Christoph Moehle

Real-Time Reverse Transcription-PCR Expression Profiling of the Complete Human ATP-Binding Cassette Transporter Superfamily in Various Tissues

BACKGROUND ATP-binding cassette (ABC) transporters are involved in many physiologic processes, such as lipid transport, sterol homeostasis, immune mechanisms, and drug transport, and cause various human inherited diseases. Thus, the analysis of ABC transporter mRNA expression profiles for basic research, especially in the field of lipid metabolism, for clinical diagnosis, and for monitoring of drug effects is of great interest. METHODS We have developed a rapid, accurate, and highly sensitive real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of all 47 currently known members of the ABC transporter superfamily. Our expression analysis is based on relative quantification using a calibration curve method. With our assay, expression monitoring of a large number of RNA samples in a 384-well format with only 50 ng of total RNA is possible. RESULTS In contrast to previous expression analyses of single ABC genes, our method allows the rapid and complete analysis of all ABC transporters in given RNA samples. We used our newly established expression panel to study the gene expression of all human ABC transporters in 20 different human tissues. As a result, we identified tissues with high transcriptional activity for ABC transporters. These organs are mainly involved in secretory function (adrenal gland), metabolic function (liver), barrier function (lung, trachea, small intestine), and tropic function (placenta, uterus). CONCLUSIONS Our RT-PCR assay allows rapid, high-throughput transcriptional profiling of the complete ABC transporter superfamily and thus provides a new enabling tool for research, clinical diagnosis of disease, and drug testing and development.

Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease

Loss of intestinal mucosa integrity is an important factor in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to characterize expression changes and allelic variants of genes related to intestinal epithelial barrier function in this disease. Therefore, ileal and colonic mucosal biopsies from nonaffected regions of patients with ulcerative colitis (UC) and Crohn’s disease (CD), as well as non-IBD probands, were subjected to Affymetrix DNA-microarray analysis. Real-time reverse transcription polymerase chain reaction was used for verification in larger IBD sample numbers. Disturbed mRNA expression was identified for several mucin genes in both disease groups and tissues. A significant downregulation in the colon was obtained for MUC2 in CD and MUC12 in CD and UC. Expression analysis of all dysregulated mucins in a broad human tissue panel revealed dominant epithelial tissue-specific transcription. In silico analysis of the regulatory regions of these mucins indicated nuclear factor κB (NFκB) binding sites in each promoter. Furthermore, NFκB was overrepresented in mucin promoters and a component of a specific combination of transcription factors (composite module). In vivo stimulation experiments in the adenocarcinoma cell line LS174T showed inducible mucin expression by the cytokines tumor necrosis factor-α and transforming growth factor-β, which could be blocked by NFκB signaling inhibitors. Allelic discrimination screening obtained statistically significant associations for the MUC2–V116M (P = 0.003) polymorphism with CD and for MUC4–A585S (P = 0.025), as well as MUC13–R502S (P = 0.0003) with UC. These data suggest that the disturbed expression of mucin genes and the connection to the NFκB pathway may influence the integrity of the intestine and therefore contribute to the pathophysiology of IBD.

Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype

BackgroundLuteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization.MethodsResting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium.ResultsLuteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures.ConclusionsOur findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.

Validation of microarray‐based resequencing of 93 worldwide mitochondrial genomes

The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip® Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of ≥99.98% for the MitoChip. The good performance was achieved by using in‐house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of ≥4 C bases or a sequence variant within 12 bases up‐ or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing. Hum Mutat 0,1–8, 2008.

The Drosophila Carbonyl Reductase Sniffer Prevents Oxidative Stress-Induced Neurodegeneration

A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimers, Huntingtons, Creutzfeld-Jakob and Parkinsons diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

Curcumin is a potent modulator of microglial gene expression and migration

BackgroundMicroglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin.MethodsResting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium.ResultsCurcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures.ConclusionsCollectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders.

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.

RBP2-H1/JARID1B is a transcriptional regulator with a tumor suppressive potential in melanoma cells

The RBP2‐H1/JARID1B nuclear protein belongs to the ARID family of DNA‐binding proteins and is a potential tumor suppressor that is lost during melanoma development. As we have recently shown, one physiological function of RBP2‐H1/JARID1B is to exert cell cycle control via maintenance of active retinoblastoma protein. We now add new evidence that RBP2‐H1/JARID1B can also directly regulate gene transcription in a reporter assay system, either alone or as part of a multimolecular complex together with the developmental transcription factors FOXG1b and PAX9. In melanoma cells, chromatin immunoprecipitation combined with promoter chip analysis (ChIP‐on‐chip) suggests a direct binding of re‐expressed RBP2‐H1/JARID1B to a multitude of human regulatory chromosomal elements (promoters, enhancers and introns). Among those, a set of 23 genes, including the melanoma relevant genes CDK6 and JAG‐1 could be confirmed by cDNA microarray analyses to be differentially expressed after RBP2‐H1/JARID1B re‐expression. In contrast, in nonmelanoma HEK 293 cells, RBP2‐H1/JARID1B overexpression only evokes a minor transcriptional response in cDNA microarray analyses. Because the transcriptional regulation in melanoma cells is accompanied by an inhibition of proliferation, an increase in caspase activity and a partial cell cycle arrest in G1/0, our data support an anti‐tumorigenic role of RBP2‐H1/JARID1B in melanocytic cells.

Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype

A disaccharide degradation product of chondrotin sulfate proteoglycan‐disaccharide (CSPG‐DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome‐wide microarray analysis was used to identify specific gene expression profiles of CSPG‐DS‐stimulated BV‐2 microglia‐like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN‐γ‐stimulated primary microglia using real‐time quantitative RT‐PCR validation. Importantly, a unique transcriptional phenotype with anti‐inflammatory and IFN‐γ counter‐regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG‐DS‐stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG‐DS‐treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN‐γ‐induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG‐DS‐activated microglia. These might be starting points to explore the in vivo role of CSPG‐DS as a bioactive microglia regulator and its potential, therapeutic application in immune‐related, neurodegenerative disorders.

Glia-induced neuronal differentiation by transcriptional regulation

There is increasing evidence that different phases of brain development depend on neuron–glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia‐derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia‐conditioned medium (GCM) and cholesterol up‐ and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes.