Inflammatory Stimuli Increase Progesterone Receptor-A Stability and Transrepressive Activity in Myometrial Cells

Abstract

The steroid hormone progesterone acting via the nuclear progesterone receptor (PR) isoforms, progesterone receptor A (PR-A) and progesterone receptor B (PR-B), is essential for the maintenance of uterine quiescence during pregnancy. Inhibition of PR signaling augments uterine contractility and induces labor. Human parturition is thought to be triggered by modulation of PR signaling in myometrial cells to induce a functional progesterone withdrawal. One mechanism for functional progesterone withdrawal is increased abundance of PR-A, which decreases progesterone responsiveness by inhibiting the transcriptional activity of PR-B. Human parturition also involves tissue-level inflammation within the myometrium. This study examined the control of PR-A abundance and transrepressive activity in myometrial cells and the role of the inflammatory stimuli in the form of interleukin-1β (IL-1β) and lipopolysaccharide (LPS) in these processes. We found that abundance of PR-A was markedly increased by progesterone and by exposure to IL-1β and LPS via posttranslational mechanisms involving increased PR-A protein stability. In contrast, progesterone decreased abundance of PR-B by increasing its rate of degradation. Together, progesterone and proinflammatory stimuli induced a PR-A-dominant state in myometrial cells similar to that observed in term laboring myometrium. IL-1β and LPS also increased the capacity for PR-A to inhibit the transcriptional activity of PR-B. Taken together, our data suggest that proinflammatory stimuli increase the steady-state levels of PR-A and its transrepressive activity in myometrial cells and support the hypothesis that tissue-level inflammation triggers parturition by inducing PR-A-mediated functional progesterone withdrawal.

Figure 1.

Effect of progesterone PR-A and PR-B abundance in hTERT-HM^A/B cells. (A) Representative immunoblot analysis of PR-A, PR-B, and GAPDH in whole cell lysate from hTERT-HM^A/B cells treated with DOX (30 ng/mL) or DAH (400 nM) for 48 hours to induce PR-A or PR-B, respectively, and then exposed to vehicle or progesterone (P4; 1 nM, 10 nM, or 100 nM) for the final 16 hours. Histogram shows relative levels of PR-A and PR-B in each condition. (B) Immunoblot analysis of PR-A, PR-B, and GAPDH in whole cell lysate from hTERT-HM^A/B cells treated with DOX (80 ng/mL) or DAH (200 nM) for 48 hours to induce PR-A and PR-B, respectively, and exposed to vehicle or progesterone (P4) for the final 24 hours. Histograms show relative levels of PR-A and PR-B in each condition. Note that progesterone treatment induced transition from PR-B dominance to PR-A dominance.

Figure 2.

Effect of progesterone on PR-A and PR-B turnover. (A) Representative immunoblot analyses of PRs and GAPDH in hTERT-HM^A/B cells induced to express PR-A (left) by exposure with DOX (100 ng/mL) or PR-B (right) by exposure to DAH (300 nM) for 48 hours and then, after removal of the inducers, exposed to progesterone or vehicle for the indicated times. Histograms show mean (± standard error of the mean; n = 3) PR/GAPDH levels between vehicle- and progesterone-treated cells at various times. Decay curves show the half-life (T_1/2) of the PRs.

Figure 3.

Role of the 26S proteosome in PR-A and PR-B degradation. Immunoblot analysis of PRs and GAPDH in hTERT-HM^A/B cells induced to express PR-A and PR-B with DOX (100 ng/mL) and DAH (300 nM), respectively, and then washed and exposed to progesterone (100 nM) ± the 26S proteasome inhibitor MG132 (10 µM) for 16 hours and 24 hours.

Figure 4.

Effect of IL-1β and LPS PR-A and PR-B abundance in hTERT-HM^A/B cells. (A) Representative immunoblot analysis of lysate from hTERT-HM^A/B cells treated with DOX (100 ng/mL) and DAH (300 nM) for 48 hours to induce expression of PR-A and PR-B and then washed and exposed to progesterone (P4; 100 nM) and IL-1β (1 ng/mL) or LPS (1 μg/mL) for various times. Histogram shows mean (± SEM; n = 3) relative abundance of PR-A and PR-B at various times after inducer withdrawal (*P < 0.05). (B) Immunoblot analysis of lysate from hTERT-HM^A/B cells treated with DOX and DAH to express PR-A and PR-B in the presence of vehicle or 10 nM R5020 with increasing concentrations of IL-1β for 24 hours. Histogram shows effect of R5020 and IL-1β on the relative abundance of PR-A and PR-B and the PR-A:PR-B ratio. Immunoblotting for tubulin was used to monitor protein loading.

Figure 5.

Effect of IL-1β and LPS on PR-A abundance in myometrium explants. Immunoblot analysis of PR-A and PR-B levels in response to P4 (100 nM) and IL-1β (1 ng/mL) or LPS (1 µg/mL) in myometrial explants after 30 minutes, 6 hours, or 24 hours of treatment. Each time represents myometrium from different women. Histogram shows relative levels of PR-A at the 24-hour time point.

Figure 6.

Effect of IL-1β and LPS on PR-A transrepression of PR-B. (A) Relative luciferase activity [mean ± standard error of the mean (SEM); n = 3] in hTERT-HM^A/B cells transiently transfected with PRE₂-LUC and REN-LUC, and then treated with DAH ± DOX to induce PR-B alone and PR-B and PR-A expression, respectively, for 48 hours. Progesterone (P4; 100 nM) ± increasing levels of IL-1β (25 pg/mL, 100 pg/mL, 1 ng/mL, or 100 ng/mL) or LPS (1 ng/mL, 10 ng/mL, 100 ng/mL, or 1 µg/mL) were added for the final 24 hours. (B) Relative luciferase activity (mean ± SEM; n = 3) in hTERT-HM^A/B cells transiently transfected with PRE₂-LUC and REN-LUC, and then treated with DAH (200 nM) and increasing levels of DOX (1× = 10 ng/mL; 2× = 50 ng/mL; or 20× = 200 ng/mL) to induce a fixed amount of PR-B with increasing amounts of PR-A, respectively, for 48 hours. Progesterone (P4; 100 nM) was added for the final 24 hours, and vehicle, IL-1β (1 ng/mL), or LPS (1 µg/mL) for the final 30 minutes (*P < 0.05). Immunoblot shows PR-A and PR-B levels achieved at the end of the experiment. (C) Effect of progesterone (100 nM for 24 hours) and IL-1β (1 ng/mL for the final 4 hours of incubation) on the abundance of FKBP5 mRNA in hTERT-HM^A/B cells conditioned to be PR-B dominant (left), PR-A dominant (middle), or PR-A/B equivalent (right). Upper panel shows qRT-PCR analysis of FKBP5 mRNA relative abundance. Lower panel show immunoblot analysis of PR isoforms and GAPDH in each experimental condition (n = 3; *P < 0.05).

Figure 7.

Working model. During most of pregnancy, progesterone via PR-B promotes myometrial cell quiescence in part by repressing responsiveness to proinflammatory stimuli (left). With advancing gestation, prolabor signals increase the inflammatory load on the uterus until a threshold is reached. The threshold is the point at which inflammatory stimuli augment PR-A stability and transrepressive activity to trigger PR-A–mediated (via transrepression of PR-B) functional progesterone withdrawal (right), which causes a proinflammatory tissue-level state that leads to the local production of PGs that increase myometrial contraction and promote cervical softening to facilitate labor.