Huiqing Tan

Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition

CONTEXT Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. OBJECTIVE Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. DESIGN Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. RESULTS PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. CONCLUSIONS Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone.

Estrogen receptor (ER) expression and function in the pregnant human myometrium: estradiol via ERα activates ERK1/2 signaling in term myometrium

Estrogens are thought to promote labor by increasing the expression of pro-contraction genes in myometrial cells. The specific estrogen receptors ((ERs: ERα and ERβ (also known as ESR1 and ESR2)) and G protein-coupled receptor 30 (GPR30; also known as G protein-coupled estrogen receptor 1)) and signaling pathways that mediate these actions are not clearly understood. In this study, we identified the ERs expressed in the pregnant human myometrium and determined a key extranuclear signaling pathway through which estradiol (E(2)) modulates expression of the gene encoding the oxytocin receptor (OXTR), a major pro-contraction protein. Using quantitative RT-PCR, we found that ERα and GPR30 mRNAs were expressed in the human pregnant myometrium while ERβ mRNA was virtually undetectable. While mRNA encoding ERα was the predominant ER transcript in the pregnant myometrium, ERα protein was largely undetectable in myometrial tissue by immunoblotting. Pharmacological inhibition of 26S proteasome activity increased ERα protein abundance to detectable levels in term myometrial explants, however, indicating rapid turnover of ERα protein by proteasomal processing in the pregnant myometrium. E(2) stimulated rapid extranuclear signaling in myometrial explants, as evidenced by increased extracellularly regulated kinase (ERK1/2) phosphorylation within 10 min. This effect was inhibited by pre-treatment with an ER antagonist, ICI 182 780, indicating the involvement of ERα. Inhibition of ERK signaling abrogated the ability of E(2) to stimulate OXTR gene expression in myometrial explants. We conclude that estrogenic actions in the human myometrium during pregnancy, including the stimulation of contraction-associated gene expression, can be mediated by extranuclear signaling through ERα via activation of the ERK/mitogen-activated protein kinase pathway.

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

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.

Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression.

Control of Progesterone Receptor-A Transrepressive Activity in Myometrial Cells: Implications for the Control of Human Parturition

Uterine quiescence during pregnancy is maintained by progesterone primarily via signaling mediated by the type-B progesterone receptor (PR-B) in myometrial cells. Withdrawal of PR-B-mediated progesterone activity is a principal trigger for labor. One mechanism for PR-B withdrawal is by inhibition of its activity by the type-A PR (PR-A) isoform in myometrial cells. We hypothesized that human parturition involves hormonal interactions that induce the capacity for PR-A to inhibit PR-B in myometrial cells and that pro-inflammatory cytokines are major regulators of this process. We tested this hypothesis in an immortalized human myometrial cell line, hTERT-HMA/B, in which levels of PR-A and PR-B can be experimentally controlled. We found that the capacity for PR-A to repress PR-B, assessed by activity of a transiently transfected reporter DNA controlled by the progesterone response element, and expression of FK506 binding protein 5 (FKBP5) an endogenous PR-B responsive gene, was increased by serum supplementation and interleukin-1β. In pregnant uterus, FKBP5 was detected exclusively in myometrial cells and its expression decreased with advancing gestation and in association with the onset of labor at term. These findings suggest that in myometrial cells the repressive activity of PR-A on PR-B increases with advancing gestation and is induced by pro-inflammatory cytokines. This may be a key mechanism linking inflammation with the onset of labor.

Progesterone and cAMP synergize to inhibit responsiveness of myometrial cells to pro-inflammatory/pro-labor stimuli

Progesterone (P4) acting through the P4 receptor (PR) isoforms, PR-A and PR-B, promotes uterine quiescence for most of pregnancy, in part, by inhibiting the response of myometrial cells to pro-labor inflammatory stimuli. This anti-inflammatory effect is inhibited by phosphorylation of PR-A at serine-344 and -345 (pSer344/345-PRA). Activation of the cyclic adenosine monophosphate (cAMP) signaling pathway also promotes uterine quiescence and myometrial relaxation. This study examined the cross-talk between P4/PR and cAMP signaling to exert anti-inflammatory actions and control pSer344/345-PRA generation in myometrial cells. In the hTERT-HMA/B immortalized human myometrial cell line P4 inhibited responsiveness to interleukin (IL)-1β and forskolin (increases cAMP) and 8-Br-cAMP increased this effect in a concentration-dependent and synergistic manner that was mediated by activation of protein kinase A (PKA). Forskolin also inhibited the generation of pSer344/345-PRA and expression of key contraction-associated genes. Generation of pSer344/345-PRA was catalyzed by stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Forskolin inhibited pSer344/345-PRA generation, in part, by increasing the expression of dual specificity protein phosphatase 1 (DUSP1), a phosphatase that inactivates mitogen-activated protein kinases (MAPKs) including SAPK/JNK. P4/PR and forskolin increased DUSP1 expression. The data suggest that P4/PR promotes uterine quiescence via cross-talk and synergy with cAMP/PKA signaling in myometrial cells that involves DUSP1-mediated inhibition of SAPK/JNK activation.