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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.

Tomasicchio M, Avenant C, Du Toit A, Ray RM, Hapgood JP - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Cape Town, Cape Town, Western Province, South Africa.

ABSTRACT
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.

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Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR.(A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p<0.001. (B) Western analysis of lysates prepared from approximately 4×106 PBMCs. Whole cell lysates of COS-1 cells overexpressing the relevant steroid receptor (+ve) or empty vector (−ve) served as the controls. GAPDH was used as a loading control. Note that the upper strong band on the MR blot is a COS-1 cell-derived non-specific signal which is absent for PBMCs, while the MR signal is the faint band just below the non-specific band which is only seen in the positive control. We were unable to obtain a more-specific anti-MR antibody. (C) Conventional PCR of cDNA prepared from human PBMCs using primers specific for the relevant steroid receptor. The controls were prepared by PCR amplification of the relative steroid receptor cDNA from plasmid DNA. GAPDH served as a control for mRNA levels. MW: molecular weight; NTC: no template control. Error bars represent standard deviation.
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pone-0062895-g002: Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR.(A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p<0.001. (B) Western analysis of lysates prepared from approximately 4×106 PBMCs. Whole cell lysates of COS-1 cells overexpressing the relevant steroid receptor (+ve) or empty vector (−ve) served as the controls. GAPDH was used as a loading control. Note that the upper strong band on the MR blot is a COS-1 cell-derived non-specific signal which is absent for PBMCs, while the MR signal is the faint band just below the non-specific band which is only seen in the positive control. We were unable to obtain a more-specific anti-MR antibody. (C) Conventional PCR of cDNA prepared from human PBMCs using primers specific for the relevant steroid receptor. The controls were prepared by PCR amplification of the relative steroid receptor cDNA from plasmid DNA. GAPDH served as a control for mRNA levels. MW: molecular weight; NTC: no template control. Error bars represent standard deviation.

Mentions: Next we sought to provide evidence that the increase in apoptosis observed with Dex and MPA was mediated via the GR and did not involve other steroid receptors. Since MPA is a PR agonist, and a partial agonist for the androgen receptor (AR) and a partial to full agonist for the GR [29], the possibility that MPA exerts its apoptotic effects via the PR or AR was investigated indirectly by using receptor-selective agonists. In order to determine whether other steroid receptors (apart from the GR) could induce apoptosis, PBMCs were treated with agonists that are selective for the AR (100 nM Mib), estrogen receptor (ER) (100 nM E2), mineralocorticoid receptor (MR) (10 nM Ald) and PR (100 nM R5020), as well as 100 nM Dex, 100 nM MPA, 10 µM NET-A or 10 µM NET for 24 hrs, and apoptosis was detected using flow cytometry as described previously. The ligands were used at saturating concentrations for each steroid receptor to control for the differences in relative binding affinities of each ligand for their respective receptors [33], [34], [94]. As found earlier, Dex significantly induced apoptosis by about 2-fold and about 3-fold in CD3+ and CD4+ T-cells, respectively (Figure 2A). MPA significantly induced apoptosis by about 1.5-fold compared to untreated control cells in the CD3+ T-cells and appeared to increase apoptosis in CD4+ T-cells to a similar extent as observed before (Figure 1B). In both CD3+ and CD4+ T-cells, the other steroid receptor-selective agonists did not induce apoptosis in a statistically significant manner (Figure 2A). Therefore, it is likely that the effects of MPA on apoptosis are mediated via the GR in T-cells. In support of these findings, PBMCs expressed GR protein whereas AR, PR, MR or ER protein expression was not detected by Western blot analysis (Figure 2B). The ER and MR mRNAs, but not AR or PR mRNAs, were however detected by PCR, indicating that ER and MR proteins may be expressed, but at a level undetectable by Western blot analysis (Figure 2C). Together with the results presented in Figure 2A, these data show that if low levels of ER and MR protein are expressed, they have no effect on apoptosis in CD3+ or CD4+ T-cells (Figure 2A). Taken together, these results strongly support the finding that the PR, AR, MR and ER do not induce apoptosis in these cells, and that MPA acts primarily via the GR to induce apoptosis in PBMCs. It is noteworthy that NET was included in this experiment as a control to exclude the possibility that the acetate form (NET-A) may be less active. However, similarly to NET-A, NET does not result in apoptosis.


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.

Tomasicchio M, Avenant C, Du Toit A, Ray RM, Hapgood JP - PLoS ONE (2013)

Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR.(A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p<0.001. (B) Western analysis of lysates prepared from approximately 4×106 PBMCs. Whole cell lysates of COS-1 cells overexpressing the relevant steroid receptor (+ve) or empty vector (−ve) served as the controls. GAPDH was used as a loading control. Note that the upper strong band on the MR blot is a COS-1 cell-derived non-specific signal which is absent for PBMCs, while the MR signal is the faint band just below the non-specific band which is only seen in the positive control. We were unable to obtain a more-specific anti-MR antibody. (C) Conventional PCR of cDNA prepared from human PBMCs using primers specific for the relevant steroid receptor. The controls were prepared by PCR amplification of the relative steroid receptor cDNA from plasmid DNA. GAPDH served as a control for mRNA levels. MW: molecular weight; NTC: no template control. Error bars represent standard deviation.
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Related In: Results  -  Collection

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pone-0062895-g002: Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR.(A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p<0.001. (B) Western analysis of lysates prepared from approximately 4×106 PBMCs. Whole cell lysates of COS-1 cells overexpressing the relevant steroid receptor (+ve) or empty vector (−ve) served as the controls. GAPDH was used as a loading control. Note that the upper strong band on the MR blot is a COS-1 cell-derived non-specific signal which is absent for PBMCs, while the MR signal is the faint band just below the non-specific band which is only seen in the positive control. We were unable to obtain a more-specific anti-MR antibody. (C) Conventional PCR of cDNA prepared from human PBMCs using primers specific for the relevant steroid receptor. The controls were prepared by PCR amplification of the relative steroid receptor cDNA from plasmid DNA. GAPDH served as a control for mRNA levels. MW: molecular weight; NTC: no template control. Error bars represent standard deviation.
Mentions: Next we sought to provide evidence that the increase in apoptosis observed with Dex and MPA was mediated via the GR and did not involve other steroid receptors. Since MPA is a PR agonist, and a partial agonist for the androgen receptor (AR) and a partial to full agonist for the GR [29], the possibility that MPA exerts its apoptotic effects via the PR or AR was investigated indirectly by using receptor-selective agonists. In order to determine whether other steroid receptors (apart from the GR) could induce apoptosis, PBMCs were treated with agonists that are selective for the AR (100 nM Mib), estrogen receptor (ER) (100 nM E2), mineralocorticoid receptor (MR) (10 nM Ald) and PR (100 nM R5020), as well as 100 nM Dex, 100 nM MPA, 10 µM NET-A or 10 µM NET for 24 hrs, and apoptosis was detected using flow cytometry as described previously. The ligands were used at saturating concentrations for each steroid receptor to control for the differences in relative binding affinities of each ligand for their respective receptors [33], [34], [94]. As found earlier, Dex significantly induced apoptosis by about 2-fold and about 3-fold in CD3+ and CD4+ T-cells, respectively (Figure 2A). MPA significantly induced apoptosis by about 1.5-fold compared to untreated control cells in the CD3+ T-cells and appeared to increase apoptosis in CD4+ T-cells to a similar extent as observed before (Figure 1B). In both CD3+ and CD4+ T-cells, the other steroid receptor-selective agonists did not induce apoptosis in a statistically significant manner (Figure 2A). Therefore, it is likely that the effects of MPA on apoptosis are mediated via the GR in T-cells. In support of these findings, PBMCs expressed GR protein whereas AR, PR, MR or ER protein expression was not detected by Western blot analysis (Figure 2B). The ER and MR mRNAs, but not AR or PR mRNAs, were however detected by PCR, indicating that ER and MR proteins may be expressed, but at a level undetectable by Western blot analysis (Figure 2C). Together with the results presented in Figure 2A, these data show that if low levels of ER and MR protein are expressed, they have no effect on apoptosis in CD3+ or CD4+ T-cells (Figure 2A). Taken together, these results strongly support the finding that the PR, AR, MR and ER do not induce apoptosis in these cells, and that MPA acts primarily via the GR to induce apoptosis in PBMCs. It is noteworthy that NET was included in this experiment as a control to exclude the possibility that the acetate form (NET-A) may be less active. However, similarly to NET-A, NET does not result in apoptosis.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Cape Town, Cape Town, Western Province, South Africa.

ABSTRACT
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.

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Related in: MedlinePlus