Browsing by Author "Hapgood, Janet"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Open Access
    Antiretroviral drugs differentially modulate glucocorticoid activity via the glucocorticoid receptor in vitro
    (2019) Kuipa, Michael; Hapgood, Janet; Moliki, Mosoko; Maritz, Michelle
    Concurrent use of anti-retroviral drugs (ARVs) and progestin-based hormonal contraceptives is widespread. During times of stress and during glucocorticoid (GC) therapy, intracellular ARVs are in the presence of high concentrations of GCs, which regulate all aspects of immunity and inflammation via the glucocorticoid receptor (GR). However, the reciprocal modulation of ARV and steroid intracellular functions is relatively unexplored. In this study, the effects of the ARVs tenofovir disoproxil fumarate (TDF), dapivirine (DPV), and maraviroc (MVC) on activation of the GR and GR-regulated mRNA expression were investigated, in the absence and presence of select GR ligands. The effects of TDF and DPV on GR protein levels and phosphorylation were also determined. The inhibitory activity of these ARVs on HIV-1 infection in the presence of the progestins medroxyprogesterone acetate (MPA) and levonorgestrel (LNG), and a GR agonist, dexamethasone (DEX) was also assessed. This study shows that (0.01 nM-10 µM) TDF, DPV and MVC do not transactivate reporter gene expression via the unliganded GR exogenously expressed in the steroid receptor-deficient U2OS human osteosarcoma cell line, or alter the reporter gene transcriptional activity of (100 nM) MPA or LNG via the GR in these cells. However, (1 µM) TDF and DPV modulate the reporter gene transcriptional efficacy of (0.01 nM-10 µM) DEX via the GR. In the U2OS cell line model, (1 µM) TDF, but not DPV significantly decreased (1µM and 10µM) DEX-induced mRNA expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. TDF also appeared to decrease (1 µM) cortisol (CORT)- and MPA-induced GILZ mRNA expression. This may be mediated by the apparent increase in (100 nM and 1µM) DEXinduced phosphorylation at Serine 226 on the GR, observed in the presence of (1µM) TDF in this study. DPV and TDF (at 1µM) did not significantly alter GR protein levels, or cell-viability in the absence and presence of (100 nM) DEX, CORT or MPA in U2OS cells. However, (1 µM) DPV and TDF alone, significantly altered cell viability in peripheral blood mononuclear cells (PBMCs). In PBMCs, (1 µM) TDF, MVC and DPV alone altered basal GILZ mRNA expression and had variable, donor-specific effects on interleukin (IL)-6, IL-8, and interferon (IFN)-γ gene expression. In PBMCs from some of the nine donors tested, these ARVs had proinflammatory effects which may undermine their efficacy at preventing HIV-1 acquisition in pre-exposure prophylaxis products. Moreover, the ARVs proinflammatory effects may negatively impact HIV-1 disease progression and increase the risk of non-AIDS mortality in individuals using the ARVs therapeutically. Neither (1 µM) DPV, TDF nor MVC significantly altered the effects of (100 nM) DEX on the immunomodulatory genes assessed in PBMCs. DEX, MPA and LNG (at 100 nM) did not affect the anti-HIV-1 activity of the ARVs (at 1 µM) in PBMCs from the majority of the three donors tested in this study. Taken together, the results show that ARVs can modulate GR activity in an ARV-, steroid-, gene- and cell-specific manner, while the steroids investigated did not modulate ARV anti-HIV-1 activity.
  • Thumbnail Image
    Item
    Open Access
    Cross talk between the glucocorticoid receptor and the progesterone receptor in modulation of progestin responses and HIV-1 infection
    (2018) Bick, Alexis J; Hapgood, Janet; Avenant, Chanel
    Current epidemiological data showing that the use of the injectable contraceptive progestin Depotmedroxyprogesterone acetate (DMPA) is associated with increased HIV-1 acquisition is controversial. However, animal and ex vivo data reveal plausible biological mechanisms whereby MPA may increase HIV-1 acquisition. Relatively high levels of endogenous progesterone (P4) found in the luteal phase of the menstrual cycle have also been linked to increased HIV-1 acquisition in animal, clinical and ex vivo models. One of the central hypotheses of the present study was that the mechanism of MPA-induced increase in HIV-1 infection occurs via a different mechanism to that of the luteal phase. Furthermore, MPA has been shown to activate both the glucocorticoid receptor (GR) and its target, the progesterone receptor (PR) isoform B (PR-B), which are both transcription factors and regulate genes involved in immune function. Both the GR and PR are expressed in the cervix, the primary site of heterosexual HIV-1 infection. PR is regulated by endogenous estrogen (E2), of which the concentrations fluctuate throughout the menstrual cycle, and GR expression also varies in response to stress hormones, leading to conditions of varied relative levels of GR/PR. The immune-related consequences of changing the relative levels of GR and PR-B are not well understood. Therefore another hypothesis of this study was that changing the relative levels of GR/PR-B modulates HIV-1 infection and immunomodulatory gene expression in response to the GR/PR agonist, MPA. Since GR and PR-B recognize similar DNA target sequences and may regulate the same genes at the same time, the final hypothesis of the present study was that GR and PR-B reciprocally modulate each other’s activity, through possible association. To investigate the effects of exogenous hormones on HIV-1 infection and mechanisms thereof, peripheral blood mononuclear cells (PBMCs) and TZM-bl cervical cells were used as model systems for HIV-1 infection. These cells were stimulated with P4 and E2 at concentrations mimicking the menstrual cycle phases or with levels of MPA at the upper range of peak serum levels detected in DMPA users. Cells were infected with the R-tropic HIV-1 infectious molecular clone, HIV-1Bal_Renilla and luciferase assays were used to measure HIV-1 infection. Levels of HIV-1 CD4 receptor and CCR5 co-receptor protein or mRNA were measured by flow cytometry or qPCR, respectively, while activation of CD4+ T cells using the activation marker CD69 was measured by flow cytometry in PBMCs. To investigate the effects of changing GR/PR-B levels on HIV-1 infection and immune gene regulation, GR/PR levels were altered in End1/E6E7 immortalized endocervical and HeLa/TZM-bl cervical carcinoma cells by GR siRNA knockdown with or without the simultaneous over-expression of PR-B, and cells were stimulated with MPA or the GR agonist Dexamethasone. mRNA expression iii of key immunomodulatory genes in End1/E6E7 and HeLa cells was measured by qPCR. The modulation of GR activity by PR-B was assessed by promoter-reporter assay in COS1 and U2OS cells over-expressing GR and PR and stimulated with GR- and/or PR-specific ligands. Association of GR and PR-B was measured by co-immunoprecipitation in COS1 and MCF-7 cells, while co-localization of GR and PR-B was measured by confocal microscopy and super-resolution structured illumination microscopy in COS1 cells. MPA significantly increased HIV-1 infection in both PBMCs and TZM-bl cells, while luteal phase hormones did so to a lesser extent. However, MPA but not luteal phase hormones increased the ratio of CD4+/CD8+ T cells in PBMCs. MPA but not luteal phase hormones also increased CCR5 protein expression on CD4+ T cells in PBMCs and total CCR5 mRNA expression in TZM-bl cells. In addition, MPA but not luteal phase hormones increased activation of CD4+ T cells in PBMCs. Using a GR antagonist or GR siRNA, it was shown that the GR but not PR-B is required for MPA-, but not luteal phase hormone-induced increased HIV-1 infection in PBMCs and TZM-bls. The presence of PR-B altered the anti-inflammatory, GR-mediated regulation of some key immunomodulatory genes, including GILZ and IL-6, in End1/E6E7 and HeLa cells in response to MPA. In general, basal (unliganded) expression of immunomodulatory genes exhibited a pro-inflammatory profile in the presence of PR-B. Co-immunoprecipitation assays showed that GR and PR-B appeared to associate. Confocal microscopy suggested GR and PR co-localized in the nucleus in response to GR- and/or PRspecific ligands, while super-resolution microscopy showed that co-localization occurred in select regions within the nucleus. Taken together, MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving increased CD4+ T cell frequency (CD4+/CD8+ ratio), activation and increased expression of CCR5 on CD4+ T cells, and requiring the GR. Furthermore, PR-B modulates GR-mediated immune function gene regulation, via potential association and region-specific nuclear co-localization. This suggests that the relative levels of GR/PR may play an important role in determining the inflammatory and immune responses and HIV-1 infection in HIV-1 target cells, both in DMPA users and women not using hormonal contraception.
  • Thumbnail Image
    Item
    Open Access
    Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ
    (Public Library of Science, 2014) Verhoog, Nicolette; Allie-Reid, Fatima; Berghe, Wim Vanden; Smith, Carine; Haegeman, Guy; Hapgood, Janet; Louw, Ann
    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.
  • Thumbnail Image
    Item
    Open Access
    An investigation into the role of acetylation and ligand-dependent nuclear localisation in glucocorticoid receptor transcriptional regulation
    (2010) Hadley, Katie Emma; Hapgood, Janet
    The glucocorticoid receptor (GR) is a ligand-activated transcription factor which, due to its central role in anti-inflammatory responses, is a target of many therapeutically prescribed drugs. The GR undergoes multiple post-translational modifications, including phosphorylation and acetylation; however the role of GR acetylation in transactivation is unclear.
  • Thumbnail Image
    Item
    Open Access
    Investigation of differential TNFα-induced interleukin-6 gene regulation by synthesis progestins medroxyprogesterone acetate (MPA) and norethindrone acetate (NET-A) in human endocervical epithelial cells and the role of the unliganded glucocorticoid receptor
    (2010) Verhoog, Nicolette Jeanette Dorothy; Hapgood, Janet
    The endocervical mucosae of the female reproductive tract (FRT) not only serve as a physical barrier against microbial infection, but they also express a wide variety of immune mediators. The endocervical epithelial cells express a distinct profile of immune-regulators, which is higher than vaginal and ectocervical epithelial cells. Constant cytokine production would ensure rapid responses to infections and maintenance of the sterility of the upper genital tract. However, overproduction of cytokines could inhibit normal reproductive processes and stimulate excess growth and cell proliferation. The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)) are synthetic steroidal hormones designed to elicit progestational effects similar to those of the endogenous hormone progesterone (P4). They are extensively used as contraceptives and in hormone replacement therapy (HRT). Numerous studies, however, have reported that synthetic progestins affect immune function, increase the risk of sexually transmitted infections (STIs) and also change the morphology of the cervicovaginal mucosa. Despite these findings little is known about the molecular mechanisms of action of MPA and NET, in particular their differential effects on gene expression.
  • Thumbnail Image
    Item
    Open Access
    The partial characterisation of an NFKB homologue from the South African abalone haliotis midae utilising in vivo and in vitro techniques
    (2010) Ray, Roslyn Michelle; Coyne, Vernon; Hapgood, Janet
    Haliotis midae is an important marine gastropod that is commercially farmed along the South African coastline. The demand for the edible foot of the abalone far exceeds the supply, as such monitoring the health status of commercially farmed abalone is important if the demand is to be met. In farming conditions, bacterial infections can spread rapidly leading to mass mortalities amongst the abalone population. In order for treatment to be effective, there needs to be an effective monitoring system in place that can assess the health status of the abalone. This study sought to address these issues by identifying a candidate gene that could be an ideal biomarker with respect to a bacterial stress.