Medroxyprogesterone acetate differentially regulates interleukin (IL)-12 and IL-10 in a human ectocervical epithelial cell line in a glucocorticoid receptor (GR)-dependent manner

Abstract

Medroxyprogesterone acetate (MPA), designed to mimic the actions of the endogenous hormone progesterone (P4), is extensively used by women as a contraceptive and in hormone replacement therapy. However, little is known about the steroid receptor-mediated molecular mechanisms of action of MPA in the female genital tract. In this study, we investigated the regulation of the pro-inflammatory cytokine, interleukin (IL)-12, and the anti-inflammatory cytokine IL-10, by MPA versus P4, in an in vitro cell culture model of the female ectocervical environment. This study shows that P4 and MPA significantly increase the expression of the IL-12p40 and IL-12p35 genes, whereas IL-10 gene expression is suppressed in a dose-dependent manner. Moreover, these effects were abrogated when reducing the glucocorticoid receptor (GR) levels with siRNA. Using a combination of chromatin immunoprecipitation (ChIP), siRNA, and re-ChIP assays, we show that recruitment of the P4- and MPA-bound GR to the IL-12p40 promoter requires CCAAT enhancer-binding protein (C/EBP)-β and nuclear factor κB (NFκB), although recruitment to the IL-10 promoter requires signal transducer and activator of transcription (STAT)-3. These results suggest that both P4 and MPA may modulate inflammation in the ectocervix via this genomic mechanism.

Keywords: Cervical Epithelium; Contraception; Cytokines; Gene Regulation; Glucocorticoid; Glucocorticoid Receptor; Inflammation; Medroxyprogesterone Acetate; Progesterone; Progestin.

FIGURE 1.

Effect of P₄ and MPA on the TNF induced expression of IL-12p40, IL-12p35, and IL-10 in the human ectocervical cell line. The human Ect1/E6E7 cell line was incubated for 6 h with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (vehicle control) or increasing concentrations of P₄ or MPA. Total RNA was isolated and reverse-transcribed to cDNA. Real time qPCR was performed to determine the mRNA expression levels of IL-12p40 (A), IL-12p35 (B), and IL-10 (C), using GAPDH as the internal standard. Results shown are the average of at least four independent experiments (±S.E.). Relative IL-12p40, IL-12p35, and IL-10 mRNA expression of treated samples was calculated relative to the vehicle control (EtOH), which was set as 1. D, relative potency (EC₅₀ ± S.E.) for each ligand for activation of the IL-12p40 and IL-12p35 genes and repression of the IL-10 gene was obtained from the data shown in A–C. Statistical analysis of the EC₅₀ values for all genes indicated P₄ versus MPA (p > 0.05).

FIGURE 2.

Decreasing GR protein levels by siRNA indicates a role for the GR in mediating the effects of P₄ and MPA on IL-12p40, IL-12p35, and IL-10 mRNA expression in the Ect1/E6E7 cell line. A, human Ect1/E6E7 cell line was incubated with 10 nm [³H]dexamethasone in the absence (total binding) and presence of 1 μm unlabeled (nonspecific binding) P₄, MPA, or cortisol for 6 h. The percentage of specific binding (total binding minus nonspecific binding) is plotted. Binding of the test compounds to the GR is shown relative to binding of cortisol set as 100%. One-way ANOVA analysis of variance and Dunnett's test (compare all columns versus control (cortisol) column) were performed as post-test. B–E, untransfected human Ect1/E6E7 cells, as well as cells transfected with 10 nm NSC or two GR siRNA oligonucleotides, were either left untreated or treated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (control) or 1 μm P₄, MPA, or cortisol for 6 h. B, for verification of GR knockdown, total protein from the untreated cells was harvested to perform Western blotting, using antibodies specific for the GR and GAPDH. The latter was used as a loading control. A representative blot is shown. GR expression levels relative to GAPDH were quantified using UN-SCAN-IT. Western blots of three independent experiments were quantified to determine the percentage GR protein knockdown. C–E, total RNA was isolated and reverse-transcribed to cDNA. Thereafter, real time qPCR was performed to determine the mRNA expression levels of IL-12p40 (C), IL-12p35 (D), and IL-10 (E), using GAPDH as the internal standard. Relative IL-12p40, IL-12p35, and IL-10 gene expression of treated samples was calculated relative to vehicle control (EtOH) of the NSC siRNA, which was set as 1. Statistically significant differences are indicated by *, **, or *** p < 0.05, p < 0.01, or p < 0.001, respectively, for GR6; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, respectively, for GR5; ns, no statistical significance; UT, untransfected.

FIGURE 3.

MPA regulates IL-12p40 and IL-10, but not IL-12p35, mRNA levels in the MDA-MB-231 cell line in a GR-dependent manner. Human MDA-MB-231 cells transfected with 10 nm NSC or GR6 siRNA oligonucleotides were either left untreated or treated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (control) or 1 μm P₄, MPA, or cortisol for 6 h. A, for verification of GR knockdown, total protein from the untreated cells was harvested to perform Western blotting, using antibodies specific for the GR and GAPDH. The latter was used as a loading control. A representative blot is shown. GR expression levels relative to GAPDH were quantified using UN-SCAN-IT. Western blots of at least two independent experiments were quantified to determine the percentage of GR protein knockdown. B–D, total RNA was isolated and reverse-transcribed to cDNA. Thereafter, real time qPCR was performed to determine the mRNA expression levels of IL-12p40 (B), IL-12p35 (C), and IL-10 (D) using GAPDH as the internal standard. Relative IL-12p40, IL-12p35, and IL-10 gene expression of treated samples was calculated relative to vehicle control (EtOH) of the NSC siRNA, which was set as 1. Results shown are the average (± S.E.) of at least two independent experiments. One-way ANOVA and Dunnett's (compares all pairs of columns versus control column) post-tests were used for statistical analysis. ns, no statistical significance; *, **, and ***, p < 0.05, p < 0.01, or p < 0.001, respectively.

FIGURE 4.

GR occupies the NFκB/C/EBPβ region of the IL-12p40 promoter in response to P₄, MPA, and cortisol. Human Ect1/E6E7 cells were incubated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH or 1 μm P₄, MPA, or cortisol for 2 h, followed by the ChIP assay. A, schematic illustration of the IL-12p40 promoter, indicating all the cis-elements investigated in this study and position of the primers. B–D, cell lysates were subjected to immunoprecipitation with the GR-specific antibody or anti-IgG (negative control). The immunoprecipitated DNA fragments and input DNA were analyzed by real time qPCR. Data shown are normalized to input and expressed as the fold-response relative to EtOH (IgG control), which was set as 1. Results shown are the average (± S.E.) of at least four independent experiments. One-way ANOVA analysis of variance and Dunnett's (compares all columns versus control (IgG EtOH) column) post-tests were used for statistical analysis. ns, no statistical significance; *, **, and ***, p < 0.05, p < 0.01, or p < 0.001, respectively.

FIGURE 5.

GR occupies the Sp1/STAT-3 region of the IL-10 promoter in response to P₄, MPA, and cortisol. Human Ect1/E6E7 cells were incubated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH or 1 μm P₄, MPA, or cortisol for 2 h, followed by the ChIP assay. A, schematic illustration of the IL-10 promoter, indicating all the cis-elements investigated in this study and position of the primers. B–D, cell lysates were subjected to immunoprecipitation with the GR-specific antibody or anti-IgG (negative control). The immunoprecipitated DNA fragments and input DNA were analyzed by real time qPCR. Data shown are normalized to input and expressed as the fold-response relative to EtOH (IgG control), which was set as 1. Results shown are the average (± S.E.) of at least four independent experiments. One-way ANOVA analysis of variance and Dunnett's test (compares all columns versus control (IgG EtOH) column) post-tests were performed as post-test. ns, no statistical significance; *, p < 0.05; **, p < 0.01.

FIGURE 6.

Recruitment of the progestogen-bound GR to the IL-12p40 promoter is dependent on both the transcription factors C/EBPβ and NFκB. A and B, human Ect1/E6E7 cells were incubated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH or 1 μm P₄, MPA, or cortisol for 2 h, followed by the re-ChIP assay. Cell lysates were subjected to immunoprecipitation with the GR-specific antibody and then with the C/EBPβ (A) or NFκB (B) antibody or anti-IgG (negative control). The immunoprecipitated DNA fragments and input DNA were analyzed by real time qPCR. Data shown are normalized to input and expressed as the fold-response relative to EtOH (IgG control), which was set as 1. Results shown are the average (± S.E.) of at least three independent experiments. One-way ANOVA analysis of variance and Dunnett's (compares all columns versus control (IgG EtOH) column) post-tests were used for statistical analysis. C and D, human Ect1/E6E7 cells, transfected with 10 nm NSC, C/EBPβ, or NFκB siRNA oligonucleotides, were treated for 6 h with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (control) or 1 μm P₄, MPA, or cortisol. For verification of C/EBPβ and NFκB knockdown, total protein from the untreated cells was harvested to perform Western blotting using antibodies specific for C/EBPβ and Hsp90 (C) and NFκB and GAPDH (D). Hsp90 and GAPDH were used as loading controls. A representative blot is shown for each knockdown. Total RNA was isolated and reverse-transcribed to cDNA. Thereafter, real time qPCR was performed to determine the mRNA expression levels of IL-12p40, using GAPDH as the internal standard. Relative IL-12p40 gene expression of treated samples was calculated relative to vehicle control (EtOH) of the NSC siRNA, which was set as 1. Results shown are the average (± S.E.) of at least three independent experiments. Two-way ANOVA analysis of variance and Bonferroni (compares all pairs of columns) post-tests were used for statistical analysis. ns, no statistical significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

STAT-3 plays a role in the GR-mediated down-regulation of IL-10 gene expression in the Ect1/E6E7 cell line. A, human Ect1/E6E7 cells were incubated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH or 1 μm P₄, MPA, or cortisol for 2 h, followed by the re-ChIP assay. Cell lysates were subjected to immunoprecipitation with the GR-specific antibody and then with the STAT-3-specific antibody or anti-IgG (negative control). The immunoprecipitated DNA fragments and input DNA were analyzed by real time qPCR. Data shown are normalized to input and expressed as the fold-response relative to EtOH (IgG control), which was set as 1. Results shown are the average (± S.E.) of at least three independent experiments. One-way ANOVA and Dunnett's (compares all columns versus control (IgG EtOH) column) post-tests were used for statistical analysis. B, human Ect1/E6E7 cells, transfected with 10 nm NSC or STAT-3 siRNA oligonucleotides, respectively, were stimulated for 6 h with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (control) or 1 μm P₄, MPA, or cortisol. For verification of STAT-3 knockdown, total protein from the untreated cells was harvested to perform Western blotting using antibodies specific for STAT-3. GAPDH was used as a loading control. A representative blot is shown. Total RNA was isolated and reverse-transcribed to cDNA. Thereafter, real time qPCR was performed to determine the mRNA expression levels of IL-10, using GAPDH as the internal standard. Relative IL-10 gene expression of treated samples was calculated relative to vehicle control (EtOH) of the NSC siRNA, which was set as 1. Results shown are the average (± S.E.) of at least three independent experiments. Two-way ANOVA and Bonferroni (compares all pairs of columns) post-tests were used for statistical analysis. ns, no statistical significance; ***, p < 0.001.

FIGURE 8.

Human Ect1/E6E7 cells transfected with 10 nm NSC or GR6 siRNA oligonucleotides were either left untreated or treated with 0.02 μg/ml TNF in the absence or presence of 0.1% EtOH (control) or 1 μm P₄, MPA, or cortisol for 24 h. For verification of GR knockdown, total protein from the untreated cells was harvested to perform Western blotting, using antibodies specific for the GR and GAPDH. The latter was used as a loading control, and a representative blot is shown. Cell culture supernatants were collected and the protein levels of IL-12p70 (A) and IL-10 (B) were measured using ELISA. The relative protein levels of the NSC siRNA vehicle control (EtOH) for IL-12p70 (∼1.5 pg/ml) and IL-10 (∼5 pg/ml) were set as 1, and the relative IL-12p70 and IL-10 protein levels of treated samples were calculated relative to this. Results shown are the average (± S.E.) of at least three independent experiments. Two-way ANOVA and Bonferroni (compares all pairs of columns) post-tests were used for statistical analysis. ns, no statistical significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 9.

Schematic model for the progestogen-induced up-regulation of IL-12p40 and down-regulation of IL-10 gene expression via the GR in the ectocervical epithelial cell line. Upon P₄ or MPA binding to the GR, the GR undergoes a conformational change and translocates to the nucleus where it occupies the IL-12p40 promoter to activate transcription of the IL-12p40 gene or occupies the IL-10 promoter to suppress IL-10 gene transcription. In response to P₄ and MPA, the GR forms a complex with the transcription factors C/EBPβ and NFκB and occupies the NFκB/C/EBPβ region of the IL-12p40 promoter to activate transcription of this gene, although to decrease transcription of the IL-10 gene, the P₄- and MPA-bound GR and the DNA-bound transcription factor STAT-3 bind as a complex to the IL-10 promoter. The questions marks indicate signaling pathways that are uncertain (see under “Discussion”). Hsp90, heat shock protein-90; STAT-3, signal transducer and activator of transcription-3.