Molecular mechanism of action of the glucocorticoid receptor:Role of ligand-dependent receptor phosphorylation and half-life in determination of ligand-specific transcriptional activity. : Contents Pages

Glucocorticoids mediate their effects by binding to the glucocorticoid receptor (GR), resulting in modulation of transcription of target genes via direct binding to DNA or tethering via proteinprotein interactions. A central question is what determines the rank order of ligand-selective transcription with different GR ligands for the same gene in the same cell. Using a panel of twelve GR ligands, including agonists, partial agonists and antagonists, the relationship between the extent of GR phosphorylation at S226, GR turnover and transcriptional response, was investigated using a variety of biochemical approaches. Using a phospho-S226-specific GR antibody, ligand-selective S226 phosphorylation was shown to occur in both COS-1 and U2OS cells, while GR phosphorylation at S226 was shown to inhibit maximal transactivation and transrepression efficacy. Attempts to identify the kinases responsible for this interaction were inconclusive but suggested a combination of kinases is responsible for the in vivo phosphorylation of the hGR in these cells. Similarly the rate of GR degradation was different for the different ligands. Interestingly, both ligand-selective GR phosphorylation and half-life were found to correlate with efficacy for transactivation and transrepression of model synthetic reporter genes, where agonists resulted in the greatest extent of phosphorylation and the fastest vii rate of GR turnover, suggesting a link between these functions. Furthermore experiments where transcription was blocked suggest that GR turnover does not require transcription. However, using a S226A GR mutant, as well as in experiments where GR turnover was blocked, it was established that neither phosphorylation of the GR at S226 nor GR degradation rate determines the rank order of ligand-selective GR transactivation. The mechanisms whereby GR phosphorylation influence GR-mediated transcription was further investigated using a triple phosphorylation deficient mutant. It was shown that phosphorylation at one or more of residues S203/S211/S226 is required for transactivation of a MMTV promoter but does not affect unliganded or agonist-induced GR degradation and acetylation. Additionally, it was shown that phosphorylation at S203/S211/S226 is not the sole determinant of co-activator p300 recruitment to the GR. Interestingly, GR-mediated transrepression via AP-1 is less sensitive to GR phosphorylation than GR-mediated transactivation, indicating different mechanisms in the role of GR phosphorylation on transactivation vs. transrepression. Pull-down and chromatin immunoprecipitation assays showed that phosphorylation of the GR at one or more of these residues are required for interaction of the GR with the co-activator GRIP-1 in vitro and for maximal recruitment of GR and GRIP-1 to the MMTV promoter in intact cells. Cellular fractionation showed that phosphorylation at these residues is not however required for GR nuclear localisation. Taken together these results support the conclusion that phosphorylation at one or more of S203/S211/S226 of the hGR is required for maximal transactivation response to enable GRIP-1 recruitment to the hGR

The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor

The glucocorticoid receptor (GR) regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr), can modulate the transcriptional response of the GR. Glucocorticoids (GCs) and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA) is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A). We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4 + T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex), cortisol and MPA induce apoptosis in primary CD4 + T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4 + T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4 + T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to susceptibility and progression of diseases such as HIV-1

The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor

Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ∼24 nM for transactivation of the anti-inflammatory GILZ gene and ∼4-20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells

Role of ligand-dependent GR phosphorylation and half-life in determination of ligand-specific transcriptional activity

A central question in glucocorticoid mechanism of action via the glucocorticoid receptor (GR) is what determines ligand-selective transcriptional responses. Using a panel of 12 GR ligands, we show that the extent of GR phosphorylation at S226 and S211, GR half-life and transcriptional response, occur in a ligand-selective manner. While GR phosphorylation at S226 was shown to inhibit maximal transcription efficacy, phosphorylation at S211 is required for maximal transactivation, but not for transrepression efficacy. Both ligand-selective GR phosphorylation and half-life correlated with efficacy for transactivation and transrepression. For both expressed and endogenous GR, in two different cell lines, agonists resulted in the greatest extent of phosphorylation and the greatest extent of GR downregulation, suggesting a link between these functions. However, using phosphorylation-deficient GR mutants we established that phosphorylation of the GR at S226 or S211 does not determine the rank order of ligand-selective GR transactivation. These results are consistent with a model whereby ligand-selective GR phosphorylation and half-life are a consequence of upstream events, such as ligand-specific GR conformations, which are maintained in the phosphorylation mutants

Ligand-selective transactivation and transrepression via the glucocorticoid receptor: Role of cofactor interaction

The mechanisms that determine ligand-selective transcriptional responses by the glucocorticoid receptor (GR) are not fully understood. Using a wide panel of GR ligands, we investigated the relationships between the potency and maximal response for transactivation via a glucocorticoid response element (GRE) and transrepression via both nuclear factor кB (NFкB) and activator protein-1 (AP-1) sites, relative binding affinity for the GR, as well as interaction with both coactivators and corepressors. The results showed ligand-selective differences in potency and efficacy for each promoter, as well as for a particular ligand between the three promoters. Ligand potency correlated with relative affinity for the GR for agonists and partial agonists in transactivation but not for transrepression. Maximal response was unrelated to relative affinity of ligand for GR for both transactivation and transrepression. A good and significant correlation between full length coactivator binding in two-hybrid assays and efficacy as well as potency of different receptor–steroid complexes for both transactivation and transrepression supports a major role for coactivator recruitment in determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to GRIP-1, as determined by both two-hybrid and DNA pull down assays, correlated positively with ligand-selective efficacy for transactivation of both a synthetic GRE reporter with expressed GR as well as of an endogenous gene via endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited strong interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. However, there was no correlation between ligand affinity for the GR and cofactor interaction. These results provide strong quantitative biochemical support for a model in which GR-mediated ligand-selective differential interaction with GRIP-1, SRC-1A, NCoR and SMRT is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression

Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR.

<p>(A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062895#pone-0062895-g001" target="_blank">Figure 1</a>. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p<0.001. (B) Western analysis of lysates prepared from approximately 4×10⁶ PBMCs. Whole cell lysates of COS-1 cells overexpressing the relevant steroid receptor (+ve) or empty vector (−ve) served as the controls. GAPDH was used as a loading control. Note that the upper strong band on the MR blot is a COS-1 cell-derived non-specific signal which is absent for PBMCs, while the MR signal is the faint band just below the non-specific band which is only seen in the positive control. We were unable to obtain a more-specific anti-MR antibody. (C) Conventional PCR of cDNA prepared from human PBMCs using primers specific for the relevant steroid receptor. The controls were prepared by PCR amplification of the relative steroid receptor cDNA from plasmid DNA. GAPDH served as a control for mRNA levels. MW: molecular weight; NTC: no template control. Error bars represent standard deviation.</p

GC and Vpr differentially regulate key genes involved in apoptosis.

<p>PBMCs were treated with or without 5 µM Vpr peptide as described previously and treated with or without 100 nM Dex, MPA, NET-A or P4 for 24 hrs. After treatment, RNA was extracted, reverse transcribed, and Bcl-2 (A) or, Bim (B) mRNA expression was measured by real time PCR, normalising to GAPDH expression levels. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p<0.05, 0.01 and 0.005 respectively. Error bars represent standard deviation.</p

Primers used for real time PCR.

<p>F = Forward; R = Reverse.</p

The GR is involved in MPA- and Vpr-mediated apoptosis in CD4⁺ T-cells.

<p>PBMCs were treated with vehicle (EtOH), 100 nM MPA, 1 µM RU486 or 100 nM MPA plus 1 µM RU486, in the absence (A) or presence (B) of 5 µM Vpr peptide for 24 hrs. Cells were stained and acquired by flow cytometry as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062895#s2" target="_blank">methods</a>. In A cells were not incubated with balanced isotonic glucose-HEPES buffer while in B, this buffer was used and a tryptic BSA digest served as a control wherever Vpr peptide was not added, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062895#s2" target="_blank">Methods</a>. The histograms show pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p<0.05, 0.01 and 0.005 respectively. Error bars represent standard deviation.</p