Assessment of the Link of ABCB1 and NR3C1 gene polymorphisms with the prednisolone resistance in pediatric nephrotic syndrome patients of Bangladesh: A genotype and haplotype approach.

Introduction: Nephrotic syndrome is a common pediatric kidney disease. Investigations on several genetic polymorphisms revealed an inconsistent influence on the resistance of patients to steroids. Objectives: This study aimed to identify the association of ABCB1 (1236C > T, 2677G > T, 3435C > T), NR3C1 (rs10482634, rs6877893), and CYP3A5 (CYP3A5*3) gene polymorphism as well as sociodemographic and clinicopathological parameters with the risk of developing prednisolone resistance in pediatric patients with nephrotic syndrome. Methods: A case-control analysis was performed on 180 nephrotic syndrome patients. Among them, 30 patients were classified as prednisolone resistant group, and 150 were classified as prednisolone sensitive group. Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: No significant association of 1236C > T polymorphism with the risk of prednisolone resistance (p > 0.05) was found. The GT heterozygous of 2677G > T was found to be significantly associated with the development of prednisolone resistance (OR = 3.9, p = 0.034). In the case of 3435C > T, a statistically significant association was observed in TC heterozygous and TT mutant homozygous genotypes (OR = 0.38, p = 0.047; OR = 3.06, p = 0.038, respectively) with prednisolone resistance. For rs10482634 polymorphism, the AG heterozygous and AG+GG genotypes were significantly linked with prednisolone resistance (OR = 2.40, p = 0.033; OR = 2.36, p = 0.034, respectively). We found no association with the risk of prednisolone resistance with rs6877893 and CYP3A5*3 polymorphism (p > 0.05). CTC and TGT haplotypes of ABCB1 and GA haplotype of NR3C1 were also associated with the increased risk of pediatric prednisolone resistance (OR = 4.47, p = 0.0003; OR = 2.71, p = 0.03; and OR = 4.22, p = 0.022, consecutively). We also observed the correlation of different sociodemographic and clinicopathological factors with prednisolone resistance in pediatric nephrotic syndrome. Conclusion: Our findings showed a significant association of ABCB1 and NR3C1 gene polymorphisms with prednisolone resistant pediatric nephrotic syndrome.

In vitro assessment of corticosteroid effects of eight chiral herbicides.

Information regarding the enantioselective endocrine disruption of chiral herbicides is scarce. This study assessed the disrupting effects of eight typical chiral herbicides on corticosteroids (including glucocorticoids and mineralocorticoids). Enantioselectivity of eight chiral herbicides were evaluated for their agonistic/antagonistic effects on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) with CHOK1 cell line using reporter gene assay. Their influence on the production of corticosteroids were further investigated in H295R cell line using enzyme-linked immunosorbent assay (ELISA). None of the racemates or enantiomers of eight chiral herbicides exhibited GR or MR agonistic activity at non-cytotoxic concentrations. However, rac-propisochlor and S-imazamox antagonized cortisol-induced transactivation of GR by 21.79% and 38.73% at the concentration of 1.0 × 10-7 M and 1.0 × 10-6 M, respectively, and R-napropamide remarkably attenuated aldosterone-induced MR transactivation by 68.78% at 1.0 × 10-6 M. The secretion of cortisol was significantly restrained after treated with 1.0 × 10-6 M rac-propisochlor and rac-/R-napropamide at the concentration of 1.0 × 10-6 M by 26.49%, 30.10% and 35.27%, respectively, while this glucocorticoid was remarkably induced by 1.0 × 10-5 M rac-diclofop-methyl and its two enantiomers at the concentration of 1.0 × 10-5 M by 75.60%, 100.1% and 68.78%, respectively. Exposure to rac-propisochlor (1.0 × 10-6 M), S-diclofop-methyl (1.0 × 10-5 M) or rac-/S-/R- acetochlor (1.0 × 10-6 M) and rac-/S-/R-lactofen (1.0 × 10-6 M) inhibited the secretion of aldosterone by approximately 40%. Our findings suggested that chiral herbicides disrupted corticosteroid homeostasis in an enantioselective way. Therefore, more comprehensive screening is required to better understand the ecological and health risks of chiral pesticides.

Repetitive switching between DNA-binding modes enables target finding by the glucocorticoid receptor.

Transcription factor mobility is a determining factor in the regulation of gene expression. Here, we have studied the intranuclear dynamics of the glucocorticoid receptor (GR) by using fluorescence recovery after photobleaching and single-molecule microscopy. First, we have described the dynamic states in which the GR occurs. Second, we have analyzed the transitions between these states by using a continuous-time Markov chain model and functionally investigated these states by making specific mutations in the DNA-binding domain. This analysis revealed that the GR diffuses freely through the nucleus and, once it leaves this free diffusion state, most often enters a repetitive switching mode. In this mode it alternates between slow diffusion as a result of brief nonspecific DNA-binding events, and a state of stable binding to specific DNA target sites. This repetitive switching mechanism results in a compact search strategy that facilitates finding of DNA target sites by the GR.This article has an associated First Person interview with the first author of the paper.

Assessment of endocrine disruption potential of essential oils of culinary herbs and spices involving glucocorticoid, androgen and vitamin D receptors.

Essential oils (EOs) of culinary herbs and spices are consumed on a daily basis. They are multicomponent mixtures of compounds with already demonstrated biological activities. Taking into account regular dietary intake and the chemical composition of EOs, they may be considered as candidates for endocrine-disrupting entities. Therefore, we examined the effects of 31 EOs of culinary herbs and spices on transcriptional activities of glucocorticoid receptor (GR), androgen receptor (AR) and vitamin D receptor (VDR). Using reporter gene assays in stably transfected cell lines, weak anti-androgen and anti-glucocorticoid activity was observed for EO of vanilla and nutmeg, respectively. Moderate augmentation of calcitriol-dependent VDR activity was caused by EOs of ginger, thyme, coriander and lemongrass. Mixed anti-glucocorticoid and VDR-stimulatory activities were displayed by EOs of turmeric, oregano, dill, caraway, verveine and spearmint. The remaining 19 EOs were inactive against all receptors under investigation. Analyses of GR, AR and VDR target genes by means of RT-PCR confirmed the VDR-stimulatory effects, but could not confirm the anti-glucocorticoid and anti-androgen effects of EOs. In conclusion, although we observed minor effects of several EOs on transcriptional activities of GR, AR and VDR, the toxicological significance of these effects is very low. Hence, 31 EOs of culinary herbs and spices may be considered safe, in terms of endocrine disruption involving receptors GR, AR and VDR.

Application of Monte Carlo cross-validation to identify pathway cross-talk in neonatal sepsis.

To explore genetic pathway cross-talk in neonates with sepsis, an integrated approach was used in this paper. To explore the potential relationships between differently expressed genes between normal uninfected neonates and neonates with sepsis and pathways, genetic profiling and biologic signaling pathway were first integrated. For different pathways, the score was obtained based upon the genetic expression by quantitatively analyzing the pathway cross-talk. The paired pathways with high cross-talk were identified by random forest classification. The purpose of the work was to find the best pairs of pathways able to discriminate sepsis samples versus normal samples. The results found 10 pairs of pathways, which were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways were identified according to analysis of extensive literature. Impact statement To find the best pairs of pathways able to discriminate sepsis samples versus normal samples, an RF classifier, the DS obtained by DEGs of paired pathways significantly associated, and Monte Carlo cross-validation were applied in this paper. Ten pairs of pathways were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways ((7) IL-6 Signaling and Phospholipase C Signaling (PLC); (8) Glucocorticoid Receptor (GR) Signaling and Dendritic Cell Maturation) were identified according to analysis of extensive literature.

Epigenetic modification of the oxytocin and glucocorticoid receptor genes is linked to attachment avoidance in young adults.

Attachment in the context of intimate pair bonds is most frequently studied in terms of the universal strategy to draw near, or away, from significant others at moments of personal distress. However, important interindividual differences in the quality of attachment exist, usually captured through secure versus insecure - anxious and/or avoidant - attachment orientations. Since Bowlby's pioneering writings on the theory of attachment, it has been assumed that attachment orientations are influenced by both genetic and social factors - what we would today describe and measure as gene by environment interaction mediated by epigenetic DNA modification - but research in humans on this topic remains extremely limited. We for the first time examined relations between intra-individual differences in attachment and epigenetic modification of the oxytocin receptor (OXTR) and glucocorticoid receptor (NR3C1) gene promoter in 109 young adult human participants. Our results revealed that attachment avoidance was significantly and specifically associated with increased OXTR and NR3C1 promoter methylation. These findings offer first tentative clues on the possible etiology of attachment avoidance in humans by showing epigenetic modification in genes related to both social stress regulation and HPA axis functioning.

Assessment of leukocyte activity in mice devoid of the glucocorticoid receptor in the noradrenergic system (GRDBHCre).

Disturbances in brain monoamines, overactivity of the hypothalamo-pituitary adrenal (HPA) axis and pro-inflammatory tendency in the immune system are the key features of depressive disorders. Recently, several murine lines with mutations in glucocorticoid receptors (GRs) have been generated and these animals may be utilized for study depressive-like disorders. In the present study, we have investigated whether selective ablation of GRs in noradrenergic neurons affects functional properties of leukocytes and redirects them towards pro-inflammatory activity. Transgenic mice selectively devoid of GRs on noradrenergic cells were constructed using the Cre/loxP approach. Peritoneal leukocytes were collected from mutant and wild type (WT) animals of both sexes and were cultured in vitro for 24h both in basal conditions and after application of selected pro- or anti-inflammatory stimuli. Metabolic activity and adherence were measured in basal conditions. Nitric oxide (NO) synthesis and arginase (ARG) activity were assessed as the markers of functional status of the cells. Because adult mutant mice lack adrenal medulla and thereby peripheral adrenaline, we modulated pro- and anti-inflammatory culture conditions by addition of noradrenaline (10-6M). Finally, effects of in vivo pro-inflammatory challenge (with intraperitoneal administration of lipopolysaccharide) on properties of leukocytes were assessed 24h (in both sexes) and 48h later (in males only). The experiments indicated that selective ablation of GR in noradrenergic neurons did not affect fundamental properties of peritoneal leukocytes and exerted effects only under conditions of selected pro- or anti-inflammatory stimuli in vitro. Stronger response to pro-inflammatory stimulation in terms of NO synthesis and ARG activity may suggest pro-inflammatory tendency in mutant mice. In vivo inflammatory challenge failed to show any effect of GR ablation on selected parameters of leukocyte activity. Both in vitro studies and in vivo challenge revealed mainly sex-related differences in leukocyte activity.

Income inequality, gene expression, and brain maturation during adolescence.

Income inequality is associated with poor health and social outcomes. Negative social comparisons and competition may involve the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes in underlying some of these complex inter-relationships. Here we investigate brain maturation, indexed by age-related decreases in cortical thickness, in adolescents living in neighborhoods with differing levels of income inequality and household income. We examine whether inter-regional variations relate to those in glucocorticoid receptor (HPA) and androgen receptor (HPG) gene expression. For each sex, we used a median split of income inequality and household income (income-to-needs ratio) to create four subgroups. In female adolescents, the high-inequality low-income group displayed the greatest age-related decreases in cortical thickness. In this group, expression of glucocorticoid and androgen receptor genes explained the most variance in these age-related decreases in thickness across the cortex. We speculate that female adolescents living in high-inequality neighborhoods and low-income households may experience greater HPA and HPG activity, leading to steeper decreases in cortical thickness with age.

Childhood abuse, promoter methylation of leukocyte NR3C1 and the potential modifying effect of emotional support.

AIM: To investigate childhood abuse victimization in relation to adult DNA methylation levels in a novel region of NR3C1, with emotional support as a possible modifier. MATERIALS & METHODS: 295 participants from the Black Women's Health Study. Multivariable linear regression models were used to compute differences in mean percent methylation levels. RESULTS: Women reporting childhood abuse victimization exhibited higher mean NR3C1 methylation levels than nonabused women, with a clear dose-response relationship. Childhood emotional support appeared to attenuate associations only among women with the highest levels of physical and sexual abuse. CONCLUSION: NR3C1 mean methylation was higher among women who reported childhood abuse. Further research is warranted to clarify whether or the extent to which childhood emotional support buffers the association.

AgRP Neuron-Specific Deletion of Glucocorticoid Receptor Leads to Increased Energy Expenditure and Decreased Body Weight in Female Mice on a High-Fat Diet.

Agouti-related protein (AgRP) expressed in the arcuate nucleus is a potent orexigenic neuropeptide, which increases food intake and reduces energy expenditure resulting in increases in body weight (BW). Glucocorticoids, key hormones that regulate energy balance, have been shown in rodents to regulate the expression of AgRP. In this study, we generated AgRP-specific glucocorticoid receptor (GR)-deficient (knockout [KO]) mice. Female and male KO mice on a high-fat diet (HFD) showed decreases in BW at the age of 6 weeks compared with wild-type mice, and the differences remained significant until 16 weeks old. The degree of resistance to diet-induced obesity was more robust in female than in male mice. On a chow diet, the female KO mice showed slightly but significantly attenuated weight gain compared with wild-type mice after 11 weeks, whereas there were no significant differences in BW in males between genotypes. Visceral fat pad mass was significantly decreased in female KO mice on HFD, whereas there were no significant differences in lean body mass between genotypes. Although food intake was similar between genotypes, oxygen consumption was significantly increased in female KO mice on HFD. In addition, the uncoupling protein-1 expression in the brown adipose tissues was increased in KO mice. These data demonstrate that the absence of GR signaling in AgRP neurons resulted in increases in energy expenditure accompanied by decreases in adiposity in mice fed HFD, indicating that GR signaling in AgRP neurons suppresses energy expenditure under HFD conditions.

The Association of Neighborhood Gene-Environment Susceptibility with Cortisol and Blood Pressure in African-American Adults.

BACKGROUND: African-American adults are disproportionately affected by stress-related chronic conditions like high blood pressure (BP), and both environmental stress and genetic risk may play a role in its development. PURPOSE: This study tested whether the dual risk of low neighborhood socioeconomic status (SES) and glucocorticoid genetic sensitivity interacted to predict waking cortisol and BP. METHODS: Cross-sectional waking cortisol and BP were collected from 208 African-American adults who were participating in a follow-up visit as part of the Positive Action for Today's Health trial. Three single-nucleotide polymorphisms were genotyped, salivary cortisol samples were collected, and neighborhood SES was calculated using 2010 Census data. RESULTS: The sample was mostly female (65 %), with weight classified as overweight or obese (M BMI = 32.74, SD = 8.88) and a mean age of 55.64 (SD = 15.21). The gene-by-neighborhood SES interaction predicted cortisol (B = 0.235, p = .001, r (2) = .036), but not BP. For adults with high genetic sensitivity, waking cortisol was lower with lower SES but higher with higher SES (B = 0.87). Lower neighborhood SES was also related to higher systolic BP (B = -0.794, p = .028). CONCLUSIONS: Findings demonstrated an interaction whereby African-American adults with high genetic sensitivity had high levels of waking cortisol with higher neighborhood SES, and low levels with lower neighborhood SES. This moderation effect is consistent with a differential susceptibility gene-environment pattern, rather than a dual-risk pattern. These findings contribute to a growing body of evidence that demonstrates the importance of investigating complex gene-environment relations in order to better understand stress-related health disparities.

Assessment of the role of cortisol and corticosteroid receptors in epidermal ionocyte development in the medaka (Oryzias latipes) embryos.

Cortisol is a pleiotropic glucocorticoid hormone that acts through the intracellular glucocorticoid receptors (GR). Cortisol affects many important biological functions in mammals, including immune function, behavior, stress, metabolism, growth and organogenesis. In fishes, cortisol has an additional function in the osmoregulatory activity of ionocytes (ICs). Although much progress has been made toward understanding cortisol action at the levels of adult osmoregulatory tissues, the developmental functions of cortisol and its receptors in ICs remain to be clarified. We first analyzed the total contents of both cortisol and corticosteroid receptor mRNAs (GR1, GR2 and MR) during medaka development. Although low levels of cortisol were detected during development of the medaka embryo, maternal GR1, GR2 and MR transcripts were detected at higher levels than zygotic transcripts. We investigated the effect of exogenous cortisol on IC number during medaka embryogenesis. We observed that cortisol treatment induced an earlier expansion of the IC population but did not modify the final IC number. Using functional genomic approaches, we also tested the involvement of GR1, GR2 and mineralocorticoid receptor (MR) in IC development by systematic knock-down with translation-blocking morpholinos. Only GR2 knock-down led to a reduction of the total number of ICs in the epidermis. In addition, a GR2 splice-blocking morpholino did not have any effect on the biogenesis of ICs, underscoring the importance of maternally inherited GR2 mRNAs. We propose that maternal GR2, but not GR1 or MR, is a major pathway in the IC biogenesis in medaka most likely through cortisol activation, and that cortisol exposition fine-tunes their developmental timing. These findings provide a framework for future research on the regulatory functions of corticosteroids in euryhaline fishes and provide medaka as an advantageous model to further elucidate the underlying molecular regulatory mechanisms of IC development.

Associations between DNA methylation of a glucocorticoid receptor promoter and acute stress responses in a large healthy adult population are largely explained by lifestyle and educational differences.

BACKGROUND: Glucocorticoids are the key regulators of the biological stress response and act by binding to glucocorticoid receptors (GR). Expression of GR is altered by DNA methylation. Methylation patterns in GR promoters have been shown to be highly variable between individuals, but little is known about the functional consequences of this variation for the acute stress response. The present study investigated associations between methylation status of the GR 1-C promoter and cortisol, cardiovascular and perceived stress responses to a psychosocial stress protocol in a large healthy adult population. METHODS: A total of 725 overall healthy men and women, aged 55-60 years, participated in a standardized psychosocial stress protocol consisting of three different stressors. At different stages during the stress protocol, salivary cortisol levels, continuous blood pressure and heart rate (HR) levels as well as perceived stress were measured. Stress reactivity was calculated as the increase between basal and peak measurements. Methylation status of the GR 1-C promoter was assessed in DNA isolated from peripheral blood samples using a methylation sensitive PCR assay for 675 of the 725 participants. RESULTS: A decrease in methylation of the GR 1-C promoter was associated with a decrease in stress reactivity as indicated by lower cortisol and lower HR reactivity. A 1% decrease in GR 1-C methylation corresponded with a cortisol decrease by 0.14% (95% CI: 0.03-0.25, p=0.02) and an HR decrease by 0.10 bpm (0.03-0.16, p=0.003). Adjusting for sex, lifestyle and education largely abolished these associations. A decrease in methylation of the GR 1-C promoter was also associated with an increase in stress perception as indicated by higher perceived stress (0.03 points [0.00-0.06, p=0.05]), lower perceived performance (-0.03 points [-0.05 to -0.01], p=0.02), and lower perceived control (-0.03 points [-0.05 to 0.00], p=0.04). After adjusting for sex and educational level the associations were no longer statistically significant. GR 1-C methylation status was not associated with blood pressure responses to the stress protocol. DISCUSSION: Although effects were small, variation in methylation status in the GR 1-C promoter was associated with physical and perceived acute stress responses. Interestingly, these associations could largely be explained by differences in lifestyle and education.

Assessment of SNPs associated with the human glucocorticoid receptor in primary open-angle glaucoma and steroid responders.

PURPOSE: While chronic glucocorticoid (GC) therapy leads to ocular hypertension in about one third of individuals, almost all primary open-angle glaucoma (POAG) patients show this response and are called "steroid responders." Two differentially spliced isoforms of the glucocorticoid receptor (GR), GRalpha and GRbeta, regulate GC responsiveness in trabecular meshwork (TM) cells. GRbeta acts as a dominant negative regulator of GC activity and is expressed at lower levels in glaucomatous TM cells, making them more sensitive to GCs. Several arginine/serine-rich splicing factor (SR) proteins have been implicated in alternative splicing of the GR. We have previously demonstrated that immunophilins FKBP5 and FKBP4 are required for GRalpha and GRbeta translocation into the nucleus, which is essential for their biologic activity. The purpose of the present study was to use single nucleotide polymorphism (SNP) genotyping to determine whether there are any allele frequency differences in GR, FKBP4/5, or SR genes between normal control, POAG, and steroid responder populations. METHODS: Clinically characterized individuals (400 normal controls, 197 POAG, and 107 steroid responders) were recruited from the U. Iowa Ophthalmology Clinics after IRB approved consent. Genotyping of DNA samples for 48 SNPs in SFRS3, SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 was done at GeneSeek using a mass spectroscopy based system. RESULTS: All 48 SNPs displayed high call rates (99%). There were no significant differences in allele frequencies or genotypes in SNPs for SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 between the 3 groups. Up to three SNPs in SFRS3 had p-values <0.05 when comparing controls to POAG or steroid responders, but this statistical significance was lost when the p values were adjusted for multiple measures. CONCLUSIONS: Although these 6 genes may be involved in the pathogenesis of GC-induced ocular hypertension, it does not appear that major heritable risk alleles in these genes are responsible for the development of GC-induced ocular hypertension or POAG.

Attenuating corticosterone levels on the day of memory assessment prevents chronic stress-induced impairments in spatial memory.

This study investigated whether chronic stress-induced spatial memory deficits were caused by changes in the hypothalamic-pituitary-adrenal axis, such as corticosterone (CORT) elevations on the day of memory assessment, rather than the consequence of structural changes in the hippocampus. Male Sprague-Dawley rats were restrained for 6 h/day/21 days, and spatial memory was assessed on the Y-maze on day 22. Ninety minutes before training, rats received a subcutaneous injection of vehicle or metyrapone, a CORT synthesis inhibitor, and then spatial memory was determined 4-h later. The highest dose of metyrapone (75 mg/kg, s.c.) was most effective at preventing stress-induced spatial memory deficits. Chronic stress increased total CORT levels following Y-maze exposure, while acute metyrapone treatment dose-dependently attenuated total and free (unbound) CORT levels in both stress and control conditions. Blood samples taken from a separate subset of chronically stressed rats showed that baseline CORT levels were similar across the restraint period. Finally, chronic stress down-regulated glucocorticoid, but not mineralocorticoid, receptor mRNA expression within the hippocampus (dentate gyrus, CA1, CA2, CA3). These findings suggest that chronic stress-induced spatial memory deficits may be mediated by hypothalamic-pituitary-adrenal axis dysregulation. Specifically, CORT elevations and reductions in hippocampal glucocorticoid receptor expression, at the time of behavioural assessment may be involved, as opposed to a direct effect that is solely dependent upon hippocampal structural changes. These results have significance for treating cognitive decline in conditions associated with elevated glucocorticoids that include subpopulations in ageing, depression, Cushing's disease and Alzheimer's disease.

A stochastic version of corticosteriod pharmacogenomic model.

The purpose of this study was to develop a stochastic version of corticosteriod fifth generation pharmacogenomic model. The Gillespie algorithm was used to generate the independent time courses of the receptor messenger RNA (mRNA). Initial parameters for the stochastic simulation were adapted from the study by Jin et al. The result obtained from the proposed stochastic model showed an overall agreement with the deterministic fifth generation model. This study suggested that because the stochastic model takes into account the "noise" nature of gene regulation, it would have potential application in pharmacogenomic modeling.

Association of estrogen receptor and glucocorticoid receptor gene polymorphisms with sporadic breast cancer.

We have utilized a cross-sectional association approach to investigate sporadic breast cancer. Polymorphisms in 2 candidate genes, ESRalpha and GRL, were examined in an unrelated breast cancer-affected and age-matched control population. Several polymorphic regions within the ESRalpha gene have been identified, and some alleles of these polymorphisms have been found to occur at increased levels in breast-cancer patients. Additionally, variations in GRL have the potential to disrupt cell transcription and may be associated with cancer formation. We analyzed 3 polymorphisms, from codons 10 (TCT to TCC), 325 (CCC to CCG) and 594 (ACA to ACG) of ESRalpha, and a highly polymorphic dinucleotide repeat, D5S207, located within 200 kb of the GRL. When allelic frequencies of the codon 594 (exon 8) ESR polymorphism were compared between affected and unaffected populations, a significant difference was observed (p = 0.005). Results from the D5S207 dinucleotide repeat located near GRL also indicated a significant difference between the tested case and control populations (p = 0.001). Allelic frequencies of the codon 10 and codon 325 ESR polymorphisms were not significantly different between populations (p = 0.152 and 0.181, respectively). Our results indicate that specific alleles of the ESR gene (alpha subtype) and a marker for the GRL gene locus are associated with sporadic breast-cancer development in the tested Caucasian population and justify further investigation of the role of these and other nuclear steroid receptors in the etiology of breast cancer.

Assessment of glucocorticoid receptor-heat shock protein 90 interactions in vivo during nucleocytoplasmic trafficking.

Unliganded glucocorticoid receptors (GRs) that reside in the cytoplasm exist as heteromeric complexes comprised minimally of 90-kDa heat shock protein (hsp90) hsp70 and p56, a 56-kDa immunophilin. The binding of hsp90 to the GR occurs primarily through its carboxy-terminal, ligand-binding domain. Dissociation of GR-associated proteins accompanies hormone binding and leads to the exposure of its various functional domains. Although an association with hsp90 presumably masks the GR nuclear localization signal sequence, the recent demonstration of the coimport of GR and hsp90 into nuclei has led to the hypothesis that hsp90 facilitates GR interactions with the nuclear transport machinery. In this report we examined whether the dynamics of GR/hsp90 interactions in vivo influences its trafficking both into and out of the nucleus. GR/hsp90 complexes were stabilized in vivo by the introduction of sodium molybdate to cultured cells using a liposome-mediated delivery system. In agreement with previous in vitro studies, we found that stabilization of GR/hsp90 complexes in live cells severely restricts hormone-dependent nuclear import of GR. Constitutive nuclear import of a GR deletion derivative that does not bind hsp90 is unaffected by intracellular administration of molybdate, demonstrating that the inhibitory effects of molybdate require the coupling of the nuclear localization signal sequence to the GR ligand-binding domain. Interestingly, molybdate treatment traps both GR and progesterone receptor in the cytoplasm of cells chronically exposed to hormone, indicating that shuttling GRs and progesterone receptors can export, but not reimport into nuclei in the presence of molybdate. This result implies that the reassociation of recycled receptors with hsp90 must be an obligatory step for receptors that exit the nucleus to reacquire the capacity for nuclear import.