No abstract

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 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 GnRH receptor (GnRHR), a member of the G protein-coupled receptor family, is a central regulator of reproductive function in all vertebrates. The peptide hormone GnRH exerts its effects via binding to the GnRHR in pituitary gonadotropes. We investigated the mechanisms of regulation of transcription of the mGnRHR gene in the mouse pituitary gonadotrope L beta T2 cell line by GnRH and dexamethasone (dex). Reporter assays with transfected mGnRHR promoter show that both dex and GnRH increase transcription of the mGnRHR gene via an activating protein-1 (AP-1) site. Real-time PCR confirmed this on the endogenous mGnRHR gene, and small interfering RNA experiments revealed a requirement for the glucocorticoid receptor (GR) for both the dex and GnRH response. Chromatin immunoprecipitation (ChIP) and immunofluorescence assays provide evidence that both GnRH and dex up-regulate the GnRHR gene via nuclear translocation and interaction of the GR with the AP-1 region on the mGnRHR promoter. We show that GnRH activates the unliganded GR by rapid phosphorylation of the GR at Ser-234 in a GnRHR-dependent fashion to transactivate a GRE reporter gene in L beta T2 and COS-1 cells. Using kinase inhibitors, we established a direct link between GnRH-induced protein kinase C and MAPK activation, leading to unliganded GR phosphorylation at Ser-234 and transactivation of the glucocorticoid response element. Furthermore, we show that GnRH and dex synergistically activate the endogenous GnRHR promoter in L beta T2 cells, via a mechanism involving steroid receptor coactivator-1 recruitment to the GnRHR AP-1 region. Our results suggest a novel mechanism of rapid nongenomic cross talk between the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes via GnRHR-dependent phosphorylation and activation of the unliganded GR in response to GnRH.

No abstract

Pharmacological doses of glucocorticoids (GCs), acting via the glucocorticoid receptor (GR) to repress inflammation and immune function, remain the most effective therapy in the treatment of inflammatory and immune diseases. Since many patients on GC therapy exhibit GC-resistance and severe side-effects, much research is focussed on developing more selective GCs and combination therapies, with greater anti-inflammatory potency. GCs mediate their classical genomic transcriptional effects by binding to the cytoplasmic GR, followed by nuclear translocation and modulation of transcription of target genes by direct DNA-binding of the GR or its tethering to other transcription factors. Recent evidence suggests, however, that the responses mediated by the GR are much more complex and involve multiple parallel mechanisms integrating simultaneous signals from other receptors, both in the absence and presence of GCs, to shift the sensitivity of a target cell to GCs. The level of cellular stress, immune activation status, or the cell cycle phase may be crucial for determining GC sensitivity and GC responsiveness as well as subcellular localization of the GR and GR levels. Central to the development of new drugs that target GR signalling alone or as add-on therapies, is an in-depth understanding of the molecular mechanisms of GC-independent GR desensitization, priming and activation of the unliganded GR, as well as synergy and cross-talk with other signalling pathways. This review will discuss the information currently available on these topics and their relevance to immunotherapy, as well as identify unanswered questions and future areas of research.

TNF␣ signaling and cytokine levels play a crucial role in cervical immunity and the host response to infections. We investigated the role of liganded and unliganded glucocorticoid receptor (GR) in IL-6 and IL-8 gene regulation in response to TNF␣ in the End1/E6E7 immortalized human endocervical epithelial cell line. In the absence of glucocorticoids, both decreasing GR protein levels by an siRNA strategy and results with the GR antagonist RU486 suggest a role for the unliganded GR in reduction of TNF␣-induced IL-6 and IL-8 mRNA levels in End1/E6E7 cells. Moreover, GR-dependent repression of endogenous IL-6 mRNA as well as a minimal IL-6 promoter-reporter gene is also demonstrated in COS-1 cells in the absence of GR ligand, suggesting a transcriptional mechanism that is not cell-specific. TNF␣ induced recruitment of both the unliganded GR and GRinteracting protein type 1 (GRIP-1) to the IL-6 promoter. This, together with GRIP-1 overexpression studies, suggests a function for GRIP-1 as a GR co-repressor in this context. TNF␣ was shown to induce phosphorylation of the unliganded human GR at Ser-226 but not Ser-211, unlike dexamethasone, which induced hyperphosphorylation at both serine residues. Ser-226 is shown to be required for the ligand-independent GR-mediated repression of IL-6 in response to TNF␣. Taken together, these results support a model whereby the unliganded GR attenuates TNF␣-stimulated IL-6 transcription by a mechanism involving selective phosphorylation and recruitment of the unliganded GR and GRIP-1 to the IL-6 promoter. These findings suggest the presence of a novel autoregulatory mechanism that may prevent overproduction of IL-6 in the endocervix, possibly protecting against negative effects of excessive inflammation.

High usage of progestin-only injectable contraceptives, which include the intramuscular injectables depo-medroxyprogesterone acetate (DMPA-IM, Depo-Provera) and norethisterone (NET) enanthate (NET-EN or Nur-Isterate), correlates worldwide with areas of high HIV-1 prevalence. Epidemiological data show a significant association between usage of DMPA-IM and increased HIV-1 acquisition but no such association from limited data for NET-EN. Whether MPA and NET have similar effects on HIV-1 acquisition and pathogenesis, and the relationship between these effects and the dose of MPA, are critical issues for women’s health and access to suitable and safe contraceptives. We show for the first time that MPA, unlike NET, significantly increases HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and a cervical cell line model. The results provide novel evidence for a biological mechanism whereby MPA, acting via the glucocorticoid receptor (GR), increases HIV-1 replication by at least in part increasing expression of the CCR5 HIV-1 coreceptor on target T-lymphocytes. MPA, unlike NET, also increases activation of T-cells and increases the CD4/CD8 ratio, suggesting that multiple mechanisms are involved in the MPA response. Our data offer strong support for different biological mechanisms for MPA versus NET, due to their differential GR activity. The dose-dependence of the MPA response suggests that significant effects are observed within the range of peak serum levels of progestins in DMPA-IM but not NET-EN users. Dose-response results further suggest that effects of contraceptives containing MPA on HIV-1 acquisition and disease progression may be critically dependent on dose, time after injection and intrinsic factors that affect serum concentrations in women.