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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.04-1684fje. |
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suppresses the expression of steroid receptor coactivator-1 and -2: A possible mechanism contributing to changes in steroid hormone responsiveness
Center for Research on Reproduction and Women’s Health, University of Pennsylvania, Philadelphia, Pennsylvania, USA
2Correspondence: Center for Research on Reproduction and Women’s Health, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: jfs3{at}mail.med.upenn.edu
SPECIFIC AIMS
We hypothesized that proinflammatory cytokines may regulate steroid hormone activity in part by altering the levels of nuclear coactivator proteins. We proposed that tumor necrosis factor-
(TNF-) might decrease coactivator levels in uterine smooth muscle cells (UtSMC), ultimately resulting in decreased progesterone receptor trans-activation. A proinflammatory cytokine-mediated decline in steroid responsiveness in the uterus may play a role in the physiological changes associated with the onset of parturition.
PRINCIPAL FINDINGS
1. TNF- decreases SRC-1 and -2 mRNA abundance and protein levels in UtSMC
To pursue the idea that TNF- administration alters cofactor levels, UtSMC monolayers were treated with TNF- (10 ng/mL) or diluent and incubated for 0–48 h. After treatment, total RNA was isolated, cDNA synthesized, and quantitative real-time RT-PCR performed. TNF- did not affect mRNA abundance of the coactivator p300/cyclic AMP response element binding protein (CBP) or of the corepressors, silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (NCoR). Steroid hormone receptor coactivator-3 (SRC-3) mRNA was reduced in TNF--treated cells but was not statistically significant. (Fig. 1
A–D). However, TNF- treatment did decrease steroid hormone receptor coactivator-1 and -2 (SRC-1 and -2) mRNA abundance by 
42% (P<0.01) and 47% (P<0.03), respectively (Fig. 1E, F
, respectively). Western blot analysis revealed a decline in SRC-1 and -2 protein levels in nuclear extracts from UtSMC treated with TNF-. To further examine the functional relationship between coactivator reduction and nuclear receptor activity, UtSMC were screened for progesterone receptor expression. UtSMC were found to express PR-A and PR-B isoforms but not PR-C under both basal and treatment conditions. TNF- did not influence expression of PR-A or PR-B or alter the isoform distribution in this cell type.
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2. TNF--induced reductions of SRC-1 and -2 interferes with progesterone-stimulated PR-B-mediated trans-activation
Reduction of SRC-1 and -2 expression may alter the ability of nuclear receptors to trans-activate target genes. Therefore, we chose to examine the effects of TNF- on PR activity in the UtSMC model system (Fig. 2
A). Cells were transfected with a PR-B expression plasmid and a PRE(2)-luciferase reporter vector. Cells were subsequently treated with increasing doses of progesterone (0.01–100 nM) with 10 ng/mL TNF- or appropriate diluent and luciferase activity was measured. In cells transfected with PR-B, TNF- (10 ng/mL) decreased progesterone-stimulated (100 nM) PRE trans-activation by 60% (P<0.02). TNF- had a statistically significant inhibitory effect on PRE trans-activation at doses as low as 0.1 ng/mL (P<0.0001, data not shown).
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To examine the link between the decline in SRC-1 and -2 levels and the TNF--dependent decrease in PR-mediated trans-activation, SRC-1 and -2 expression plasmids and empty vectors were transfected into UtSMC in addition to PR-B and the PRE(2)-luciferase reporter. This model system allowed us to test the effects of TNF- on a background of fixed PR-B and model promoter levels. Cells were subsequently treated with progesterone (100 nM) and/or TNF- (10 ng/mL) and luciferase assays were performed. Exogenous SRC-1 administration partially prevented TNF-’s inhibitory effect on progesterone-activated PR-B trans-activation of the PRE reporter. However, SRC-2 fully prevented the TNF--induced decline in PRE-luciferase activity, suggesting that SRC-2 plays a dominant role in PR-mediated trans-activation (Fig. 2B
).
CONCLUSIONS AND SIGNIFICANCE
The biochemical events preceding uterine contraction and subsequent normal and preterm parturition have yet to be fully elucidated. In most mammalian species, high progesterone levels maintain uterine quiescence and labor is precipitated by a decline of progesterone within the bloodstream. In humans and higher primates, progesterone levels remain elevated throughout pregnancy; therefore, labor is not initiated by a decline in circulating progesterone. The onset of parturition in humans may involve a "functional progesterone withdrawal" or loss of progesterone sensitivity in the uterus. This loss of progesterone responsiveness in target tissues—specifically, the myometrium—may be mediated by an inflammatory reaction. Inflammation has been found to play a role in the physiological processes surrounding parturition: ripening of the cervix, rupturing of the fetal membranes, and ensuing uterine contractions. Due to the association between inflammation and ensuing uterine contraction, we investigated the effect of inflammatory cytokines on steroid hormone function, exploring the impact of TNF- on cofactor transcription and its ultimate effect on the loss of progesterone sensitivity at the myometrium. The present study demonstrated that a proinflammatory cytokine could suppress progesterone-stimulated PR-B trans-activation in an in vitro system. Primary cultures of UtSMC derived from a nonpregnant uterus, while providing an easily manipulatable system, are not a perfect representation of the pregnant human myometrium. However, these in vitro studies demonstrate that TNF- affects cellular sensitivity to steroid hormones in ways previously unrecognized.
Coactivators are members of a class of coregulatory proteins that interact with nuclear receptors to enhance their transcriptional activities by acting as a bridge between the receptor and the transcriptional machinery and facilitating the interaction of this complex with the target gene promoter. We have shown for the first time that proinflammatory cytokines can regulate SRC-1 and -2 expression and affect cellular responses to steroid hormones, a novel feature of cytokine action. Others have demonstrated that SRC-2, -3, and CBP decline in the fundal uterine tissue of women in labor, further supporting the theory that a reduction of coactivators could explain progesterone insensitivity in the uterus at term. This TNF--induced reduction in coactivator levels most likely is not specific to a single nuclear receptor, as cofactors have been found to interact with multiple nuclear receptor targets, potentially affecting the transcription of a large number of genes. To further validate an ultimate physiological response of the coactivator reduction, functional studies were performed using UtSMC and a model PRE(2)-luciferase reporter construct. Our goal was to investigate a potential ramification of coactivator reduction by exploring the mechanisms involved in inflammation-induced "functional progesterone withdrawal."
The TNF--induced reduction of SRC-1 and -2 resulted in diminishment of the ability of the PR to trans-activate a model promoter, potentially disrupting progesterone’s immunosuppressive activity and enabling the biochemical events that initiate parturition. This effect could be fully rescued by exogenous administration of SRC-2 and partially rescued with exogenous SRC-1, validating the integral involvement of SRC-1 and -2 in the PR trans-activation pathway. We have provided evidence for one avenue by which inflammatory cytokines can affect tissue steroid sensitivity and potentially initiate functional progesterone withdrawal at the myometrium. The modulation of coactivator expression by cytokines is a novel mechanism of cross-talk between the immune and endocrine systems.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1684fje; doi: 10.1096/fj.04-1684fje
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B pathway, suppresses SCR-1 and -2 levels within the nucleus. This decline in coactivator levels ultimately results in decreased PR trans-activation, precipitating "functional progesterone withdrawal." The release of progestational dominance within the fundus allows for subsequent uterine contraction, followed by parturition.