Role of the glucocorticoid receptor and HIV-1 Vpr in inflammatory gene expression and HIV-1 LTR transcription in response to dexamethasone and progestogens
Doctoral Thesis
2015
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University of Cape Town
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The relationship between progestin-only injectable contraception and risk of HIV-1 acquisition is controversial. Most clinical data suggests that the injectable contraceptive medroxyprogesterone acetate (MPA), unlike norethisterone enanthate (NET-EN), increases susceptibility to infections such as HIV-1. The first part of this thesis investigated the differential effects, molecular mechanisms of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 and HeLa cell line models for the endocervical epithelium, a key point of entry for pathogens in the lower female genital tract (FGT). Quantitative real-time PCR analysis showed that 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 cervical cell lines and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR siRNA 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 (DEX). This is at least in part consistent with direct effects on transcription, without a requirement for new protein vi synthesis. This is the first study to show direct proof for a GR-mediated mechanism of action in anti-inflammatory effects of MPA. 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 MPA effects on genital mucosal immune function and susceptibility to infections are likely to be very different to those of NET and P4, when mediated by the GR The second part of this thesis investigated the effects of the virion associated HIV-1 protein, Vpr, on GR-regulated inflammatory genes in the presence of the ligands.
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Govender, Y. 2015. Role of the glucocorticoid receptor and HIV-1 Vpr in inflammatory gene expression and HIV-1 LTR transcription in response to dexamethasone and progestogens. University of Cape Town.