Mechanisms of brain glucocorticoid resistance in stress-induced psychopathologies

Exposure to stress activates the hypothalamic–pituitary–adrenal axis and leads to increased levels of glucocorticoid (GC) hormones. Prolonged elevation of GC levels causes neuronal dysfunction, decreases the density of synapses, and impairs neuronal plasticity. Decreased sensitivity to glucocorticoids (glucocorticoid resistance) that develops as a result of chronic stress is one of the characteristic features of stress-induced psychopathologies. In this article, we reviewed the published data on proposed molecular mechanisms that contribute to the development of glucocorticoid resistance in brain, including changes in the expression of the glucocorticoid receptor (GR) gene, biosynthesis of GR isoforms, and GR posttranslational modifications. We also present data on alterations in the expression of the FKBP5 gene encoding the main component of cell ultra-short negative feedback loop of GC signaling regulation. Recent discoveries on stressand GRinduced changes in epigenetic modification patterns as well as normalizing action of antidepressants are discussed. GR and FKBP5 gene polymorphisms associated with stress-induced psychopathologies are described, and their role in glucocorticoid resistance is discussed.     

Bombesin attenuates pre-mRNA splicing of glucocorticoid receptor by regulating the expression of serine-arginine protein p30c (SRp30c) in prostate cancer cells

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.     

Interindividual glucocorticoid sensitivity in young healthy subjects: the role of glucocorticoid receptor alpha and beta isoforms ratio.

Only few studies have addressed the interindividual variation and tissue specificity of glucocorticoid (GC) sensitivity in healthy individuals, a phenomenon observed in pathological conditions. Alternative splicing of the glucocorticoid receptor (GR) produces alpha and beta isoforms. GRbeta has dominant-negative effects on hormone-induced GRalpha effects, and an increased expression of the GRbeta has been associated with glucocorticoid resistance. We determined, using a simple, rapid, and accurate Real-Time PCR assay, the individual mRNAs expression of GRalpha and GRbeta in 26 normal subjects (mean+/-SE, age 30+/-6 years; 12 males and 14 females), in order to evaluate the role of these isoforms in glucocorticoid sensitivity in health. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeper gene. GRalpha/GAPDH, GRbeta/GAPDH and GRalpha/GRbeta ratios showed a normal distribution. We observed a higher expression of GRalpha compared to GRbeta and an interindividual variability in the GRalpha, GRbeta, and GAPDH gene expressions in the young healthy population. In addition, no correlation was observed between GRalpha/GRbeta ratio and the dexamethasone (DEX) doses needed to suppress plasma cortisol, GRalpha/GRbeta ratio and the concentration of DEX that caused inhibition of Con-A stimulated cell proliferation, and GRalpha/GRbeta ratio and the affinity of GR (Kd) of each subject. Therefore, the variability of GC sensitivity observed in normal subjects can not be ascribed to the variation in the GRalpha and GRbeta expression.     

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.     

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.