Browsing by Author "Hapgood, Janet"

Now showing 1 - 9 of 9
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    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.
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    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.
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    Epidermal growth factor-mediated activation of the unliganded glucocorticoid receptor in A549 cells
    (2025) Kemp, Calvin; Hapgood, Janet; Avenant, Chanel
    Multiple studies have described ligand-independent glucocorticoid receptor (GR) activation in which non-steroidal ligands are able to activate the GR in the absence of glucocorticoids. This study investigates the regulatory effects of epidermal growth factor (EGF) on cytokine gene expression in order to determine whether previously published examples of EGF repressing the expression of interleukin 6 (IL-6) mRNA occur in other cell types and whether the effects of EGF are mediated via the GR. This study provides the first evidence of a single ligand (EGF) causing ligand-independent GR activation, resulting in the regulation of cytokine genes in the lung epithelium fibroblast A549 cell line. Evidence suggests that the EGF-induced repression acts on signalling via the TLR-2 receptor but not via the protein kinase C (PKC) pathway. The EGF response is shown to act via the nuclear factor kappa B (NF-κB) promoter element in a GR-dependent manner. Some mechanistic insights into the regulation are provided by demonstrating that EGF results in site-specific GR phosphorylation, while EGF does not induce GR turnover or alter GR protein expression levels. A novel finding is that regulation of IL-6 mRNA expression is shown to require protein translation. Taken together, this study provides evidence that expands on the knowledge of how EGF may affect immune function and provides proof of the mechanisms likely involved.
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    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.
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    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.
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    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.
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    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.
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    Relative level of glucocorticoid and progesterone receptor: implications for immune and reproductive functions in women
    (2025) Appadoo, Prettysha; Hapgood, Janet; Avenant, Chanel
    Background: The glucocorticoid receptor (GR) and progesterone receptor (PR) are very closely related in terms of their amino acid sequence and three-dimensional structure. PR ligands have been previously reported to cross-react with the GR. The cross-talk effects of certain PR ligands via the GR may impact immune function and risk of acquiring sexually transmitted infections (STIs). This suggests that GR and PR expression levels are relevant to immunity against pathogens in the female genital tract (FGT) and systemic immunity. PR protein expression is well characterised ex vivo in FGT tissues while very little is known about GR protein expression ex vivo in the FGT. Immune cells patrol the FGT but the expression of GR as well as PR proteins in resident immune cells of the FGT has not been previously reported. Aims and Hypotheses: This study aims to assess potential cross-reactivity of GR and PR primary antibodies as it is hypothesised that the antibodies exhibit cross-receptor binding, confounding data interpretation. Another aim is to evaluate GR and PR protein expression in FGT epithelial and stromal cells, with the hypothesis that both GR and PR proteins are expressed in FGT epithelial and stromal cells. The study also seeks to determine and compare GR and PR expression in FGT-resident CD4+ and CD8+ immune cells, hypothesising that these cells express more GR than PR proteins. Finally, the study aims to determine and compare GR and PR protein expression in systemic immune cells, hypothesising that systemic CD3+, CD4+, and CD8+ T lymphocytes, as well as CD14+ monocytes, express higher levels of GR than PR. Models and methodologies: PR-negative COS1 cells overexpressing exogenous GR or PR proteins were used to assess cross-reactivity of primary antibodies by western blot analysis, immunofluorescence staining and flow cytometry. FGT ectocervical tissue explants were used as a model for the FGT. These were stained by immunofluorescence for GR and PR as well as for CD4+ and CD8+ markers. Immunofluorescence staining in ectocervical tissue samples was visualised by confocal microscopy. Peripheral blood mononuclear cells (PBMCs) were used as a model to investigate protein expression levels of GR and PR in systemic CD3+, CD4+, CD8+ and CD14+ cells by flow cytometry. Results: Western blotting, immunofluorescence staining and flow cytometry in COS1 cells overexpressing GR and/or PR confirmed that the anti-GR antibodies did not cross-react with PR proteins and the anti-PR antibodies did not cross-react with GR proteins. Confocal imaging of ectocervical tissue sections demonstrated relatively high levels of expression of GR in epithelial cells, whereas PR expression was predominantly observed in stromal cells. GR was also expressed in some, but not all, FGT CD4+ and CD8+ cells. In contrast, PR fluorescence was absent in CD4+ and CD8+ cells within the FGT. Additionally, no notable trend was observed in GR expression density, PR expression density and the relative expression density of GR versus PR between CD4+ and CD8+ cells. Flow cytometric analysis of PBMCs revealed a high frequency of GR+ cells among the systemic immune cells including CD3+, CD4+ and CD8+ T cells, with significantly lower frequency of CD14+GR+ cells. There was no significant difference in the expression density of GR among systemic CD3+, CD4+, CD8+, and CD14+ cells. Conversely, PR detection in systemic CD3+, CD4+, CD8+, and CD14+ cells was problematic due to non-specific signals and gating challenges. After adjusting for these issues with appropriate controls and gating strategies, flow cytometry data indicated a lack of PR protein expression in systemic CD3+, CD4+, CD8+, and CD14+ cells. Conclusion: The lack of cross-reactivity of GR and PR primary antibodies ensures the reliability of antibody-based protein detection assays employed in the thesis. The widespread expression of GR across different FGT cells, including epithelial, stromal and specific T lymphocytes as well as systemic T lymphocytes and monocytes, signifies its diverse roles throughout the body. These findings suggest a pivotal role of GR in cells regulating FGT barrier function and systemic immunity. In contrast to the GR, PR expression is more restricted and is primarily found in FGT stromal cells. Its presence in FGT epithelial cells is minimal and absent in FGT and systemic T lymphocytes and/or monocytes. The limited expression of PR compared to GR suggests that certain progestogens, such as progesterone and medroxyprogesterone acetate (MPA), may exert off-target effects through the GR in cells crucial for women's immune and reproductive systems. Collectively these findings provide insights on the potential implications of progestins-based hormonal contraceptives (HCs) such as depot MPA (DMPA) for women's health.
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    The regulation of the luteinizing hormone (LH) and the follicle stimulating hormone (FSH) by glucocorticoids and progestins
    (2025) Capitaine, Carole-Keza; Hapgood, Janet; Bick, Alexis
    Estradiol (E2) plays a crucial role in female reproduction and in the defense against HIV-1 in the female genital tract (FGT). Medroxyprogesterone acetate (MPA) intramuscular (DMPA-IM) (Depo-Provera) and norethisterone (NET) enanthate (NET-EN) are injectable progestin only contraceptives. DMPA-IM is commonly used in sub-Saharan Africa, while NET-EN is commonly used in South Africa. Controversial observational studies have demonstrated a heightened susceptibility to HIV-1 acquisition associated with the use of DMPA-IM but not NET-EN. The recent Women's Health, Injectable Contraception and HIV (WHICH) clinical trial compared E2 levels in equal numbers of women randomized to DMPA-IM (n=262) and NET-EN (n=259) found that both DMPA-IM and NET-EN use caused hypoestrogenism. Considering the widespread use of DMPA-IM and NET-EN among South African women, who are disproportionately affected by HIV-1, it is important to explore the mechanisms through which these contraceptives induce hypoestrogenism. These could potentially involve luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH), all hormones involved in the hypothalamic-pituitary-ovarian (HPO) axis and key regulators of E2 synthesis and release. This thesis analyzed the serum concentrations of E2, LH, FSH, and GnRH in women randomized to DMPA-IM (n = 100) or NET-EN (n = 93) in a subpopulation of the WHICH cohort. This thesis also involved a translational approach, comparing clinical data with mechanistic data from an in vitro model. Towards understanding the mechanisms whereby MPA and NET may regulate gonadotropin levels, it was hypothesized that these progestins exert direct actions on pituitary gonadotropes. It was further hypothesized that while both progestins are progestogenic and could act via the progesterone receptor (PR), MPA would most likely also exert glucocorticoid like actions via the glucocorticoid receptor (GR), unlike NET. These hypotheses were tested in the LβT2 mature mouse pituitary gonadotrope cell line. This in vitro model allowed the effects of MPA and NET on the gonadotropins at a transcriptional, post-transcriptional and secretion level to be tested. This study also investigated the effects of MPA, NET, dexamethasone (DEX, a GR agonist) and progesterone (P4, a PR agonist), in the absence and presence of GnRH, on LH and FSH regulation in the LβT2 cells. 3 Clinical results showed that E2 levels were similarly suppressed to postmenopausal levels by both contraceptives in the subpopulation of women. No effects were detected for either contraceptive on GnRH levels. LH levels decreased in both contraceptive groups, whereas FSH levels decreased in the NET-EN group and increased in the DMPA-IM group. The results in LβT2 cells showed that MPA and DEX both suppressed the GnRH-induced promoter-reporter activity of the beta subunit of LH (LHβ), with a non-significant decrease in GnRH-induced LH protein secretion. MPA and DEX increased the promoter-reporter activity and mRNA level of the beta subunit of FSH (FSHβ), while NET and P4 had no detectable change on FSH beta expression. Additionally, both MPA and DEX enhanced basal and GnRH-induced FSHβ promoter activity and mRNA levels. Taken together with GR antagonist experiments, the effects of MPA and DEX on FSHβ gene expression are likely mediated by the GR. Attempts to measure secreted FSH protein were unsuccessful. In summary, the combined clinical and in vitro data suggest that the hypoestrogenic effect caused by DMPA-IM and NET-EN in women is likely through direct actions of MPA and NET on pituitary gonadotropes to decrease LH levels. The decrease in LH levels in DMPA-IM users occurs most likely both at the level of gene transcription, as well as at the level of protein secretion, while this does not appear to be the case for NET-EN users. The increase in FSH levels in DMPA-IM users is likely due to direct effects by MPA on pituitary gonadotropes to increase FSHβ transcription and mRNA levels. This increase in FSH levels is likely due to the GR-mediated transcriptional regulation of FSHβ by MPA and not NET. The in vitro data do not explain the decrease in FSH levels detected in NET-EN users. However, there may be other unexplored mechanisms used by MPA and NET at the level of FSH secretion. The data also do not exclude that DMPA-IM and NET-EN may have other mechanisms that act at the ovarian level or in the hypothalamus or above the hypothalamus, to affect GnRH pulsatility. This study gives insight into previously unknown mechanisms involved in the regulation of gonadotropins by MPA and NET. This research also enhances our understanding of the potential mechanisms behind the hypoestrogenic effects caused by progestin-only injectable contraceptives.