantities in monocytes and M-CSF differentiated macrophages. Importantly, ABCA10 expression is suppressed by cholesterol import into macrophages, indicating that it is a cholesterol-responsive gene. Our results identify ABCA10 as a novel member of the group of ABCA6-like transporters and suggest its involvement in macrophage lipid homeostasis.

Zinc finger protein ZNF202 structure and function in transcriptional control of HDL metabolism.

Purpose of review The zinc finger protein ZNF202 is a transcriptional repressor controlling promoter elements predominantly found in genes involved in lipid metabolism and energy homeostasis. Here we summarize the structure, regulation and modulation of ZNF202 function by protein interactions. Recent findings We review recent data and discuss the importance of the steadily growing list of ZNF202 target genes, defining a central role for ZNF202 as a key transcriptional regulator in metabolic disorders. Furthermore, we provide an interlink between transcriptional repression by ZNF202 and enhancement of gene activation via nuclear receptor coactivation by SCAN domain protein 1. Summary The novel findings suggest that ZNF202 together with other SCAN domain proteins orchestrates a complex transcriptional regulatory network, which justifies a further exploration of its potential as a therapeutic target in lipid disorders such as atherosclerosis and associated metabolic syndromes.

Novel sphingolipid derivatives promote keratinocyte differentiation

Abstract:  Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence‐induced differentiation model of normal human keratinocytes was established to allow evaluation of pro‐ and anti‐differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (–C6), stearoyl (–C18) and salicyl (–SLC) derivatives, C12‐alkylamine‐salicylate (C12‐SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA‐microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation‐related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment‐induced transcriptional changes were analysed by the ExPlain™ software (BIOBASE GmbH). Five of the assayed substances (SA, SO‐C6, PS‐C6, SO‐SLC, PS‐SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12‐SLC revealed potential anti‐differentiation properties. ExPlain™ analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their –C6 and especially –SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.

Caveolin-1 deficiency alters plasma lipid and lipoprotein profiles in mice

Caveolae are specialized membrane microdomains formed as the result of local accumulation of cholesterol, glycosphingolipids, and the structural protein caveolin-1 (Cav-1). To further elucidate the role of Cav-1 in lipid homeostasis in-vivo, we analyzed fasting and post-prandial plasma from Cav-1 deficient mice on low or on high fat diet. In total plasma analysis, an increase in ceramide and hexosylceramide was observed. In cholesteryl ester (CE), we found an increased saturated+monounsaturated/polyunsaturated fatty acid ratio in fasting plasma of low fat fed Cav-1(-/-) mice with increased proportions of CE16:1, CE18:1, CE20:3, and decreased proportions of CE18:2 and CE22:6. Under high fat diet HDL-CE, free cholesterol and pre-beta-HDL were increased accompanied by a shift from slow to fast migrating alpha-HDL and expansion of apoE containing HDL. Our results demonstrate a significant role of Cav-1 in HDL-cholesterol metabolism and may reflect a variety of Cav-1 functions including modulation of ACAT activity and SR-BI function.

Lipid profiling of lipoprotein X: Implications for dyslipidemia in cholestasis.

Lipoprotein X (Lp-X) is an abnormal lipoprotein that may typically be formed in intra- and extrahepatic cholestasis and potentially interfere with lipid analysis in the routine lab. To gain insight into lipid class and species composition, Lp-X, LDL and HDL from cholestatic and control serum samples were subjected to mass spectrometric analysis including phospholipids (PL), sphingolipids, free cholesterol (FC), cholesteryl esters (CE) and bile acids. Our analysis of Lp-X revealed a content of 46% FC, 49% PL with 34% phosphatidylcholine (PC) as main PL component. The lipid species pattern of Lp-X showed remarkable high fractions of mono-unsaturated species including PC 32:1 and PC 34:1 and phosphatidylethanolamine (PE) 32:1 and 34:1. LDL and HDL lipid composition in the same specimens strongly reflected the lipid composition of Lp-X with increased PC 32:1, PC 34:1, PE 32:1, PE 34:1 and FC accompanied by decreased CE compared to controls. Comparison of Lp-X and biliary lipid composition clearly indicates that Lp-X does not originate from a sole release of bile lipids. Moreover, these data present evidence for increased hepatic fatty acid and PL synthesis which may represent a reaction to high hepatic FC level observed during cholestasis.