Glucocorticoid Receptor pre-mRNA Research Articles

Transcriptional and metabolic effects of glucocorticoid receptor α and β signaling in zebrafish.

In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRβ. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRβ. In addition, specific GRβ overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRβ may act as a dominant-negative inhibitor of GRα when GRβ is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRβ. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRβ had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.

Super-High-Dose Methylprednisolone Does Not Improve Efficacy or Induce Glucocorticoid Resistance in Experimental Allergic Encephalomyelitis

Objective: To investigate whether a super-high dose (SHD) of methylprednisolone (MP) improves its efficacy or induces glucocorticoid (GC) resistance, and to explore the potential mechanisms of GC resistance in experimental allergic encephalomyelitis (EAE). Methods: The therapeutic effects of SHD and low-dose MP were evaluated in EAE by analyzing clinical scores, pathological changes and cytokine production. Immunohistochemistry and RT-PCR were used to investigate the expression of GC receptor (GR) isoforms and splicing factor SRp30c. Results: Both MP doses had similar therapeutic effects. The ratio of GRα to GRβ was positively correlated with clinical score changes. However, there was no difference in the GRα/GRβ ratio between SHD and low-dose MP groups. SRp30c mRNA was correlated with GRβ expression. Conclusion: This study indicates that the GRα/GRβ ratio is associated with GC sensitivity, and SRp30c may play an important role in promoting alternative splicing of GR pre-mRNA to generate GRβ in EAE rats. Compared with low-dose MP, SHD MP does not improve efficacy or induce GC resistance.

Alternative splicing in exon 9 of glucocorticoid receptor pre-mRNA is regulated by SRp40

Increasing evidence indicates that alternative splicing of human glucocorticoid receptor (GR) transcripts is implicated in the development of glucocorticoid resistance but the underlying mechanism was not well known. Serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 play an important role in the spliceosome assembly. In this study, we analyzed the effects of different SR proteins and hnRNP A1 on the alternative splicing of GR pre-mRNA in HeLa and 293T cells using a minigene transfection assay. Our results revealed that only SRp40 could induce a GRalpha to GRbeta shift of pre-mRNA splicing in exon 9 in HeLa cells and this effect induced by SRp40 was further confirmed by small interfering RNA study. However, in 293T cells, SRp40 could not induce this shift. These results indicated that SRp40 may influence the alternative splicing of GR pre-mRNA to regulate the ratio of GRalpha to GRbeta, and this effect is cell-dependent.

High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRalpha and GRbeta, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform. The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRalpha was 475 +/- 62 and 985 +/- 107 for PBMCs and neutrophils, respectively. For GRbeta, the MFI was 350 +/- 60 and 1,389 +/- 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRbeta staining to 2,497 +/- 140 (P < 0.05). No change in GRalpha expression was observed. This inversion of the GRalpha/GRbeta ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRbeta mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRalpha/GRbeta heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

An ACTH-producing small cell lung cancer expresses aberrant glucocorticoid receptor transcripts from a normal gene

ACTH production by non-pituitary tumors is generally not suppressible by exogenous glucocorticoid administration. We had postulated that defects in the glucocorticoid receptor (GR) signaling system might be responsible for this apparent glucocorticoid resistance and had previously demonstrated that DMS-79 cells, derived from an ectopic ACTH-producing tumor, express an abnormal GR mRNA. In this DMS-79 cell GR the sequence normally derived from exons 8 and 9 is replaced by sequence unmatched in the DNA databases. The protein encoded by this mRNA lacks the steroid-binding domain and does not function as a ligand-activated transcription factor. In the present work, we sought to identify the origin of the novel GR mRNA sequence. Southern blot analysis of DMS-79 genomic DNA showed no major structural alteration of the GR gene. Southern blotting of cosmid clones of the normal GR gene revealed that the novel DMS-79 GR mRNA sequence is derived from intron G, between exons 7 and 8. No splice site mutations were found in PCR-amplified DMS-79 DNA fragments surrounding the downstream splice junctions. Further sequencing indicated that the aberrant GR transcript appears to be generated by use of a consensus cleavage/polyadenylation signal found 3650 base pairs into the normal intron G. We conclude that abnormal GR pre-mRNA processing rather than a GR gene mutation confers glucocorticoid resistance on DMS-79 cells.