As the leader of the PsychoImmunology group, my interest is in explaining the transcriptional regulation of the glucocorticoid receptor. Aberrant GR levels are associated with stress-related disorders such as depression, and affect social behaviour, mood, learning and memory. Dissecting how tissue-specific GR levels are regulated, in particular in the brain, is the first step towards understanding the mechanisms underlying aberrant GR levels in disease and altered behaviour.
Over the last few years we have shown that the unique variability in the 5’ region of the GR gene, with 9 alternative first exons and 13 splice variants plays a critical role in transcriptional control maintaining homeostasis of the gluco-corticoid receptor (GR). This 5’mRNA heterogeneity, common to all species investigated, remains untrans-lated since the alternative first exons are spliced to exon 2 immediately upstream of the translation initia-tion codon. These alternative first exons are located either immediately upstream of the coding exons in the CpG island (exons B-H and J), or further upstream (exons 1A and 1I). The mechanisms regulating the differential usage of these first exons in different tissues and individuals, and the role of the 5’UTR in the splicing of the coding exons are still poorly understood. Data from our laboratory and others have shown that the multiple first exons represent only a first layer of complexity orchestrated probably by tissue-specific transcription factors. Modulation of alternative first exon activity by epigenetic methylation of their promoters represents a second layer of complexity at least partially controlled by perinatal programming. The alternative promoter usage also appears to affect the 3’ splicing generating the different GR coding variants, GRalpha, GRbeta, and GR-P.
It is anticipated that the group will continue working on understanding the transcriptional control of the GR, including investigating the role of microRNA, and detailed epigenetic studies to determine their effects on GR transcription.
Abstract: In order to investigate rapid non-genomic effects of acute stress, rats were restrained for 15 min which was sufficient to activate the hypothalamus-pituitary-adrenal (HPA) axis but too short to induce massive genomic effects of cortisol. Subcellular fractions of thymocytes (cytosol, nucleus, membrane) were investigated using quantitative 2D DIGE with MALDI-TOF/TOF mass spectrometry. In total, 108 proteins with differential subcellular localizations were identified. The specificity of the changes induced by psychological stress was reflected by the prominent modulation of proteins involved in the HPA and sympathoadrenal medullar (SAM) axis such as HMGB1 and NHERF1. Intracellular trafficking was characterized by a dominant protein exodus from the cytosol. Real translocation was observed for 9 proteins with 6 that shuttled from the cytosol to the nucleus (HYOU1, HNRPF, HNRPC, STRAP, PSA1, PPA1) and 3 from the nucleus to the cytosol (HMGB1, NHERF1, PSMA1). Proteins showing subcellular reshuffling were largely involved in transcription and translation processes (39 of 108) with a significant enrichment of RNA splicing factors. Bioinformatics analysis revealed significant enrichment for protein kinase A and 14-3-3 signaling, probably reflecting real non-genomic effects. This is the first study investigating rapid effects of stress-induced HPA activation in vivo at the proteome level.
Abstract: Appropriate signaling in the brain by the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is critical in regulation of the hypothalamic-pituitary-adrenal (HPA) axis, emotional arousal and cognitive performance. To date, few data exist on MR (and GR) expression in the brain of patients suffering from major depressive disorder (MDD).
With the help of quantitative PCR we assessed MR and GR mRNA expression, including the splice variants MRï¡ and MRï¢, in tissue samples from the hippocampus, amygdala, inferior frontal gyrus, cingulate gyrus and nucleus accumbens. Expression levels were compared between tissue samples from six MDD patients and six non-depressed subjects.
Relative to total GR, total MR mRNA expression was higher in hippocampus and lower in the amygdala, inferior frontal gyrus and nucleus accumbens. Both MRï¡ and MRï¢ could be detected in all brain regions that were analyzed, although MRï¢ expression was low. Significantly lower expression levels (30â50%) were detected for MR or GR in hippocampal, inferior frontal gyrus and cingulate gyrus tissue from MDD patients (p< .05), while no differences were found in amygdala or nucleus accumbens.
The data show that both MRï¡ and MRï¢ mRNA are expressed throughout the human limbic brain with highest expressions in the hippocampus. A decreased expression of corticosteroid receptors in specific brain regions of MDD patients could underlie HPA hyperactivity, mood and cognitive disturbances often observed in patients suffering from stress-related psychopathologies.
Abstract: The effects of cortisol (CORT) on resting and lipopolysaccharide (LPS)-activated monocyte-derived THP-1 macrophages were investigated by proteomics. Forty-seven proteins were found to be modulated, 20 by CORT, 11 by LPS, and 16 by CORT and LPS. Cortisol-sensitive chaperones and cytoskeletal proteins were mostly repressed. HCLS1, MGN, and MX1 were new proteins identified to be under the transcriptional control of this steroid and new CORT-sensitive variants of MX1, SYWC and IFIT3 were found. FKBP51, a known CORT target gene, showed the strongest response to CORT and synergism with LPS. In resting THP-1 macrophages, 18 proteins were modulated by CORT, with 15 being down-regulated. Activation of macrophages by LPS was associated with enhanced expression of immune response and metabolic proteins. In activated macrophages, CORT had a more equilibrated effect and almost all metabolism-related proteins were up-regulated, whereas immune response proteins were mostly down-regulated. The majority of the LPS up-regulated immune response-related proteins are known interferon (IFN) target genes (IFIT3, MX1, SYWC, PSME2) suggesting activation of the IRF3 signaling pathway. They were all suppressed by CORT. This is the first proteomics study to investigate the effects of CORT on activated immune cells.
Abstract: The megalencephalic leucoencephalopathy with subcortical cyst 1 (MLC1) gene is mainly expressed in astrocytes. Homozygous mutations cause MLC, an autosomal recessive neurological disorder characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly) in humans. Furthermore, variants of MLC1 have been associated with psychiatric disorders like schizophrenia and bipolar disorder. Currently, little is known about the MLC1 protein. Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of MLC1 and its gene regulation has not been investigated successfully so far. We investigated the 5â region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.
Abstract: Glucocorticoid receptor levels are thought to be controlled by multiple alternative first exons. Seven of these exons are located in an upstream CpG island. In this study, we investigated the promoter activity of the intronic regions between these exons, and their susceptibility to CpG methylation and sequence variability. The seven promoters were cloned into luciferase reporter genes, and their activity measured in ten cell lines. CpG islands of 221 donors were genotyped and the effects of these SNPs were investigated in a reporter gene assay. We showed that each of the first exons was independently controlled by a unique promoter located directly upstream. Promoter activities were cell type-specific, and varied considerably between cell types. Irrespective of the cell type, in vitro methylation effectively silenced all reporter constructs. Eleven SNPs were observed within the CpG island of 221 donors, and a new promoter-specific haplotype was revealed. Four of the minor alleles reduced the reporter gene activity, with cell type specific effects. This complexity within the CpG island helps to explain the variable, tissue-specific transcriptional control of the GR, and provides insight into the mechanisms underlying tissue specific deregulation of GR levels.
Abstract: The unique variability in the 5' region of the GR gene, with 9 alternative first exons and 13 splice variants plays a critical role in transcriptional control maintaining homeostasis of the glucocorticoid receptor (GR). This 5'm RNA heterogeneity, common to all species investigated, remains untranslated since the alternative first exons are spliced to exon 2 immediately upstream of the translation initiation codon. These alternative first exons are located either immediately upstream of the coding exons in the CpG island (exons B-H and J), or further upstream (exons 1A and 1I). The mechanisms regulating the differential usage of these first exons in different tissues and individuals, and the role of the 5' UTR in the splicing of the coding exons are still poorly understood. Here we review some of the mechanisms that have so far been identified. Data from our laboratory and others have shown that the multiple first exons represent only a first layer of complexity orchestrated probably by tissue-specific transcription factors. Modulation of alternative first exon activity by epigenetic methylation of their promoters represents a second layer of complexity at least partially controlled by perinatal programming. The alternative promoter usage also appears to affect the 3' splicing generating the different GR coding variants, GRalpha, GRbeta, and GR-P. Aberrant GR levels are associated with stress-related disorders such as depression, and affect social behaviour, mood, learning and memory. Dissecting how tissue-specific GR levels are regulated, in particular in the brain, is a first step to understand the significance of aberrant GR levels in disease and behaviour.
Abstract: Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is one of the most consistent findings in major depressive disorder (MDD). Impaired HPA feedback may be due to the lower glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) levels in the forebrain. GR levels are transcriptionally controlled by multiple untranslated alternative first exons, each with its own promoter providing a mechanism for tissue-specific fine-tuning of GR levels. Recently epigenetic methylation of these GR promoters was shown to modulate hippocampal GR levels. Here we investigate in post-mortem brain tissues whether in MDD HPA axis hyperactivity may be due to epigenetic modulation of GR transcript variants. Levels of GRalpha, GRbeta and GR-P transcripts were homogeneous throughout the limbic system, with GRalpha being the most abundant (83%), followed by GR-P (5-6%) while GRbeta was barely detectable (0.02%). Among the alternative first exons, 1B and 1C were the most active, while 1E and 1J showed the lowest expression and transcript 1F expressed intermediate levels of about 1%. In MDD, total GR levels were unaltered, although GRalpha was decreased in the amygdala and cingulate gyrus (p<0.05); transcripts containing exons 1B, 1C and 1F were lower, and 1D and1J were increased in some regions. NGFI-A, a transcription factor of exon 1F was down-regulated in the hippocampus of MDD patients; concomitantly exon 1F expression was reduced. Bisulphite sequencing of the alternative promoters showed low methylation levels in both MDD and control brains. Promoter 1F was uniformly unmethylated, suggesting that reduced 1F transcript levels are not linked to promoter methylation but to the observed dearth of NGFI-A. Previous studies showed high methylation levels in the 1F promoter, associated with childhood abuse. Provided our donors were not abused, our results suggest that the pathomechanism of MDD is similar but nevertheless distinct from that of abuse victims, explaining the clinical similarity of both conditions and that susceptibility to depression may be either predisposed by early trauma or developed independent of such a condition. However, this should be further confirmed in dedicated studies in larger cohorts.
Abstract: In fibromyalgia (FM) patients, differences in glucocorticoid receptor (GR) affinity and disturbances associated with loss of hypothalamic-pituitary-adrenal (HPA) axis resiliency have been observed. Based on these studies, we investigated whether FM would be associated with abnormalities in glucocorticoid (GC) sensitivity. Salivary and blood samples were collected from 27 FM patients and 29 healthy controls. Total plasma cortisol and salivary free cortisol were quantified by ELISA and time-resolved fluorescence immunoassay, respectively. GR sensitivity to dexamethasone was evaluated through IL-6 inhibition in stimulated whole blood. The corticosteroid receptors, GR alpha and mineralocorticoid receptor, as well as the glucocorticoid-induced leucine zipper (GILZ) and the FK506 binding protein 5 mRNA expression were assessed in peripheral blood mononuclear cells (PBMCs) by real-time RT-PCR. Furthermore, the corticosteroid receptors were analysed for polymorphism. We observed lower basal plasma cortisol levels (borderline statistical significance) and a lower expression of corticosteroid receptors and GILZ in FM patients when compared to healthy controls. The MR rs5522 (I180V) minor allele was found more often in FM patients than in controls and this variant was recently associated with a mild loss of receptor function. The lower GR and MR expression and possibly the reduced MR function may be associated with an impaired function of the HPA axis in these patients which, compounded by lower anti-inflammatory mediators, may sustain some of symptoms that contribute to the clinical picture of the syndrome.
Abstract: The transcription start sites (TSS) and promoters of many genes are located in upstream CpG islands. Methylation within such islands is known for both imprinted and oncogenes, although poorly studied for other genes, especially those with complex CpG islands containing multiple first exons and promoters. The glucocorticoid receptor (GR) CpG island contains seven alternative first exons and their promoters. Here we show for the five GR promoters activated in PBMCs that methylation patterns are highly variable between individuals. The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS. We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique. The majority of these contain methylatable CpG sites, suggesting that methylation may orchestrates alternative first exon usage, silencing and controlling tissue-specific expression. The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events. With 78% of evolutionarily conserved alternative first exons falling into such complex CpG islands, their internal structure and epigenetic modifications are bound to be biologically important, and may be a common transcriptional control mechanism used throughout many phyla.
Abstract: All human glucocorticoid receptor (hGR) isoforms are encoded by the NR3C1 gene consisting of seven core exons (exons 2-8) common to all protein isoforms. The gene has two major exon 8-9 splice variants and a 5'-UTR consisting of 11 alternative splice variants. The N-terminal region of the hGR includes a tau 1 transactivation domain that interacts with proteins in the basal transcriptional apparatus, including the TATA box-binding protein. Here, we report the existence and the tissue distribution of a novel splice variant, hGRDelta313-338, with a 26 residue (78 bp) deletion in this N-terminal region encoded by exon 2, between amino acids 313 and 338. The hGRDelta313-338 observed at the mRNA level represents a transcript variant encoding a smaller protein isoform detected by WB with a predicted deletion between the tau 1 domain and the DNA-binding domain (DBD) encoded by exons 3 and 4. Previous studies in transgenic mice showed that the removal of the entire exon 2 covering both the tau 1 transactivation domain and our deleted region produced a functional receptor albeit with an altered glucocorticoid-induced gene transcription pattern. Interestingly, the deleted residues show a number of potential phosphorylation sites including serine 317, known to be phosphorylated. It is thought that phosphorylation plays an important role in transactivation action of hGR. Thus, we hypothesize that hGRDelta313-338 represents a hGR isoform with an altered glucocorticoid-induced transactivation profile.
Abstract: The glucocorticoid (GC) cortisol, the main mediator of the hypothalamic-pituitary-adrenal axis has many implications in metabolism, stress response and the immune system. Its function is mediated via binding to the glucocorticoid receptor (GR), a member of the superfamily of ligand-activated nuclear hormone receptors. The activity of the ligated GR results from its binding as a transcription factor to glucocorticoid response elements (GREs). Two-dimensional gel electrophoresis with DIGE (fluorescence difference gel electrophoresis) technology was applied to study the effects of cortisol on the human THP-1 monocytic cell line. A total of 28 cortisol-modulated proteins were identified belonging to five functional groups: cytoskeleton (8), chaperones (9), immune response (4), metabolism (3) and transcription/translation (4). Their corresponding genes were screened for putative GREs in their + 10 kb/- 0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS). FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein, and contains the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggests GC inducibility of all transcripts. Additionally, proteomics (2D DIGE and 2D immunoblotting) revealed the existence of several FKBP51 isoforms, which were not previously described. To our knowledge this is the first proteomic study that addresses the effects of cortisol on immune cells. FKBP51 isoforms found on the gel map were linked to alternative promoter usage on the genetic level, successfully correlating both the specific proteomic and genomic findings.
Abstract: Several lines of evidence implicate the immune system in the pathophysiology of fibromyalgia (FM). We investigated the role of cytokines and adhesion molecules involved in immune cell trafficking and the influence of 1.5 mg of dexamethasone (DEX) per os on their expression. L-selectin was elevated on monocytes and neutrophils of FM patients. Differences in group response to DEX were observed for CD11b on NK cells, sICAM-1 and IL-2. This study shows a slight disturbance in the innate immune system of FM patients, and suggests an enhanced adhesion and recruitment of leukocytes to inflammatory sites.
Abstract: The behavioral performances of adult mice exposed to sub-acute doses of benzo(a)pyrene (B(a)P) were monitored in tests related to learning and memory (Y maze and Morris water maze), locomotor activity (open-field test) and motor coordination (Locotronic apparatus). At low doses (0.02 and 0.2mg/kg), B(a)P impaired short-term learning and spatial memory performance in the Y maze and in the Morris water maze tests. Surprisingly, in the Y maze, the performances of animals exposed to the highest dose of B(a)P (200mg/kg) were quite similar to those of control animals. Hyperactivity/hyperarousal observed in both tests at this dose and attributed to an anxiolytic-like effect of B(a)P may have blurred the learning deficit in these mice faced with a new situation. These deficits seem to be unrelated to motor impairments because B(a)P had no effect on locomotor activity and motor coordination. We demonstrated that sub-acute exposure to B(a)P in adult mice also modulates gene expression of NMDA-R1 subunit in brain areas highly involved in cognitive function like the hippocampus, suggesting a relationship between the expression of functional NMDA-R1 mRNA, impairment of short-term and spatial memory and the B(a)P exposure levels.
Abstract: Nuclear receptors (NR) are key modulators of gene transcription. Their activity is ligand induced and modulates a large variety of tissue-specific cellular functions. However, for many NR little is known about their role in cells of the immune system. In this study, expression patterns and distribution of 24 NR were investigated in human peripheral blood mononuclear cells. We provide the first evidence of the expression of the 12 receptors CAR, CoupTFalpha, CoupTFbeta, FXR, GCNF, HNF4alpha, PPARbeta/delta, PXR, RevErbbeta, TR2, TR4 and TLX in highly purified CD4, CD8, CD19, CD14 cells. The expression profile of RevErbalpha and LXRalpha previously observed in B cell and macrophages, respectively, has been extended to CD4, CD8 and CD14 cells. Except for RARbeta, which was absence in any of the cells tested, our results suggest an almost ubiquitous expression of the NR in the different cell lineages of the immune system. The expression of CAR, CoupTFalpha, FXR was also confirmed at a protein level and despite conspicuous mRNA levels of HNF4alpha, only low levels of this receptor were detectable in the nuclear fraction of PBMCs. Expression of the latter receptors was mostly only a fraction (4-20%) of their expression in the thyroid gland, the adrenal gland, the lung or subcutaneous adipose tissue. The Spearman rank order correlation test was performed to examine the correlation in expression between individual nuclear receptor pairs in the four cell types for several donors. Distinct correlation patterns were observed between receptor pairs in the individual cell types. In CD4 T cells four NR, GCNF, PPARgamma, PPARalpha7 and RevErbbeta are perfectly correlated with each other (P> or =0.0167). In the other cell types correlations between NR pairs were more diverse, but also statistically highly significant. Interestingly, the relative expression level of a number of receptor pairs ranked identical or similar in at least three (CoupTFalpha and PPARbeta/delta, CoupTFbeta and HNF4alpha as well as RORbeta and PXR) or four cell types (CoupTFalpha and CoupTFbeta, PPARgamma and RevErbbeta). Despite the variability of NR expression in immune cells, these results suggest that some of the NR may be co-regulated in human immune cells.
Abstract: The CpG island upstream of the GR is highly structured and conserved at least in all the animal species that have been investigated. Sequence alignment of these CpG islands shows inter-species homology ranging from 64 to 99%. This 3.1kb CpG rich region upstream of the GR exon 2 encodes 5' untranslated mRNA regions. These CpG rich regions are organised into multiple first exons and, as we and others have postulated, each with its own promoter region. Alternative mRNA transcript variants are obtained by the splicing of these alternative first exons to a common acceptor site in the second exon of the GR. Exon 2 contains an in-frame stop codon immediately upstream of the ATG start codon to ensure that this 5' heterogeneity remains untranslated, and that the sequence and structure of the GR is unaffected. Tissue specific differential usage of exon 1s has been observed in a range of human tissues, and to a lesser extent in the rat and mouse. The GR expression level is tightly controlled within each tissue or cell type at baseline and upon stimulation. We suggest that no single promoter region may be capable of containing all the necessary promoter elements and yet preserve the necessary proximity to the transcription initiation site to produce such a plethora of responses. Thus we further suggest that alternative first exons each under the control of specific transcription factors control both the tissue specific GR expression and are involved in the tissue specific GR transcriptional response to stimulation. Spreading the necessary promoter elements over multiple promoter regions, each with an associated alternative transcription initiation site would appear to vastly increase the capacity for transcriptional control of GR.
Abstract: The 5' untranslated region (UTR) of the glucocorticoid receptor (GR) plays a key role in determining tissue-specific expression and protein isoforms. Analysis of the 5' UTR of the human GR (hGR) has revealed 11 splice variants of the hGR exon 1, based on seven exon 1s, four of which (1-D to 1-F and 1-H) were previously unknown. All of the exon 1 variants have unique splice donor sites and share a common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the predicted translation from all splice variants is identical. The four new exon 1s show remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17 and 36 bp upstream of the corresponding ends of the rat exon 1(4). Exon 1-E is only 6 bp longer than its homologue exon 1(5). Exon 1-F contains two short inserts of 11 and 6 bp when compared with the rat 1(7). 1-H is 18 bp longer than the corresponding rat 1(11). In addition to these new exons, we found that the human exon 1-C occurs as three distinct splice variants, covering the region homologous to the rat exons 1(9) and 1(10). All of the alternative hGR exons 1s presented here were found to be transcribed in human tissue. The human hippocampus expresses mRNA of all the exon 1 variants, while the expression of the other exon 1s seems to be tissue specific. While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5' region of the hGR is more complex than previously thought, and we suggest that each of these untranslated first exons have a distinct proximal promoter region, providing additional depth to the mechanisms available for tissue-specific expression of the hGR isoforms.
Abstract: Members of the Toll-like receptor (TLR) family are currently under intense scrutiny for their role in the sampling and recognition of pathogens. It has already been reported that both vaccinia virus and Yersinia spp. express proteins that help them evade the TLR mediated immune response, acting through the Toll-interleukin-1 receptor-resistance (TIR) domain and leucine-rich repeat region of the host TLRs respectively. The TIR domain is involved in the dimerisation of the TLRs and their complexation with their adapter molecules. We tested here the hypothesis that bacteria have the ability to secrete proteins containing similar motifs to the intracellular TIR domains that are involved in the TIR-TIR interaction necessary for the subsequent signal transmission. Based upon their sequence homology, proteins expressing TIRs have been divided into three sub-classes, based around the TLRs, the TLR adapter proteins, and the interleukin-1 and -18 adapter proteins. The highly conserved regions from these separate sub-families were then used to identify similar bacterial proteins. The bacterial proteins identified were then included in an iterative MEME-BLAST process to broaden the search. Tollip, a known TLR antagonist and adapter protein, was included in this investigation although it does not fit into any of the three sub-classes outlined above. If suitable bacterial proteins had been identified, it would signify that certain bacteria had evolved a mechanism to aid them in avoiding detection by the innate immune system acting through the TIR domains. At this stage one has to conclude that there is no evidence currently available suggesting such a mechanism, when using the strategy applied here.
