Jonathan D. Turner

Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed

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.

Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms

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.

Transcriptional control of the human glucocorticoid receptor: identification and analysis of alternative promoter regions

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.

Glucocorticoid sensitivity in fibromyalgia patients : Decreased expression of corticosteroid receptors and glucocorticoid-induced leucine zipper

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.

Membrane Glucocorticoid Receptor Activation Induces Proteomic Changes Aligning with Classical Glucocorticoid Effects

Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly “priming” classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.

Adhesion molecules and cytokine expression in fibromyalgia patients: Increased L-selectin on monocytes and neutrophils

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.

A New Transcript Splice Variant of the Human Glucocorticoid Receptor

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, hGRΔ313‐338, with a 26 residue (78 bp) deletion in this N‐terminal region encoded by exon 2, between amino acids 313 and 338. The hGRΔ313‐338 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 hGRΔ313‐338 represents a hGR isoform with an altered glucocorticoid‐induced transactivation profile.

The use of saliva for assessment of cortisol pulsatile secretion by deconvolution analysis

Cortisol is the key effector molecule of the HPA axis and is secreted in a pulsatile manner in all species studied. In order to understand cortisol signalling in health and disease, detailed analysis of hormone pulsatility is necessary. To dissect cortisol pulsatility in plasma deconvolution techniques have been applied. Blood sampling is a labour-intensive, expensive and invasive technique that causes stress and alters HPA axis activity. Therefore saliva has been extensively investigated as an alternative sample to measure cortisol. Here we use state of the art deconvolution algorithms to investigate cortisol pulsatility in saliva. Blood and saliva samples were obtained at 15-min intervals over an 8h period in 18 healthy men to analyse their diurnal cortisol levels. A multiparameter deconvolution technique was used to generate statistically significant models of cortisol secretion and elimination in plasma and saliva. The models consisted of estimates of the number, amplitude, duration and frequency of secretory bursts as well as the elimination half-life (t1/2) in a subject specific manner. No significant differences were noted between plasma and saliva with regard to the observed secretory bursts (7.8±1.5 vs. 7.0±1.4) and the interpeak interval (59.6±10.5 min vs. 61.0±11.5 min). Moreover a strong positive correlation between the numbers of peaks in both fluids was observed (r=0.83, P<0.0001). Monte Carlo simulations revealed an 84% temporal concordance between plasma and saliva peaks in all donors (P<0.05) with a mean of 1.3±0.8 plasma peaks unmatched in saliva. The percentage concordance increased to 90% when concording only the morning cortisol peaks in plasma and saliva up to 11:00 h. The deconvolution of the most distinct component of cortisol diurnal rhythm-cortisol awakening response (CAR), revealed an average 2.5±1.1 peaks based on the individual time for cortisol to return to baseline levels. In conclusion, deconvolution analysis of plasma and salivary cortisol concentration time series showed a close correlation and similar pulsatile characteristics between saliva and plasma cortisol. Similarly, Monte Carlo simulations revealed a high concordance between the peaks in these coupled time series suggesting that saliva is a suitable medium for subsequent deconvolution analysis yielding accurate and reliable models of cortisol secretion in particular during the morning hours.

Diurnal redistribution of human lymphocytes and their temporal associations with salivary cortisol

Immune cell trafficking is crucial for surveillance and effector functions of the immune system. Circadian rhythms of the hypothalamic-pituitary-adrenal (HPA) axis and of cortisol have been implicated in circadian redistribution of circulating lymphocytes and granulocytes. However, information regarding the diurnal redistribution of immune cells and their temporal correlations with cortisol is scarce. In this study, we investigated the diurnal redistribution of T, B, and natural killer (NK) cell subsets in relation to the endogenous cortisol rhythm. Saliva and blood samples were collected every 15 min over an 8-h period. Salivary-free cortisol was measured by enzyme-linked immunosorbent assay (ELISA). Surface markers (CD3, CD19, CD8, CD56, CD16, KIR) were measured in whole blood samples by 6-color flow cytometry and cell subsets quantified as a percentage of the total lymphocyte population. To study associations between the diurnal cortisol rhythm and the redistribution of T, B, and NK cells, we calculated cross-correlations with lag periods of 15 min. The salivary cortisol levels showed the typical diurnal variations with a significant morning cortisol awakening response (CAR) peaking around 07:30 h followed by an afternoon nadir. Whereas B cells remained stable throughout the 8 h, T cells (CD3 + CD8+ and CD3 + CD8−) showed a significant positive cross-correlation with cortisol levels when a delay of 30–105 min was taken into account. This was followed by a negative correlation covering a period of 165–285 min after the cortisol peak. Conversely, NK cells showed an initial negative correlation at 45–105 min, followed by a positive correlation at 120–285 min. The major CD56 + CD16+ subset and the CD56 − CD16+ population showed similar temporal correlation profiles. The minor CD56 + CD16− NK cell subset showed no temporal changes. The major NK subset (CD56 + CD16+) contains cells with higher cytolytic activity (KIR+) cells, whereas the single-positive subsets CD56 + CD16− and CD56 − CD16+ are mainly involved in cytokine production. Significant positive correlations were observed in KIR+ subsets coincident with this of NK cells covering a period of 105–300 min after the cortisol peak. In conclusion, our results suggest that cortisol and the HPA axis orchestrate tidal waves of immune cells that are alternatively based toward innate and acquired immune surveillance. (Author correspondence: claude.muller@CRP-SANTE.LU)

Where does transcription start? 5′-RACE adapted to next-generation sequencing

The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5′ cDNA ends (5′-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5′-m7G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R) and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5′ m7G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5′ UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GR N-terminal protein isoform levels.