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

Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ

Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg . Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ’s involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ

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

The Injectable Contraceptive Medroxyprogesterone Acetate Attenuates Mycobacterium tuberculosis-Specific Host Immunity Through the Glucocorticoid Receptor.

BACKGROUND: The effects of the widely used progestin-only injectable contraceptives, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A), on host susceptibility to Mycobacterium tuberculosis (Mtb) are unknown. METHODS: We recruited human immunodeficiency virus-uninfected females, not taking any contraceptives, from Cape Town, South Africa, to evaluate the effect of MPA, NET-A, and dexamethasone on Mtb containment in monocyte-derived macrophages co-incubated with purified protein derivative (PPD)-driven peripheral blood-derived effector cells. RESULTS: MPA (P < .005) and dexamethasone (P < .01), but not NET-A, significantly attenuated Mtb containment in Mtb-infected macrophages co-cultured with PPD-driven effector cells at physiologically relevant concentrations and in a dose-dependent manner. Antagonizing the glucocorticoid receptor with mifepristone (RU486) abrogated the reduction in Mtb containment. In PPD-stimulated peripheral blood mononuclear cells, MPA and dexamethasone, but not NET-A, upregulated (median [interquartile range]) regulatory T cells (5.3% [3.1%-18.2%]; P < .05), reduced CD4+ T-cell interferon-γ (21% [0.5%-28%]; P < .05) and granzyme B production (12.6% [7%-13.5%]; P < .05), and reduced CD8+ perforin activity (2.2% [0.1%-7%]; P < .05). RU486 reversed regulatory T-cell up-regulation and the inhibitory effect on Th1 and granzyme/perforin-related pathways. CONCLUSIONS: MPA, but not NET-A, subverts mycobacterial containment in vitro and downregulates pathways associated with protective CD8+- and CD4+-related host immunity via the glucocorticoid receptor. These data potentially inform the selection and use of injectable contraceptives in tuberculosis-endemic countries

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs

Glucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution

The original publication is available at http://www.journals.elsevier.com/steroids/Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10¯ 6 M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10 ¯10 -10 ¯ 9 M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18–27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.Post-prin

Not all progestins are the same : implications for usage

The original publication is available at www.sciencedirect.comRecent clinical evidence showing unexpected side-effects of progestins used in contraception and hormone replacement therapy has highlighted the importance of choice of synthetic progestin. The molecularmechanisms of action of the relatively nonspecific and most widely used synthetic progestins, medroxyprogesterone acetate and norethisterone, are discussed in the context of this recent clinical evidence. Future directions involving a more mechanism-based approach for improved therapeutics, with greater specificity and fewer side-effects, are discussed.Publishers' Versio

Synthetic progestins used in HRT have different glucocorticoid agonist properties

The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-EN or NET-A), are widely used as female contraceptive agents and in hormone replacement therapy (HRT). Competitive binding revealed that MPA displays a higher relative binding affinity than NET-A and progesterone (prog) for the human GR (Kd of 4.2 nM for dexamethasone (dex) and Ki's of 10.8, 270 and 215 nM for MPA, NET-A and prog, respectively). Furthermore, MPA displays much greater glucocorticoid (GC) transactivation agonist potency than NET-A or prog (EC50s of 1.1, 7.2, >1000 and 280 nM for dex, MPA, NET-A and prog, respectively) and much greater GC agonist potency for transrepression than NET-A or prog (EC50s of 0.21, 2.7, >100 and 26 nM for dex, MPA, NET-A and prog, respectively). In addition, MPA induces phosphorylation of the GR at Ser 211 to a much greater extent than NET-A or prog and protects the GR from partial trypsin digestion in vitro to a much greater extent than NET-A or prog at saturating concentrations. Together these results suggest that the differences in biological activity of the progestins are not merely due to differences in their affinity for the GR but also due to the induction of different conformational changes in the liganded-GR. MPA and NET-A therefore display very different GC-like properties compared to each other and to prog, and are likely to exhibit different side effects via the GR

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