Real sweating in a virtual stress environment: Investigation of the stress reactivity in people with primary focal hyperhidrosis.

BACKGROUND: Hyperhidrosis (excessive sweating, OMIM %114110) is a complex disorder with multifactorial causes. Emotional strains and social stress increase symptoms and lead to a vicious circle. Previously, we showed significantly higher depression scores, and normal cortisol awakening responses in patients with primary focal hyperhidrosis (PFH). Stress reactivity in response to a (virtual) Trier Social Stress Test (TSST-VR) has not been studied so far. Therefore, we measured sweat secretion, salivary cortisol and alpha amylase (sAA) concentrations, and subjective stress ratings in affected and non-affected subjects in response to a TSST-VR. METHOD: In this pilot study, we conducted TSST-VRs and performed general linear models with repeated measurements for salivary cortisol and sAA levels, heart rate, axillary sweat and subjective stress ratings for two groups (diagnosed PFH (n = 11), healthy controls (n = 16)). RESULTS: PFH patients showed significantly heightened sweat secretion over time compared to controls (p = 0.006), with highest quantities during the TSST-VR. In both groups, sweating (p < 0.001), maximum cortisol levels (p = 0.002), feelings of stress (p < 0.001), and heart rate (p < 0.001) but not sAA (p = 0.068) increased significantly in response to the TSST-VR. However, no differences were detected in subjective ratings, cortisol concentrations and heart rate between PFH patients and controls (pall > 0.131). CONCLUSION: Patients with diagnosed PFH showed stress-induced higher sweat secretion compared to healthy controls but did not differ in the stress reactivity with regard to endocrine or subjective markers. This pilot study is in need of replication to elucidate the role of the sympathetic nervous system as a potential pathway involved in the stress-induced emotional sweating of PFH patients.

Genome-wide linkage analysis of families with primary hyperhidrosis.

Primary focal hyperhidrosis (PFH, OMIM %144110) is a genetically influenced condition characterised by excessive sweating. Prevalence varies between 1.0-6.1% in the general population, dependent on ethnicity. The aetiology of PFH remains unclear but an autosomal dominant mode of inheritance, incomplete penetrance and variable phenotypes have been reported. In our study, nine pedigrees (50 affected, 53 non-affected individuals) were included. Clinical characterisation was performed at the German Hyperhidrosis Centre, Munich, by using physiological and psychological questionnaires. Genome-wide parametric linkage analysis with GeneHunter was performed based on the Illumina genome-wide SNP arrays. Haplotypes were constructed using easyLINKAGE and visualised via HaploPainter. Whole-exome sequencing (WES) with 100x coverage in 31 selected members (24 affected, 7 non-affected) from our pedigrees was achieved by next generation sequencing. We identified four genome-wide significant loci, 1q41-1q42.3, 2p14-2p13.3, 2q21.2-2q23.3 and 15q26.3-15q26.3 for PFH. Three pedigrees map to a shared locus at 2q21.2-2q23.3, with a genome-wide significant LOD score of 3.45. The chromosomal region identified here overlaps with a locus at chromosome 2q22.1-2q31.1 reported previously. Three families support 1q41-1q42.3 (LOD = 3.69), two families share a region identical by descent at 2p14-2p13.3 (LOD = 3.15) and another two families at 15q26.3 (LOD = 3.01). Thus, our results point to considerable genetic heterogeneity. WES did not reveal any causative variants, suggesting that variants or mutations located outside the coding regions might be involved in the molecular pathogenesis of PFH. We suggest a strategy based on whole-genome or targeted next generation sequencing to identify causative genes or variants for PFH.

Glucocorticoid receptor gene variants and lower expression of NR3C1 are associated with cocaine use.

Animal and cross-sectional human studies suggest that chronic cocaine use is associated with altered responsivity of the hypothalamic-pituitary-adrenal axis to stress. Moreover, increased susceptibility to stress has been proposed as an important factor for development, maintenance and relapse of cocaine addiction. As the glucocorticoid receptor gene (NR3C1) mediates genomic effects of the stress hormone cortisol, we investigated NR3C1 expression and the association of NR3C1 genotypes with cocaine use, addiction and comorbid psychiatric symptoms in 126 chronic cocaine users and 98 stimulant-naïve healthy controls. A comprehensive psychiatric assessment was performed including severity of depressive symptoms and current psychological distress. Whole blood NR3C1 mRNA levels were determined and six NR3C1 polymorphisms (rs10482605, rs41423247, rs10052957, rs6189, rs56149945 and rs6198) were genotyped. Compared to controls, cocaine users showed significantly lower NR3C1 expression (P < 0.001), which was not affected by NR3C1 genotypes. In controls, rs41423247 [P < 0.01, false discovery rate (FDR)-corrected], haplotype 2 and haplotype 3 (both P < 0.05, FDR-corrected) were associated with altered NR3C1 gene expression. Haplotype 3 (including minor alleles of rs10052957 and rs41423247) was associated with an increased risk for cocaine addiction (odds ratio = 2.74, P < 0.05, uncorrected). Moreover, addicted cocaine users carrying haplotype 3 showed higher depression scores (P < 0.01, FDR-corrected) than noncarriers. Considering possible confounding effects of alcohol and/or depression, we conclude that chronic cocaine use is associated with lower NR3C1 gene expression suggesting possible direct effects of the drug on the biological adaptation of stress-related genes. Finally, we postulate that haplotype 3 of NR3C1 might serve as a potential risk factor for stimulant addiction and associated psychiatric symptoms.

Sex, ADHD symptoms, and CHRNA5 genotype influence reaction time but not response inhibition.

People showing symptoms of attention deficit hyperactivity disorder (ADHD) often present an impairment of reaction time and response inhibition. These executive functions are influenced by nicotinergic acetylcholine receptors (nAchr) as mediators of cholinergic signaling, and show differences between both sexes. We examined the effects of two functional polymorphisms rs3841324 (S/L) and rs16969968 (G/A) of the cholinergic gene CHRNA5, ADHD symptoms and sex on response inhibition/reaction time in the Stop Signal Task. In the analyses, 183 participants (52.4% females) were included. In participants carrying the diplotype (SS_GG), men with ADHD symptoms responded faster, while men without ADHD symptoms were slower than women (F = 5.313; p = 0.023; ηp ² = 0.034). Although explorative, this threefold interaction on reaction time but not response inhibition extend previous findings, suggesting a moderating effect of ADHD symptoms in men carrying the CHRNA5 diplotype SS_GG and might inspire research on genotype- and gender-specific ADHD medication.

Promoter haplotypes of the corticotropin-releasing hormone encoding gene modulate the physiological stress response in vitro and in vivo.

The corticotropin-releasing hormone (CRH) is a neuropeptide mediating stress responses. CRH exerts effects via the hypothalamus pituitary adrenal axis as well as immediate effects on the sympathetic-adrenal-medullary system. Genetic variants of the CRH promoter were previously found to be associated with altered CRH promoter activity and physiological reactions. Functional characterization of three CRH promoter haplotypes have been performed in vitro using a reporter gene assay under different stimulation conditions. Furthermore, 232 healthy subjects were genotyped and the influence of CRH haplotypes on basal parameters such as post-awakening cortisol and blood pressure as well as on stress reactivity measured after socially evaluated cold pressor test (SeCPT) was investigated. In vitro, CRH haplotype 2 showed the highest promoter activity under baseline conditions and after forskolin stimulation compared with other haplotypes. Forskolin treatment resulted in a two fold increase of haplotype 2 promoter activity compared with the baseline condition. Cell line-dependent promoter activation was found after hydrocortisone treatment. In vivo, CRH haplotype 2 carriers showed significant higher baseline blood pressure (p = .002) and blood pressure after SeCPT (p < .001), but did not differ in cortisol levels. This study provides converging evidence for the importance of CRH promoter variants on physiological stress response parameters.

Temporal dynamics of cortisol-associated changes in mRNA expression of glucocorticoid responsive genes FKBP5, GILZ, SDPR, PER1, PER2 and PER3 in healthy humans.

Secretion of the stress hormone cortisol follows a circadian rhythm and is stimulated following stress exposure. Cortisol regulates the transcription of several genes, primarily through activation of the glucocorticoid receptor (GR). Previously, we showed an upregulation of PERIOD genes PER1 and PER3 after pharmacological/glucocorticoid challenge in vivo and in vitro. The current study aims to investigate the temporal association between unstimulated, diurnal cortisol secretion and the expression of selected GR-target genes (PER1, PER2, PER3, FKBP5, GILZ and SDPR) in vivo to determine the timing of the most pronounced coupling between cortisol and mRNA expression. Unstimulated plasma and saliva cortisol concentrations and gene expression levels in whole blood were measured every 15 min from early morning until 16:00 h in 18 healthy men. Time-lagged correlations of cortisol concentrations with mRNA expression levels were assessed allowing lags between -240 and + 240 min. Strong positive correlations at non-zero lags between cortisol levels and the expression of FKBP5 (plasma: r = 0.74 (CI = 0.65-0.81), p < 0.001, lag + 90 min; saliva: r = 0.71 (CI = 0.61-0.78), p < 0.001, lag + 75 min), and GILZ (plasma: r = 0.59 (CI = 0.46-0.69), p < 0.001, lag + 30 min; saliva r = 0.53 (CI = 0.41-0.63), p < 0.001, lag +15 min) were observed. Expressions of PERIOD genes and SDPR correlated only weakly with cortisol (all |r| < 0.25). Our findings demonstrate strong correlations between cortisol secretion and gene expression in humans under unstimulated conditions. The observed time-lags can guide future research aiming to characterize glucocorticoid-dependent gene expression in clinical samples with stress-related disorders.

Glucocorticoid receptor variants in childhood attention-deficit/hyperactivity disorder and comorbid psychiatric disorders.

Stress results in a variety of neuroendocrine, immune and behavioral responses and represents a risk factor for many disorders. Following exposure to stress, glucocorticoids are secreted from the adrenal cortex and act via the ligand-activated glucocorticoid receptor (GR). Several polymorphisms of the GR-encoding gene NR3C1 have been described and functionally investigated. However, the impact of these variants on complex diseases such as Attention-Deficit/Hyperactivity Disorder (ADHD) is still unclear. In this study, 251 children with ADHD, 19 affected and 35 unaffected siblings, and their parents were included in a family-based association study assessing seven common variants of NR3C1 (TthIIII_rs10052957; NR3C1-I_rs10482605; ER22/23EK_rs6189/rs6190; N363S_rs56149945; BclI_rs41423247; GR-9beta_rs6198). A four-marker haplotype (TthIIII-NR3C1-I-ER22/23EK) was nominally associated with ADHD. In addition, in index children with ADHD, associations with comorbid disorders, inattentive and hyperactive-impulsive symptoms were explored. N363S minor allele carriers were more likely to show comorbid conduct disorder (CD). In our study, NR3C1 variants moderately affected ADHD and had a significant effect on comorbid CD. Therefore, NR3C1 as an important gene of the hypothalamic-pituitary-adrenal axis seems to be particularly relevant for the pathophysiology of ADHD combined with comorbid CD. For a deeper understanding, investigations in larger samples of healthy, ADHD and CD individuals are warranted.

The acute and temporary modulation of PERIOD genes by hydrocortisone in healthy subjects.

The physiological stress system and the circadian clock system communicate with each other at different signaling levels. The steroid hormone cortisol, the end-effector of the hypothalamus-pituitary-adrenal axis, is released in response to stress and acts as a mediator in circadian rhythms. We determined the effect of escalating cortisol doses on the expression of PERIOD genes (PER1, PER2 and PER3) in healthy subjects and analyzed whether the glucocorticoid receptor (GR) is involved in the cortisol-mediated PERIOD gene expression. Forty participants (50% males and 50% females) were randomly assigned to groups receiving a saline placebo solution or 3 mg, 6 mg, 12 mg and 24 mg of hydrocortisone. Blood was drawn every 15 min to measure quantitative gene expression of PER1, PER2 and PER3. A potential role of the GR was determined by an ex vivo study stimulating whole blood with hydrocortisone and RU486 (a GR antagonist). As a result, moderate doses of hydrocortisone produced an acute and temporary induction of PER1 and PER3 mRNA levels, whereas PER2 was not responsive to the hormone administration. The cortisol-dependent induction of PER1 was blocked by the GR antagonist in whole blood after treatment with hydrocortisone and RU486 ex vivo. In conclusion, acute pharmacological stress modulated the expression of PER1 and PER3 in whole blood temporarily in our short-term sampling design, suggesting that these circadian genes mediate stable molecular mechanisms in the periphery.

Polymorphisms of genes related to the hypothalamic-pituitary-adrenal axis influence the cortisol awakening response as well as self-perceived stress.

The hypothalamus-pituitary-adrenal (HPA) axis is a crucial endocrine system for coping with stress. A reliable and stable marker for the basal state of that system is the cortisol awakening response (CAR). We examined the influence of variants of four relevant candidate genes; the mineralocorticoid receptor gene (MR), the glucocorticoid receptor gene (GR), the serotonin transporter gene (5-HTT) and the gene encoding the brain-derived neurotrophic factor (BDNF) on CAR and self-perceived stress in 217 healthy subjects. We found that polymorphisms of GR influenced both, the basal state of the HPA axis as well as self-perceived stress. MR only associated with self-perceived stress and 5-HTT only with CAR. BDNF did not affected any of the investigated indices. In summary, we suggest that GR variants together with the CAR and supplemented with self reports on perceived stress might be useful indicators for the basal HPA axis activity.

The cardiovascular and hypothalamus-pituitary-adrenal axis response to stress is controlled by glucocorticoid receptor sequence variants and promoter methylation.

BACKGROUND: Gender, genetic makeup, and prior experience interact to determine physiological responses to an external perceived stressor. Here, we investigated the contribution of both genetic variants and promoter methylation of the NR3C1 (glucocorticoid receptor) gene to the cardiovascular and hypothalamus-pituitary-adrenal (HPA) axis response to the socially evaluated cold pressor test (seCPT). RESULTS: Two hundred thirty-two healthy participants were recruited and underwent the experiment. They were randomly assigned to either the seCPT group (cold water) or a control group (warm water). The seCPT group had a clear stress reaction; salivary cortisol levels and peak systolic and diastolic blood pressure all increased significantly compared to the control group. GR genotype (TthIIII, NR3C1-I, 1H, E22E, R23K, BclI and 9beta) and methylation data were obtained from 218 participants. Haplotypes were built from the GR genotypes, and haplotype 2 (minor allele of BclI) carriers had a higher cortisol response to the seCPT in comparison to non-carriers (20.77 ± 13.22; 14.99 ± 8.42; p = 0.034), as well as independently of the experimental manipulation, higher baseline heart rate (72.44 ± 10.99; 68.74 ± 9.79; p = 0.022) and blood pressure (115.81 ± 10.47; 111.61 ± 10.74; p = 0.048). Average methylation levels throughout promoter 1F and 1H were low (2.76 and 1.69 %, respectively), but there was a strong correlation between individual CpGs and the distance separating them (Pearson's correlation r = 0.725, p = 3.03 × 10(-26)). Higher promoter-wide methylation levels were associated with decreased baseline blood pressure, and when incorporated into a linear mixed effect model significantly predicted lower systolic and diastolic blood pressure evolution over time in response to the experimental manipulation. The underlying genotype significantly predicted methylation levels; particularly, the homozygous BclI minor allele was associated with higher methylation in promoter 1H (p = 0.042). CONCLUSIONS: This is one of the first studies linking epigenetic modifications of the GR promoter, receptor genotype and physiological measures of the stress response. At baseline, there were clear genetic and epigenetic effects on blood pressure. The seCPT induced a strong cardiovascular and HPA axis response, and both systems were affected by the functional genetic variants, although methylation also predicted blood pressure reactivity. The return to baseline was predominantly influenced by the genomic sequence. Overall, the physiological response to the seCPT is controlled by an exquisite mix of genetic and epigenetic factors.

The role of the glucocorticoid receptor gene (NR3C1) for the processing of aversive stimuli.

The glucocorticoid receptor (GR) is a crucial component of the hypothalamus-pituitary-adrenal (HPA) axis and as such a part of the stress response system. An impairment of the GR not only alters the level of glucocorticoids, but also modulates cognitive functions and the processing of emotional stimuli. We tested the effects of functional polymorphisms of the GR-encoding gene (NR3C1) on the processing of emotional stimuli on a basal level. In a sample of n=182 participants, we found a haplotype (NR3C1-CTGGACA) to modulate the performance in an emotional reaction time task. Compared to non-carriers, participants who carried the haplotype were quicker to react after aversive stimuli had been presented. In contrast, the presence of the haplotype had no effect on the processing of neutral stimuli. We conclude that properties of the glucocorticoid receptor contribute to the processing of emotional stimuli and influence the intensity of their processing even in the absence of acute stressors.

Individual response speed is modulated by variants of the gene encoding the alpha 4 sub-unit of the nicotinic acetylcholine receptor (CHRNA4).

Acetylcholine (ACh) is a known modulator of several domains of cognition, among them attention, memory and learning. The neurotransmitter also influences the speed of information processing, particularly the detection of targets and the selection of suitable responses. We examined the effect of the rs1044396 (C/T) polymorphism of the gene encoding the nicotinic acetylcholine receptor α4-subunit (CHRNA4) on response speed and selective visual attention. To this end, we administered a Stroop task, a Negative priming task and an exogenous Posner-Cuing task to healthy participants (n = 157). We found that the CHRNA4 rs1044396 polymorphism modulated the average reaction times (RTs) across all three tasks. Dependent on the C allele dosage, the RTs linearly increased. Homozygous T allele carriers were always fastest, while homozygous C allele carriers were always slowest. We did not observe effects of this polymorphism on selective attention. In sum, we conclude that naturally occurring variations within the cholinergic system influence an important factor of information processing. This effect might possibly be produced by the neuromodulator system rather than the deterministic system of cortical ACh.

Sex matters! Interactions of sex and polymorphisms of a cholinergic receptor gene (CHRNA5) modulate response speed.

Acetylcholine influences the speed of information processing. We examined the effect of the rs3841324 polymorphism (L/S) and the rs16969968 (G/A) polymorphism on response speed in the Stroop task and the Negative priming task. These polymorphisms are located in the gene that encodes the nicotinic acetylcholine receptor α5-subunit (CHRNA5). Male carriers of the rs3841324 S/S genotype and the rs16969968 G/G genotype were faster than male carriers of at least one L allele or one A allele. In contrast, female carriers of the rs3841324 S/S genotype and the rs16969968 G/G genotype were slower than female carriers of at least one L allele or one A allele. These results indicate that the minor alleles of both polymorphisms modulate response speed in a sex-dependent, diametrically opposed manner.

Genes of the dopaminergic system selectively modulate top-down but not bottom-up attention.

Cognitive performance is modulated by the neurotransmitter dopamine (DA). Recently, it has been proposed that DA has a strong impact on top-down but not on bottom-up selective visual attention. We tested this assumption by analyzing the influence of two gene variants of the dopaminergic system. Both the catechol O-methyltransferase (COMT) protein and the dopamine transporter (DAT) protein are crucial for the degradation and inactivation of DA. These metabolizing proteins modulate the availability of DA, especially in the prefrontal cortex and basal ganglia. The functional COMT Val158Met polymorphism of the COMT gene represents two coding variants, valine and methionine. In Met allele carriers, the COMT activity is reduced three- to fourfold. A variable number of tandem repeats (VNTR) polymorphism exists in the DAT1 gene, which encodes DAT. The DAT density was reported to be about 50% higher for the DAT1 10-repeat than the DAT1 9-repeat allele. We assessed attention via two experimental tasks that predominantly measure either top-down processing (the Stroop task) or bottom-up processing (the Posner-Cuing task). Carriers of the Met allele of the COMT Val158Met polymorphism displayed better performance in the Stroop task, but did not outperform the other participants in the Posner-Cuing task. The same result was noted for carriers of the DAT1 10-repeat allele. From these findings, we suggest that normal variations of the dopaminergic system impact more strongly on top-down than on bottom-up attention.

Elevated social stress levels and depressive symptoms in primary hyperhidrosis.

Primary hyperhidrosis is defined as excessive sweating of certain body areas without physiological reasons. Hyperhidrotic individuals report a high psychological strain and an impairment of their quality of life. Thus, the aim of the study is to investigate the relation between hyperhidrosis and different psychological as well as physiological aspects of chronic stress as a co-factor for the etiology of depression. In this study, forty hyperhidrotic subjects were compared to forty age- and sex-matched healthy control subjects. The Trier Inventory of Chronic Stress ('Trierer Inventar zum chronischen Stress': TICS), the Beck Depression Inventory (BDI-II) and the Screening for Somatoform Disorders (SOMS-2) were used to examine the correlation between primary hyperhidrosis and stress as well as accompanying depressive and somatic symptoms. The cortisol awakening response of each subject was analyzed as a physiological stress correlate. In hyperhidrotics, we found a significant lack of social recognition as well as significantly more depressive symptoms compared to the control subjects. A subgroup of patients with axillary hyperhidrosis had the highest impact on these increased issues of chronic stress, pointing to a higher embarrassment in these subjects. Especially in social situations, hyperhidrotics showed higher stress levels, whereby a vicious circle of stress and sweating is triggered. However, the cortisol awakening response did not significantly differ between hyperhidrotics and controls. Moreover, affected persons suffer from more depressive symptoms, which may be caused by feelings of shame and a lack of self-confidence. This initial study provides an impetus for further investigation to reveal a causative relationship between hyperhidrosis and its psychological concomitants.

Haplotype co-segregation with attention deficit-hyperactivity disorder in unrelated German multi-generation families.

Complex disorders have proved to be elusive in the search for underlying genetic causes. In the presence of large multi-generation pedigrees with multiple affected individuals, heritable familial forms of the disorders can be postulated. Observations of particular chromosomal haplotypes shared among all affected individuals within pedigrees may reveal chromosomal regions, in which the disease-related genes may be located. Hence, the biochemical pathways involved in pathogenesis can be exposed. We have recruited eight large Attention Deficit-Hyperactivity Disorder (ADHD, OMIM: #143465) families of German descent. Densely spaced informative microsatellite markers with high heterozygosity rates were used to fine-map and haplotype chromosomal regions of interest in these families. In three subsets and one full family of the eight ADHD families, haplotypes co-segregating with ADHD-affected individuals were identified at chromosomes 1q25, 5q11-5q13, 9q31-9q32, and 18q11-18q21. Positive LOD scores supported these co-segregations. The existence of haplotypes co-segregating among affected individuals in large ADHD pedigrees suggests the existence of Mendelian forms of the disorder and that ADHD-related genes are located within these haplotypes. In depth sequencing of these haplotype regions can identify causative genetic mechanisms and will allow further insights into the clinico-genetics of this complex disorder.

The chromosome 15q14 locus for bipolar disorder and schizophrenia: is C15orf53 a major candidate gene?

Bipolar disorder (BD) and schizophrenia are complexly inherited and highly heritable disorders with currently unknown etiologies. Recently, two independent genome-wide association studies for BD identified a small region on chromosome 15q14-15.1, pointing to a locus close to the gene C15orf53. Previously, this genomic region was also found to co-segregate with periodic catatonia (SCZD10, OMIM %605419), an unsystematic schizophrenia according to Leonhard's classification, in several multiplex families, thus pointing to overlapping etiologies of both conditions. A susceptibility locus on chromosome 15q14-15.1 was narrowed down to a 4.38 Mb region in these affected families followed by mutation and segregation analyses of C15orf53. Association analysis of individuals affected by BD and/or SCZD10 (n = 274) and controls (n = 230) and expression analyses in distinct post-mortem human limbic brain tissues were conducted. C15orf53 revealed no mutations in our SCZD10 family members, but segregation of two common haplotypes was found. No association of identified haplotypes was found in our case-control samples. Gene expression could be demonstrated for immune-system-derived cells but not for the post-mortem human limbic brain tissue. Our results indicate that C15orf53 is probably neither causative for the etiology of BD nor for SCZD10 in our samples.

Genetic susceptibility for individual cooperation preferences: the role of monoamine oxidase A gene (MAOA) in the voluntary provision of public goods.

In the context of social dilemmas, previous research has shown that human cooperation is mainly based on the social norm of conditional cooperation. While in most cases individuals behave according to such a norm, deviant behavior is no exception. Recent research further suggests that heterogeneity in social behavior might be associated with varying genetic predispositions. In this study, we investigated the relationship between individuals' behavior in a public goods experiment and the promoter-region functional repeat polymorphism in the monoamine oxidase A gene (MAOA). In a dynamic setting of increasing information about others' contributions, we analyzed differences in two main components of conditional cooperation, namely the players' own contribution and their beliefs regarding the contribution of other players. We showed that there is a significant association between individuals' behavior in a repeated public goods game and MAOA. Our results suggest that male carriers of the low activity alleles cooperate significantly less than those carrying the high activity alleles given a situation where subjects had to rely on their innate beliefs about others' contributions. With increasing information about the others' cooperativeness, the genetic effect diminishes. Furthermore, significant opposing effects for female subjects carrying two low activity alleles were observed.

Proteomic analysis of the cortisol-mediated stress response in THP-1 monocytes using DIGE technology.

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.

A new transcript splice variant of the human glucocorticoid receptor: identification and tissue distribution of hGR Delta 313-338, an alternative exon 2 transactivation domain isoform.

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.