Agonist-Dependent Downregulation of Progesterone Receptors in Human Cervical Stromal Fibroblasts

Abstract

Progesterone (P(4)) maintains uterine quiescence during the majority of pregnancy, whereas diminished progesterone receptor (PR) expression and/or activity (ie, functional P(4) withdrawal) promotes parturition. To investigate the regulation of PR expression in cervical stroma, fibroblasts from premenopausal hysterectomy specimens were prepared. Greater than 99% of the cultures were vimentin positive (mesenchymal cell marker) with only occasional cytokeratin-8 positivity (epithelial cell marker) and no evidence of CD31-positive (endothelial cell marker) cells. Cells were immunolabeled with antibodies directed against PRs (PR-A and PR-B), estrogen receptor α (ER-α), and glucocorticoid receptor-α/β (GR-α/β). All cells were uniformly immunopositive for ER-α and GR-α/β but did not express PRs. Incubation of cells with 10(-8) mol/L 17β-estradiol induced a time-dependent increase in PR-A and PR-B messenger RNAs (mRNAs) by quantitative real-time polymerase chain reactions and proteins by immunoblotting and immunofluorescence. Incubation of cervical fibroblasts with PR ligands (medroxyprogesterone acetate or Org-2058) downregulated PR-A and PR-B levels. Coincubation of cells with PR ligands plus RU-486, a PR antagonist, partially abrogated agonist-induced receptor downregulation. Dexamethasone, a pure glucocorticoid, had no inhibitory effect on PR expression. These results indicate that progestins and estrogens regulate PR expression in cervical fibroblasts. We postulate that hormonal regulation of PR expression in the cervical stroma may contribute to functional P(4) withdrawal in preparation for parturition.

Keywords: cervix; estradiol; parturition; progesterone receptor; receptor downregulation.

Figure 1.

Establishment and initial characterization of cervical fibroblast cell cultures. A, Photograph depicting small fragments of explanted cervical stromal tissue held in place under glass coverslips and grown in individual wells of a 6-well tissue culture plate in complete medium. B, Low magnification phase contrast image of cellular outgrowth from explanted cervical tissue after 21 days in culture. C, Representative immunofluorescence image of cervical fibroblasts grown under basal conditions (untreated) and coimmunolabeled using antibodies directed against vimentin (green) and cytokeratin 8 (CK-8, red), followed by counterstaining of nuclei with 4′,6-diamidino-2-phenylindole (DAPI; blue). By the second passage following the initial outgrowth period, >99% of the cells demonstrated vimentin labeling (marker for mesenchymally-derived cells), whereas only an occasional cell labeled with anti-CK8 (epithelial marker). Scale bars = 20 μm. D, Cells cultivated as in panel C were coimmunolabeled using antibodies detecting CD31 (green, endothelial marker) and vimentin (red) with DAPI counterstaining. CD31 labeling was absent in cervical cells but was readily detected in human umbilical vein endothelial cells (green in D’). Scale bars = 20 μm. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 2.

Progesterone receptor (PR) expression in premenopausal cervical biopsy specimens. A, Histological section of cervical tissue immunolabeled with an antibody directed against PR (brown). B, Detail of area within box in panel A. Red arrows indicate PR immunolabeling in nuclei of cervical stromal fibroblasts, while blue arrowheads denote PR expression in basal squamous epithelial cells of the ectocervix. PR labeling was also observed in columnar epithelial cells of the endocervix and glands but was undetected in the more superficial layers of the ectocervix (asterisk). C, Serial section of the biopsy shown in previous panels immunolabeled with an isotype control antibody at a concentration equivalent to the anti-PR antibody. Scale bars = 100 μm. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 3.

Cervical fibroblasts cultured in vitro constitutively express estrogen receptor α (ERα) and glucocorticoid receptors α/β (GR-α/β), whereas progesterone receptor (PR) expression is inducible following 17β-estradiol (17β-E₂) priming. A, Immunofluorescence images demonstrating ERα and (B) GR immunolabeling (red) and DAPI-stained nuclei (blue) in cervical fibroblasts grown under basal conditions. Scale bar = 20 μm. C, Cervical fibroblasts were incubated in treatment medium (see Materials and Methods section) for 3 to 12 days containing either vehicle alone (Veh, 0.01% ethanol, gray bars) and/or 10⁻⁸ mol/L 17β-E₂ (white bars), and PR messenger RNA (mRNA) expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) using primer/probe sets detecting total PR (through amplification of the transcript region that is common to both PR-A and PR-B) or PR-B only. Samples were normalized to RPLP0 mRNA and expressed as the fold-change relative to Veh-treated cells collected after 3 days of treatment (mean ± standard error of the mean [SEM], 2 independent experiments). D, Representative immunofluorescence images demonstrating induction of nuclear PR expression (red) following a 7-day incubation in 17β-E₂. Scale bars = 20 μm. E, Nuclear extracts prepared from cervical fibroblasts following incubation in the absence or presence of 17β-E₂ for 7 days were analyzed by immunoblotting using an antibody detecting PR. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 4.

Effects of PR and GR agonists on PR expression. Cervical fibroblasts were incubated in medium with or without 10⁻⁸ mol/L 17β-estradiol (17β-E₂) in the absence or presence of medroxyprogesterone acetate (MPA, 10⁻⁸ mol/L), Org-2058 (10⁻⁸ mol/L), or Dex (10⁻⁸ mol/L) for 7 days. A, Representative immunofluorescence images demonstrating nuclear PR expression (red) and the number of nuclei per field (DAPI, blue) in each of the treatment conditions. Scale bars = 20 μm. B, Bar graphs (mean ± SEM) summarizing measurements of PR-like fluorescence intensity in 3 independent experiments (**P < .01 vs Veh; Kruskal–Wallis test with Dunn’s multiple comparison posttest). C, qRT-PCR analysis of PR-Total and PR-B mRNA expression. Samples were normalized to RPLP0 mRNA and expressed as the fold-change relative to Veh-treated cells. Bars represent mean ± SEM from 3 independent experiments (^# P < .01 vs Dex; ^§ P < .01 vs Veh; Kruskal–Wallis test with Dunn’s multiple comparison posttest). D, Representative immunoblot (from 3 separate experiments) demonstrating PR-A and PR-B protein expression in cervical fibroblasts following 7 days in each treatment condition; as a loading control, blots were reprobed using an antibody against TATA-binding protein (TBP). E, Densitometric analysis of PR-A and PR-B immunoblots, normalized to TBP and expressed as fold change relative to Veh (mean ± SD, 2 independent experiments). PR indicates progesterone receptor; GR, glucocorticoid receptor; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean; qRT-PCR, quantitative real-time polymerase chain reaction; mRNA, messenger RNA; SD, standard deviation. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 5.

RU-486 attenuates PR agonist-induced PR downregulation in the presence of 17β-E₂. A, Cervical fibroblasts were treated for 7 days in the presence of solvent control (Veh, 0.01% ethanol), 10⁻⁸ mol/L 17β-E₂, 17β-E₂₂ with 10⁻⁷ mol/L MPA in the absence or presence of 10⁻⁶ mol/L RU-486, or 17β-E₂ with 10⁻⁷ mol/L Org-2058 in the absence or presence of RU-486. Representative immunofluorescence images are shown for cervical fibroblasts immunolabeled with anti-PR (red) and counterstained with DAPI (blue). Scale bars = 20 μm. B, Bar graph (mean ± SD) summarizing fluorescence intensity measurements for total PR-like immunoreactivity for experiment described in panel A. C, Immunoblot of cervical fibroblast nuclear extracts treated identically as in panel A, probed using an anti-PR antibody. PR indicates progesterone receptor; MPA, medroxyprogesterone acetate; SD, standard deviation. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)