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Endocrinology Vol. 146, No. 11 4994-5002
Copyright © 2005 by The Endocrine Society

Expression of the GTP-Binding Protein (G{alpha}s) Is Repressed by the Nuclear Factor {kappa}B RelA Subunit in Human Myometrium

Neil R. Chapman, Ioannis Smyrnias, Dilly O. C. Anumba, G. Nicholas Europe-Finner and Stephen C. Robson

School of Surgical and Reproductive Sciences (Obstetrics and Gynecology) (N.R.C., I.S., G.N.E.-F., S.C.R.), Faculty of Medical Sciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom; and Academic Unit of Reproductive and Developmental Medicine (N.R.C., D.O.C.A.), Sheffield Teaching Hospitals NHS Trust, Sheffield S10 2SF, United Kingdom

Address all correspondence and requests for reprints to: Dr. Neil Chapman, Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessup Wing, Central Sheffield Teaching Hospitals Trust, Tree Root Walk, Sheffield, South Yorkshire S10 2SF, United Kingdom. E-mail: n.r.chapman{at}sheffield.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor {kappa}B (NF-{kappa}B) family of transcription factors. Recently, we have provided evidence that the RelA NF-{kappa}B subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the G{alpha}s/cAMP/protein kinase A pathway. TNF{alpha} is a potent activator of NF-{kappa}B, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNF{alpha} was observed to repress expression of G{alpha}s while, at the same time, stimulating NF-{kappa}B activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNF{alpha} was seen to repress endogenous G{alpha}s mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the G{alpha}s promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of G{alpha}s expression. Together, these data suggest that, in human myometrium, repression of the G{alpha}s gene by NF-{kappa}B occurs through a non-DNA binding mechanism involving competition for limiting amounts of cellular coactivator proteins including cAMP response element binding protein binding protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ONE OF THE most significant physiological adaptations of the uterus to pregnancy is the development of a relative state of myometrial smooth muscle inactivity termed quiescence. At the onset of labor, the quiescent state ends and a series of powerful, coordinated uterine contractions act to expel the infant. Premature contractions can result in preterm birth, which complicates 6–10% of all pregnancies and is responsible for over two thirds of perinatal deaths (1, 2). Moreover, surviving infants are at increased risk of major long-term mental and physical handicap (3). Importantly, current medical therapies have limited use and are associated with serious side effects for both the infant and mother (4). In addition, the United Kingdom health service spends between £42–74 million per annum on neonatal intensive care for babies weighing less than 1500 g (5).

There is growing evidence indicating that components of the cAMP signaling pathway are differentially expressed in the human myometrium during pregnancy, thereby potentiating the maintenance of uterine quiescence until term. These include calcitonin gene-related peptide receptors (6) and the adenylyl cyclase stimulatory G protein, G{alpha}s (7, 8, 9, 10, 11, 12, 13), whose levels of expression are considerably increased within the myometrium during gestation, causing an increased production of cAMP and activation of protein kinase A (PKA); these factors are reduced subsequently in labor (7, 8, 9, 10, 11, 12, 13). At present, there is a paucity of data describing how G{alpha}s expression is regulated in any system including the myometrium. Recently, our group reported that the G{alpha}s promoter was regulated by Sp-like transcription factors requiring phosphorylation by PKA (12). The precise nature of these factors and those that down-regulate G{alpha}s levels at term, however, have yet to be defined (12).

Elevation of cAMP and activation of PKA has been shown to inhibit nuclear factor {kappa}B (NF-{kappa}B)-mediated transcription in a number of cell lines, possibly through competition with other transcription factors for limiting amounts of the coactivator cAMP response element binding protein-binding protein (CBP) (14, 15, 16). However, this thesis is further complicated by reports detailing an association between RelA and PKA (17); with PKA being required to activate NF-{kappa}B DNA-binding in a cAMP-independent fashion (18). Furthermore, we have demonstrated that RelA and the PKA catalytic subunit associate within a protein complex in human myometrial homogenates from pregnant women (19). Importantly, TNF{alpha}, which serves as a potent activator of NF-{kappa}B, has been shown to reduce cAMP levels in cardiac myocytes (20) and up-regulate expression of G{alpha}i and G{alpha}q in human airway smooth muscle (21). More recently, it has been demonstrated that NF-{kappa}B is required to stimulate expression of the G{alpha}i2 gene (22).

NF-{kappa}B, which is rapidly induced by over 400 different stimuli including cytokines and growth factors (http://people.bu.edu/gilmore/nf-kb/inducers/index.html), is present in virtually every cell type within the body. NF-{kappa}B can take a number of different forms and is composed of dimeric complexes formed from five distinct subunits, RelA (p65), RelB, c-Rel, NF-{kappa}B1 (p105/p50), and NF-{kappa}B2 (p100/p52) (reviewed in Refs.23 and 24). Combinations of subunits determine the specificity of transcriptional activation and all have distinct, nonoverlapping functions (23, 24, 25, 26).

In the majority of unstimulated cell types, NF-{kappa}B is retained within the cytoplasm in an inactive form, bound to its inhibitor protein, I{kappa}B, of which there are several isoforms (23, 24). Activation of NF-{kappa}B is ultimately facilitated by the ubiquitination and degradation of I{kappa}B with the concomitant release of particular NF-{kappa}B subunits, for example, RelA:p50 heterodimers, which translocate to the nucleus where they modulate gene expression (23, 24).

A role for NF-{kappa}B in both term and preterm human labor is supported by observations that it is induced by, and subsequently activates, the cellular inflammatory pathway in all gestational tissues examined including amnion and decidua (27, 28, 29, 30, 31, 32, 33, 34), cervix (28, 35), and myometrium (36, 37). Specific NF-{kappa}B-regulated genes examined include COX-2, IL-1ß, IL-8, and phospholipase-A2 (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), Furthermore, inhibitors of NF-{kappa}B activity, including IL-10 (38), can suppress both lipopolysaccharide (LPS)-induced preterm labor in mice and rats (39) and IL-1ß-induced uterine contractions in Rhesus monkeys (40), providing further, albeit circumstantial, evidence for NF-{kappa}B function during parturition. These studies all support the contention that labor is associated with a highly regulated, inflammatory-like process that is underpinned by NF-{kappa}B-mediated gene activation.

Significantly, however, although NF-{kappa}B will generally activate transcription, the Drosophila NF-{kappa}B homolog, Dorsal, has also been documented to repress gene expression in the presence of corepressor proteins such as Groucho (41). Furthermore, mammalian NF-{kappa}B also represses transcription through interactions with other transcription factors including Egr-1 (42), c-Myc (43), histone deacetylases (HDACs) (44, 45, 46, 47), or by non-DNA-binding mechanisms that include the competition for cellular coactivators such as CBP and p300 (48, 49).

Thus, although it is clear that there is cross-talk between the NF-{kappa}B and cAMP/PKA pathways within myometrium, there are no data examining whether NF-{kappa}B may actually regulate G{alpha}s expression. Consequently, we tested the hypothesis that NF-{kappa}B represses G{alpha}s expression while concomitantly inducing the up-regulation of the proinflammatory cascade associated with parturition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Selection of patients and tissue collection
All women were recruited from the Department of Obstetrics and Gynaecology at the Royal Victoria Infirmary (Newcastle-upon-Tyne, UK). This study received approval from the Newcastle and North Tyneside Health Authority Ethics Committee and all patients gave informed, written consent.

Term pregnant myometrium (P)
Lower uterine segment myometrial samples were obtained from healthy women undergoing elective caesarean section at term (n = 14, age 16–43, gestation 37–40 weeks) as described previously (19). Myometrial smooth muscle cell cultures were then subsequently generated as detailed in Phaneuf et al. (50).

Transient transfections, plasmids, and luciferase assays
Transient transfection of primary human myometrial cells and human embryonic kidney (HEK) 293 cells was performed using the LT-1 reagent from Miras (Cambridge Biosciences, Cambridge, UK) according to the manufacturer’s guidelines. The G{alpha}s-luciferase reporter (G{alpha}s-Luc) has been described previously (12). The 3x-{kappa}B-ConA-luciferase (3x-{kappa}B-ConA-Luc) and enh-ConA-luciferase ({Delta}{kappa}B-ConA-Luc) vectors were the generous gift of Prof. Ron Hay (University of St. Andrews, Fife, UK) and have been detailed in Ref.51 . pBS-RelA, RSV-RelA, RSV-ß-galactosidase, and pcDNA3-Gal4-RelA(TAD) plasmids were the kind gift of Dr. Neil Perkins (University of Dundee, Dundee, UK) and have been described previously (44, 52, 53). The RcRSV-HA-CBP expression plasmid was supplied by Dr. Paul Hurd (University of Cambridge, Cambridge, UK), but was originally generated in the Goodman laboratory (Vollum Institute, Portland, OR), and its construction has been detailed elsewhere (54). For each transfection, 200 ng of luciferase reporter was used. Twenty-four hours after transfection, cells were then stimulated for a further 24 h with 10 ng/ml TNF{alpha} or 2 µg/ml bacterial LPS. For experiments using exogenous RelA, 250 ng RSV-RelA or 250 ng RSV-ß-galactosidase as a control were used. For those studies investigating the role of the Rel homology domain (RHD), 250 ng pcDNA3-Gal4-RelA(TAD) or 250 ng pcDNA3 were used. Transfections using CBP used 0.5, 1.0, and 1.5 µg RcRSV-CBP, or 0.5, 1.0, and 1.5 µg RcRSV-NRC-MCS to normalize the amount of DNA. Luciferase assays were performed as previously described (10).

Preparation of total RNA
Total RNA was prepared from primary human myometrial cultures using TRI-reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. The resulting pellet was resuspended in 100 µl RNase-free water. This total RNA was then applied to an RNeasy column (QIAGEN, Valencia, CA) to facilitate DNaseI treatment, thereby removing any residual genomic DNA. Again the manufacturer’s guidelines were followed. Total RNA was then eluted in 35 µl RNase-free water. Expression of G{alpha}s was then determined using RT-PCR analyses with the following primers: G{alpha}s sense, 5'-ATGGGCTGCCTCGGGAACAGTAAGACC-3' and G{alpha}s antisense, 5'-TTAGAGCAGCTCGTACTGACGAAGGTG-3'. To ensure equal loading of samples, a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using the following primers: GAPDH sense, 5'-CTGCCGTCTAGAAAAACC-3' and GAPDH antisense, 5'-CCACCTTCGTTGTCATACC-3'. All RT-PCRs used the Rapid Access RT-PCR kit from Promega (Southampton, UK). Product sizes of approximately 1.2 kB (G{alpha}s) and approximately 220 bp (GAPDH) were obtained.

Western immunodetection and precipitation analyses
Protein expression was examined using Western analysis with immunoblots probed for the RelA NF-{kappa}B subunit essentially as detailed in Chapman et al. (19). After SDS-PAGE, resolved proteins were then electroblotted onto nitrocellulose (Sigma). The gel and membrane were first briefly soaked in Toubin transfer buffer (25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol) and then blotted for 90 min at 9 V (constant current; 90 mA). For experiments examining CBP expression, gels were blotted overnight at 60 V (120 mA). The membranes were then briefly washed in PBS and then blocked in 10% blocking buffer [PBST; PBS including 0.05% (vol/vol) Tween 20 and 10% (wt/vol) low-fat dried milk powder] for two 30-min sessions.

NF-{kappa}B immunoblots were performed with antibodies that recognize either the amino-terminal or carboxyl-terminal of RelA (p65) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; nos. sc-109 and sc-372, respectively) and p50/p105 (Upstate Biotechnology, Inc., Lake Placid, NY; no. 06–886). Anti-CBP serum was obtained from Santa Cruz Biotechnology, Inc. (no. sc-369). All incubations were performed in PBST including 3% (wt/vol) low-fat dried milk powder overnight at 4 C. After incubation with an appropriate secondary antiserum (goat antirabbit-horseradish peroxidase conjugate; DakoCytomation, Denmark; no. P0448), the membrane was then washed in PBST (blocking buffer without milk powder) for two 15-min washes and developed according to the enhanced chemiluminescence detection protocol described by the manufacturer (Amersham-Pharmacia Biotech).

Chromatin immunoprecipitation assay (ChIP)
The ChIP assay was performed using the ChIP assay kit provided by Upstate Biotechnology, Inc. (no. 17-295), following the manufacturer’s guidelines but including the following modifications: formaldehyde fixation was performed at 37 C for 15 min. The immunoprecipitation buffer was supplemented with 0.01% (vol/vol) Nonidet P-40 nonionic detergent. Five micrograms of each antibody was used per immunoprecipitation. Antisera used were anti-RelA C-terminal antibody (Upstate Biotechnology, Inc., no. 06–418) or nonspecific IgG (Santa Cruz Biotechnology, Inc., no. sc-2027). Before the PCR step, the immunoprecipitated DNA was recovered by phenol:chloroform extraction, isopropanol/potassium acetate precipitated, and then resuspended in 30 µl Tris/EDTA buffer, pH 8.0.

PCR on the immunoprecipitated DNA was carried out using primers flanking the {kappa}B sites within the I{kappa}B{alpha} promoter (sense, 5'-GACGACCCCAATTCAAATCG-3' and antisense, 5'-TCAGGCTCGGGGAATTTCC-3') or the putative {kappa}B site within the G{alpha}s promoter (sense, 5'-GCGAGGGGTCGTCACTGGCGCGG-3' and antisense, 5'-CGCCGCG-GCGGGCGGGGGGAGG-3'). Reactions conditions were those described in Saccani et al. (55) and are given here: one cycle of denaturation at 94 C for 3 min, 36 cycles of 94 C for 45 sec, 60 C for 1 min, and 72 C for 1 min followed by a final elongation at 72 C for 10 min. Samples were then resolved using 1% Tris-acetate agarose gel electrophoresis, and product sizes of approximately 300 bp (I{kappa}B{alpha} promoter) and approximately 360 bp (G{alpha}s promoter) were obtained.

Nuclear extracts and EMSA
Nuclear extracts from HEK 293 and primary myometrial cells were prepared as detailed in Phillips et al. (12) and Chapman et al. (43). EMSAs were performed using in vitro-translated RelA, synthesized using the pBS-RelA plasmid in the TnT-Quick reticulocyte lysate system from Promega as described previously (42). Five microliters of RelA was added to give a 20-µl reaction volume consisting of 20 mM HEPES (pH 7.9), 1 mM dithiothreitol, 1 µg poly (dI-dC:dI-dC) and approximately 0.1 ng {alpha}-32P-labeled double-stranded HIV 3' long terminal repeat {kappa}B site (5'-GATCCGCTGGGGACTTTCCAGGC G-3'; {kappa}B site in bold) or the putative G{alpha}s {kappa}B site (5'-GTACCAGGGACCCCCTCC-3'; putative {kappa}B site in bold). Proteins were preincubated for 5 min at room temperature with reaction buffer minus probe. To assess the specificity of binding, 100 ng (1000-fold excess) of the respective cold (unlabeled) specific {kappa}B or nonspecific (5'-GATCCACTCAGACCACGTGGTCGGGTAC-3'; c-Myc binding site in bold) oligonucleotides were included with the labeled probed in each reaction. For supershift analyses, antisera were included in the preincubation step; RelA supershifts used the sc-109 antiserum. After this time, labeled probe was added and incubations continued for a further 15 min at room temperature. DNA:protein complexes were then resolved using 1x Tris-glycine-EDTA (25 mM Tris (pH 8.0), 190 mM glycine, and 1 mM EDTA)-4% nondenaturing polyacrylamide gels.

Statistical analysis
Data were compared using an unpaired, two-tailed t test; P < 0.05 was considered statistically significant. All experiments were performed three times in triplicate, and results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} and LPS repress expression of the G{alpha}s promoter while inducing NF-{kappa}B activity
A number of proinflammatory cytokines, including TNF{alpha}, are associated with the onset of both normal and preterm birth (56). When primary myometrial myocytes were transfected with the G{alpha}s-Luc reporter and stimulated with TNF{alpha} for 24 h, a significant reduction in luciferase activity was observed, indicating that TNF{alpha} could repress the G{alpha}s promoter (Fig. 1AGo). Importantly, in parallel experiments where primary myometrial cells were transfected with a NF-{kappa}B-responsive reporter (3x-{kappa}B-ConA-Luc), TNF{alpha} stimulation was seen to strongly activate NF-{kappa}B activity (Fig. 1BGo); removal of the {kappa}B sites ({Delta}{kappa}B-ConA-Luc) abrogated this response to TNF{alpha} (Fig. 1CGo). To confirm that TNF{alpha} was activating NF-{kappa}B, nuclear extracts were prepared from primary cultures of myometrial myocytes exposed to TNF{alpha} and subject to Western analysis. Immunoblots were then probed for the RelA NF-{kappa}B subunit because this subunit is rapidly activated by TNF{alpha} (24). As expected, TNF{alpha} induced a strong nuclear localization of RelA (Fig. 1DGo).



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  		FIG. 1. TNF{alpha} represses G{alpha}s expression while inducing NF-{kappa}B activity in primary myometrial myocytes. Primary myometrial cultures were transfected with 200 ng of either G{alpha}s-luc (A), 3x-{kappa}B-ConA-luc (NF-{kappa}B-responsive) (B), or {Delta}{kappa}B-ConA-luc (NF-{kappa}B unresponsive) (C). After 24 h, cells were stimulated with TNF{alpha} (10 ng/ml) for a further 24 h. After this time, luciferase activity was quantitated. TNF{alpha} repressed G{alpha}s expression (A) and activated NF-{kappa}B (3x-{kappa}B-ConA) (B). C, No NF-{kappa}B activity was observed in a control reporter lacking {kappa}B sites ({Delta}{kappa}B-ConA). D, TNF{alpha} was seen to induce RelA nuclear localization in primary myometrial myocytes.

 
Approximately 40% of cases of preterm labor have an underlying bacterial infection (57). Consequently, we tested the notion that LPS, through activation of NF-{kappa}B, may also repress G{alpha}s expression. When primary myometrial myocytes were stimulated with LPS for 24 h, G{alpha}s-Luc activity was greatly reduced (Fig. 2AGo) while NF-{kappa}B activity was induced (Fig. 2BGo). Again, removal of the {kappa}B elements abolished the LPS response (Fig. 2CGo). We confirmed that LPS was able to induce increased nuclear localization of RelA (Fig. 2DGo).



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  		FIG. 2. Bacterial LPS represses G{alpha}s expression while inducing NF-{kappa}B activity in primary myometrial myocytes. Primary myometrial cultures were transfected with 200 ng of either G{alpha}s-luc (A), 3x-{kappa}B-ConA-luc (NF-{kappa}B-responsive) (B), or {Delta}{kappa}B-ConA-luc (NF-{kappa}B unresponsive) (C). After 24 h, cells were stimulated with LPS (2 µg/ml) for a further 24 h. After this time, luciferase activity was quantitated. Similar to TNF{alpha}, LPS repressed G{alpha}s expression (A) and activated NF-{kappa}B (3x-{kappa}B-ConA) (B). C, No NF-{kappa}B activity was observed in a control reporter lacking {kappa}B sites ({Delta}{kappa}B-ConA). D, LPS was seen to induce RelA nuclear localization in primary myometrial myocytes.

 
The RelA NF-{kappa}B subunit specifically represses G{alpha}s expression
It is well documented that both TNF{alpha} and LPS are potent inducers of NF-{kappa}B activity in many cell types (24) including myometrial myocytes (Figs. 1Go and 2Go). Therefore, we addressed the question of whether expression of exogenous RelA NF-{kappa}B could also repress basal G{alpha}s promoter activity. When primary myometrial myocytes were transfected with the RelA NF-{kappa}B subunit, G{alpha}s-Luc activity was seen to be repressed (Fig. 3AGo) while activating NF-{kappa}B-responsive controls (Fig. 3BGo). No significant effect was seen on the {Delta}{kappa}B-ConA-luc control (Fig. 3CGo), thus reflecting the effect seen with TNF{alpha} and LPS and reinforcing the notion that RelA NF-{kappa}B can repress G{alpha}s expression. No repression of G{alpha}s was seen when only the transactivation domain of RelA, Gal4-RelA(TAD), was present, suggesting an intact Rel homology domain is required for this effect (Fig. 3DGo). Interestingly, down-regulation of G{alpha}s could also be induced by RelA in HEK 293 cells, suggesting this effect is a general mechanism working in other cell types (Fig. 3EGo). Furthermore, a nonspecific transcriptional activator, Gal4-VP16, could not repress G{alpha}s-luc activity (data not shown).



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  		FIG. 3. The RelA NF-{kappa}B subunit represses G{alpha}s expression in primary myometrial myocytes and HEK 293 cells. Primary myometrial cultures were transfected with 200 ng of either G{alpha}s-luc (A), 3x-{kappa}B-ConA-luc (B), or {Delta}{kappa}B-ConA-luc (C) and 250 ng of a RelA expression vector or 250 ng Gal4-RelA(TAD). Cells were harvested after 48 h, and luciferase activity was quantitated. RelA also repressed G{alpha}s expression (A) and activated the NF-{kappa}B-responsive control (B) but not the {Delta}{kappa}B-ConA control (C). D, Gal4-RelA(TAD) fails to repress G{alpha}s-luc activity. HEK 293 cells were transfected with 200 ng G{alpha}s-luc and 250 ng of a RelA expression vector. E, RelA was also seen to repress G{alpha}s in this cell line.

 
TNF{alpha} represses expression of the endogenous G{alpha}s gene but RelA NF-{kappa}B subunits do not bind to a putative {kappa}B element in the G{alpha}s promoter
We have shown that TNF{alpha} and LPS can repress the G{alpha}s gene when it is placed in a reporter plasmid (Figs. 1Go and 2Go). However, such data does not consider the natural G{alpha}s promoter context because it is unclear whether reporter plasmid DNA forms chromatin with a structure reflective of that in chromosomal DNA, i.e. any selectivity provided when the site is within its native promoter may be lost when placed into a reporter plasmid (55). To address these concerns, primary myometrial myocytes were stimulated with TNF{alpha} for 24 h, total RNA was isolated, and then the level of endogenous G{alpha}s expression was assessed using RT-PCR. Significantly, we were able to confirm, in vivo, that TNF{alpha} consistently repressed endogenous G{alpha}s mRNA expression by 50% (Fig. 4AGo).



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  		FIG. 4. TNF{alpha} represses expression of the endogenous G{alpha}s gene but RelA NF-{kappa}B subunits do not bind to a putative {kappa}B element in the G{alpha}s promoter. Primary cultures of human myometrial cells were stimulated with TNF{alpha} (10 ng/ml) for 24 h. Cells were then used for obtaining total RNA for G{alpha}s quantitation by RT-PCR. A, TNF{alpha} consistently repressed endogenous G{alpha}s expression by 50%. For EMSA analysis, in vitro-translated recombinant RelA was incubated with a radiolabeled consensus HIV-{kappa}B probe or the putative {kappa}B site from the G{alpha}s promoter. Reactions were then resolved on nondenaturing polyacrylamide gels. B, No binding to the G{alpha}s-derived sequence was observed. Primary cultures of myometrial cells were stimulated with TNF{alpha} (10 ng/ml) for 24 h. The ChIP assay was then used to determine whether RelA bound the endogenous G{alpha}s promoter (C; top panel) in a manner similar to that seen for RelA binding to a positive control (I{kappa}B{alpha}) promoter (C; lower panel).

 
The data presented thus far illustrate that TNF{alpha} and LPS can repress G{alpha}s expression through induction of the RelA NF-{kappa}B subunit. However, it was unclear from these studies how RelA was mediating this repression. A search of the G{alpha}s promoter using the AliBaba2.1 web-based software (Ref.58 and www.gene-regulation.com/cgi-bin/pub/programs/alibaba2/) illustrated an imperfect {kappa}B element centralized over position –545 with the sequence 5'-AGGGACCCCC-3'. This may permit binding of NF-{kappa}B to the promoter and facilitate recruitment of corepressor proteins, including HDACs (44, 45, 46, 47), to the G{alpha}s promoter causing its repression. Importantly, the consensus NF-{kappa}B binding site is generally viewed as 5'-GGGRNYYYCC-3' (where R = A or G; N = A, C, T or G; and Y = C or T), although there are a great many functional variations on this (59). As such, we used EMSA to determine whether RelA NF-{kappa}B could bind to this putative {kappa}B site in the G{alpha}s promoter. No binding to this site was observed compared with that seen with the positive control (3' {kappa}B site from the HIV-long terminal repeat; Fig. 4BGo).

The EMSA data illustrated in Fig. 4BGo highlighted that RelA did not bind to a putative {kappa}B element located in the G{alpha}s promoter. Because binding of a transcription factor to DNA in vivo can be influenced by juxtaposed sequences/factors that were not present within the short (24-bp) EMSA oligonucleotides used here (52, 53), the ChIP was used. Primary myometrial myocytes were stimulated with TNF{alpha} for 24 h and the ChIP assay was performed as detailed in Materials and Methods. However, the G{alpha}s promoter could not be recovered by immunoprecipitating with an anti-RelA serum (Fig. 4CGo, top panel). We confirmed that the assay was functioning through the ability of TNF{alpha} to induce RelA DNA-binding to a known NF-{kappa}B-responsive promoter, namely I{kappa}B{alpha} (Fig. 4CGo, lower panel).

CBP relieves RelA-induced repression of the G{alpha}s promoter
It has been previously shown that phosphorylation of RelA at serine 276 is critical for its interaction with CBP (18). Furthermore, a number of studies have demonstrated that competition for limiting amounts of coactivator proteins, such as CBP, is a mechanism by which the actions of some transcription factors, including NF-{kappa}B, can be regulated (48, 49, 60, 61, 62, 63). Consequently, we tested the hypothesis that the RelA-induced repression of G{alpha}s was occurring through the ability of RelA to sequester the coactivator, CBP, from the G{alpha}s-promoter. In this instance, HEK 293 cells, which are more amenable to transfection than primary myometrial cultures, were transfected with both RelA and CBP. As before, RelA repressed G{alpha}s in these cells, whereas expression of additional amounts of CBP relieved this effect in a dose-dependent manner, with 1.5 µg CBP being observed as the optimum level (Fig. 5AGo). Western blot analysis confirmed that exogenous expression of CBP did not have a negative effect on the expression of RelA (Fig. 5BGo). Finally, we were also able to confirm that CBP could relieve RelA-induced G{alpha}s repression in cultures of primary myometrial cells (Fig. 5CGo). Again, CBP had no negative effects on the expression of RelA (Fig. 5DGo).



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  		FIG. 5. RelA-induced repression of G{alpha}s in HEK 293 cells and primary myometrial cultures is relieved by expression of CBP. HEK 293 cells were transfected with 250 ng RelA and 0.5, 1.0, and 1.5 µg CBP. Cells were harvested after 48 h, and luciferase activity was quantitated. A, Repression of G{alpha}s was relieved by coexpression of CBP in a dose-dependent manner. Expression of CBP did not effect expression from the RelA plasmid (only RelA blot illustrated; B). Primary myometrial cultures were transfected with 200 ng G{alpha}s-luc, 250 ng a RelA expression vector and 1.5 µg CBP. Cells were harvested after 48 h and luciferase activity was quantitated. C, RelA-induced G{alpha}s repression was also relieved by CBP in myometrial cells. D, Again, expression of CBP had no detrimental effects on expression of RelA in myometrial cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF{alpha}, LPS, and RelA repress G{alpha}s expression
We have examined the effect of proinflammatory mediators on the expression of the G{alpha}s promoter in primary myometrial cells. Our findings illustrate, for the first time, that TNF{alpha} and LPS can repress the expression of the G{alpha}s gene. Significantly, this effect was mediated by the RelA NF-{kappa}B subunit in both human myometrial cultures and HEK 293 cells, suggesting a conserved cellular mechanism regulates G{alpha}s expression. Furthermore, repression could not be induced when the RHD of RelA was replaced with the Gal4 DNA-binding region (Gal4-RelA; Fig. 3DGo). The RHD encompasses the region of RelA that associates with other regulatory proteins including CBP (17, 18) providing further, albeit indirect, support of the notion that RelA-induced repression of G{alpha}s uses competition for limiting amounts of cellular coactivators (see below). Finally, a control transcription factor, Gal4-VP16, could not repress G{alpha}s-luc activity (data not shown).

Therefore, these data suggest a novel mode of action for NF-{kappa}B whereby it also functions to repress certain proquiescent genes within the myometrium. As such, our data would imply that NF-{kappa}B may play a critical role in regulating the events that lead to the initiation of human parturition through both transcriptional activation of well-characterized proinflammatory genes including COX-2 and IL-8 (27, 28, 29, 30, 36, 37), and the concomitant repression of quiescence-associated proteins. Down-regulation of such genes would be expected to be of equal importance for the successful induction of parturition.

Effect of TNF{alpha} on other G protein subunits
The source of proinflammatory mediators within the myometrium is unclear. There is evidence to suggest that TNF{alpha} is derived from myometrial macrophages in response to LPS (64), whereas in the mouse uterus, IL-1ß secretion is stimulated by fetal lung-derived surfactant protein-A (65). However, other data suggest smooth muscle cells can secrete inflammatory factors even in the absence of immunological challenge, thereby providing autocrine and paracrine regulatory loops within the organ itself (66).

TNF{alpha} itself serves as a potent activator of NF-{kappa}B and has been shown previously to reduce cAMP levels in cardiac myocytes (20), up-regulate expression of G{alpha}i and G{alpha}q in human airway smooth muscle (21), and activate expression of G{alpha}i2 in the preerythroblast cell line K562 (22). We did not assess the levels of cAMP in our system, but we did see a 50% reduction in G{alpha}s levels as judged by RT-PCR. Importantly, earlier studies have demonstrated that there is marked reduction in both G{alpha}s protein and intracellular cAMP from laboring myometrium (7, 8) and this would be consistent with our current observations.

Mechanism of RelA-induced G{alpha}s repression
In the present study, we have demonstrated that the RelA NF-{kappa}B subunit can repress G{alpha}s in both human myometrial cells and HEK 293 cells. This repression does not involve direct binding of RelA to the G{alpha}s promoter, but uses a non-DNA binding mechanism involving the cellular coactivator protein CBP. Consistent with this observation are those that document similar molecular circuits that serve to regulate the p53-mediated apoptotic pathway (48, 49), steroid receptor-mediated repression of inflammation (60), macrophage development controlled by the Jak/STAT and Ras/AP-1 pathways (61), and the RelA-induced repression of the cAMP response element binding protein binding protein (CREB)-responsive gene phosphoenolpyruvate carboxykinase (62).

At present, the factors that serve to increase G{alpha}s expression are not well defined and there is very little data detailing how transcription of this gene is regulated. Consequently, it is difficult to accurately describe which factors would compete for RelA in the proposed model. Putative transcription factor binding sites for proteins, including Egr-1, CREB, Sp1, and WT-1, have been identified in the G{alpha}s promoter (Ref.58 and www.gene-regulation.com/cgi-bin/pub/programs/alibaba2/), with both CREB and Egr-1 being able to associate with CBP in other cell types (Refs.67 and 68 , reviewed in Ref.69). Therefore, we speculate that CBP is recruited to and activates the G{alpha}s promoter through binding one or more of these factors. Interestingly, we observed that when CBP alone was expressed in myometrial myocytes it readily activated G{alpha}s expression (data not shown), although it remains to be defined whether this was a promoter-specific effect.

Functional significance of RelA-induced G{alpha}s repression
To ensure tight control of transcription is maintained, critical cofactors, including CBP, appear to be present within the cell in very small, hence limiting, amounts. Recently, however, we reported that levels of CBP are elevated in pregnant myometrial homogenates (70). Although the reason for this observation remains unclear, one possibility is that the specific increase in CBP expression may reflect a mechanism by which the myometrium can effectively remove intracellular competition for this coactivator ensuring the expression of essential, quiescence-promoting genes is maintained throughout pregnancy. In the context of myometrial activity, it has previously been documented that both the COX-2 and IL-8 promoters require RelA and CBP for full transactivation to be attained (37), an observation that is consistent with data from various nonmyometrial cell lines (71). Consequently, one can envisage a situation during pregnancy, illustrated in Fig. 6Go, where CBP is associated with factors that are needed to activate G{alpha}s expression. However, before induction of parturition, when levels of CBP are again limiting (72), the immediate activation of RelA NF-{kappa}B would facilitate removal of CBP from the G{alpha}s promoter, thereby permitting repression of this quiescence-associated gene, and, at the same time, inducing activation of key inflammatory mediators, for example COX-2 and IL-8, which are associated with labor (27, 28, 29, 30, 36, 37). Obviously, such sequestration of cofactors may also underpin the ability of NF-{kappa}B to repress progesterone receptor-mediated transactivation in the human myometrium at term; progesterone receptor cofactors, such as steroid receptor coactivator-1, also form large multiprotein complexes with CBP, which serve to activate progesterone-responsive genes (72, 73). Removal of CBP from these steroid receptor complexes would repress ligand-dependent, steroid receptor-induced transcription (60, 73).



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  		FIG. 6. Schematic to illustrate how RelA NF-{kappa}B and CBP may repress the G{alpha}s gene. During pregnancy, engagement of G protein-coupled trans-membrane receptor (GPCTR) activates the G{alpha}s/adenylyl cyclase (AC) pathway, which causes an increase in intracellular cAMP and activation of PKA. PKA is then able to phosphorylate a number of nuclear proteins, including CREB, which, in turn, serves as a binding partner for CBP. Once CBP is associated with CREB at a given promoter, in this case G{alpha}s, it would serve to both acetylate histones within this region, thus facilitating promoter melting and also ensure all the necessary transcription factors for G{alpha}s expression are in the correct spatial positions, thereby ensuring the promoter can be transcribed by RNA polymerase II (Pol II). Activation of NF-{kappa}B at or before parturition by proinflammatory mediators, such as TNF{alpha} (acting through the TNF{alpha} receptor; TNF{alpha}R) or bacterial LPS (binding to Toll-like receptor 4; TLR4) stimulates the phosphorylation and nuclear localization of RelA. Once inside the nucleus, RelA binds to specific target genes, for example COX-2, where it is then able to recruit CBP to the promoter region. Because there is only a finite amount of CBP, a more favorable interaction with RelA on the COX-2 promoter over CREB may result in the loss of CBP from the G{alpha}s regulatory region. Loss of CBP would then be expected to down-regulate the G{alpha}s gene, possibly through the replacement of CBP with a repressor protein/complex such as a HDAC.

 
In summary, this study illustrates that proinflammatory agents, including both LPS and TNF{alpha}, serve to repress G{alpha}s expression in human primary myometrial cells. This repression is mediated by the RelA NF-{kappa}B subunit and also occurs in other cell types. Significantly, RelA does not bind directly to the G{alpha}s promoter, suggesting repression is through a non-DNA-binding mechanism involving the coactivator, CBP, implying that competition between individual promoters for this limiting cofactor may underpin the ability of RelA to down-regulate G{alpha}s immediately before parturition in humans.


   Acknowledgments
 
We thank Richard Goodman, Ron Hay, Paul Hurd, and Neil Perkins for providing some of the plasmids used in this study. The authors are also grateful to Drs. Jarrod Bailey, Alison Tyson-Capper, and Kate Gilmore for technical assistance and comments on the manuscript, and to the patients and staff at the Royal Victoria Infirmary for providing/obtaining myometrial biopsies.


   Footnotes
 
This work was funded by the University of Newcastle-upon-Tyne (to N.R.C.). G.N.E.-F. is funded by grants from Action Medical Research and the Wellcome Trust.

First Published Online August 4, 2005

Abbreviations: CBP, cAMP response element binding protein-binding protein; ChIP, chromatin immunoprecipitation assay; CREB, cAMP response element binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; HEK, human embryonic kidney; LPS, lipopolysaccharide; NF-{kappa}B, nuclear factor {kappa}B; PKA, protein kinase A; RHD, Rel homology domain.

Received May 3, 2005.

Accepted for publication July 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lumley J 2003 Defining the problem: the epidemiology of preterm birth. Br J Obstet Gynaecol. 110(Suppl 20):3–7
  2. Macintosh MCM, Haviland J, Korkodilos M 2001 Confidential enquiry into stillbirths and deaths in infancy. 8th annual report, 1 January–31 December 1999. London: Maternal and Child Health Research Consortium; 83–100
  3. López-Bernal A, Watson SP, Phaneuf S, Europe-Finner GN 1993 Biochemistry and physiology of preterm labour and delivery. Baillieres Clin Obstet Gynaecol 7:523–552[CrossRef][Medline]
  4. Higby K, Xenakis EMJ, Pauerstein CJ 1993 Do tocolytic agents stop preterm labor? A critical and comprehensive review of efficacy and safety. Am J Obstet Gynecol 168:1247–1259[Medline]
  5. Hall MH, Daniellian P, Lamont RF 1997 The importance of preterm birth. In Eldmer ML, Romero R, Lamont RF, eds. Preterm labour. London: Churchill Livingstone; 1–28
  6. Dong YL, Fang L, Kondapaka S, Gangula PR, Wimalamansa SJ, Yallampalli C 1999 Involvement of calcitonin gene-related peptide in the modulation of human myometrial contractility during pregnancy. J Clin Invest 104:559–565[Medline]
  7. Europe-Finner GN, Phaneuf S, Watson SP, López-Bernal A 1993 Identification and expression of G-proteins in human myometrium: up-regulation of G{alpha}s in pregnancy. Endocrinology 132:2484–2490[Abstract]
  8. Europe-Finner GN, Phaneuf S, Tolkovsky AM, Watson SP, López-Bernal A 1994 Down regulation of G{alpha}s in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 79:1835–1839[Abstract]
  9. Europe-Finner GN, Phaneuf S, Mardon HJ, López-Bernal A 1996 Human myometrial G{alpha}s-small (with serine) and G{alpha}s-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G{alpha}s protein isoforms in pregnancy and their down-regulation during labor. J Clin Endocrinol Metab 81:1069–1075[Abstract]
  10. Bailey J, Sparey C, Phillips RJ, Gilmore K, Robson SC, Dunlop W, Europe-Finner GN 2000 Expression of CREB, CREM and ATF2 cyclic AMP dependent transcription factors in the human myometrium during pregnancy and labor. Mol Hum Reprod 6:648–660[Abstract/Free Full Text]
  11. Bailey J, Phillips RJ, Pollard AJ, Gilmore K, Robson SC, Europe-Finner GN 2002 Characterization and functional analysis of CREM and ATF2 isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties. J Clin Endocrinol Metab 87:1717–1728[Abstract/Free Full Text]
  12. Phillips RJ, Bailey J, Robson SC, Europe-Finner GN 2002 The differential expression of the adenylyl cyclase-stimulatory GTP-binding protein G{alpha}s in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic AMP and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter. J Clin Endocrinol Metab 87:5675–5685[Abstract/Free Full Text]
  13. MacDougall MWJ, Europe-Finner GN, Robson SC 2003 Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII{alpha}) protein kinase A holoenzyme. J Clin Endocrinol Metab 88:2194–2205[Abstract/Free Full Text]
  14. Chen D and Rothenberg EV 1994 Interleukin-2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J Exp Med 179:931–942[Abstract/Free Full Text]
  15. Neumann M, Grieshammer T, Chuvpiol S, Kneitz B, Lohoff M, Schimpl A, Franza Jr BR, Serfling E 1995 RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. EMBO J 14:1991–2004[Medline]
  16. Parry GCN and Mackman N 1997 Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-{kappa}B-mediated transcription. J Immunol 159:5450–5456[Abstract]
  17. Zhong H, SuYang H, Erdjument-Bromage H, Tempst P, Ghosh S 1997 The transcriptional activity of NF-{kappa}B is regulated by the I{kappa}B-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89:413–424[CrossRef][Medline]
  18. Zhong H, Voll RE, Ghosh S 1998 Phosphorylation of NF-{kappa}B p65 PKA stimulates transcriptional activity by promoting a novel bivalent interaction with coactivator CBP/p300. Mol Cell 1:661–671[CrossRef][Medline]
  19. Chapman NR, Europe-Finner GN, Robson SC 2004 Expression and DNA-binding activity of the nuclear factor {kappa}B (NF-{kappa}B) family in the human myometrium during pregnancy and labor. J Clin Endocrinol Metab 89:5683–5693[Abstract/Free Full Text]
  20. Gulick T, Chung MK, Pieper SJ, Lange LG, Schreiner GF 1989 Interleukin-1 and tumor necrosis factor inhibit cardiac myocyte ß-adrenergic responsiveness. Proc Natl Acad Sci USA 86:6753–6757[Abstract/Free Full Text]
  21. Hotta K, Emala CW, Hirshman CA 1999 TNF-{alpha} upregulates Gi{alpha} and Gq{alpha} protein expression and function in human airway smooth muscle cells. Am J Physiol 276:L405–L411
  22. Arinze IJ and Kawai Y 2005 Transcriptional activation of the human G{alpha}si2 gene promoter through nuclear factor-{kappa}B and antioxidant response elements. J Biol Chem 280:9786–9795[Abstract/Free Full Text]
  23. Chapman NR, Rocha S, Adcock IM, Perkins ND 2002 NF-{kappa}B function in cellular stress and human disease. In: Storey KB and Storey JM, eds. Cell and molecular responses to stress. Vol 3. Amsterdam: Elsevier Press; 61–73
  24. Hayden MS and Ghosh S 2004 Signaling to NF-{kappa}B. Genes Dev 18:2195–2224[Abstract/Free Full Text]
  25. Perkins ND, Schmid RM, Duckett CS, Leung K, Rice NR, Nabel GJ 1992 Distinct combinations of NF-{kappa}B subunits determine the specificity of transcriptional activation. Proc Natl Acad Sci USA 89:1529–1533[Abstract/Free Full Text]
  26. Gerondakis S, Grossmann M, Nakamura Y, Pohl T, Grumont R 1999 Genetic approaches in mice to understand Rel/NF-{kappa}B and I{kappa}B function: transgenics and knockouts. Oncogene 18:6888–6895[CrossRef][Medline]
  27. Allport VC, Pieber D, Slater DM, Newton R, White JO, Bennett PR 2001 Human labor is associated with nuclear factor-{kappa}B activity which mediates cyclo-oxygenase-2 expression and is involved with the "functional progesterone withdrawal". Mol Hum Reprod 7:581–586[Abstract/Free Full Text]
  28. Elliot CL, Allport VC, Loundon JAZ, Wu GD, Bennett PR 2001 Nuclear factor {kappa}-B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells. Mol Hum Reprod 7:787–790[Abstract/Free Full Text]
  29. Lee Y, Allport V, Sykes A, Lindstrom T, Slater D, Bennett PR 2003 The effects of labor and of interleukin-1ß upon the expression of nuclear factor {kappa}B related proteins in human amnion. Mol Hum Reprod 9:213–218[Abstract/Free Full Text]
  30. Yan X, Xiao CW, Sun M, Tsang BK, Gibb W 2002 Nuclear factor {kappa}B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1ß. Biol Reprod 66:1667–1671[Abstract/Free Full Text]
  31. Lappas M, Permezel M, Rice GE 2003 N-acetyl-cysteine inhibits phospholipids metabolism, proinflammatory cytokine release, protease activity and nuclear factor-{kappa}B deoxyribonucleic acid-binding activity in human fetal membranes in vitro. J Clin Endocrinol Metab 88:1723–1729[Abstract/Free Full Text]
  32. Yan X, Sun M, Gibb W 2002 Localisation of nuclear factor-{kappa}B (NF-{kappa}B) and inhibitory factor-{kappa}B (I{kappa}B) in human fetal membranes and deciduas at term and preterm delivery. Placenta 23:288–293[CrossRef][Medline]
  33. Lappas M, Permezel M, Geogiou HM, Rice GE 2004 Regulation of phospholipase isozymes by Nuclear factor-{kappa}B in human gestational tissues in vitro. J Clin Endocrinol Metab 89:2365–2372[Abstract/Free Full Text]
  34. Lappas M and Rice GE 2004 Phospholipase-A2 isozymes in pregnancy and parturition. Prostaglandins Leukotrienes Essential Fatty Acids 70:87–100[CrossRef][Medline]
  35. Stjernholm-Vladic Y, Stygar D, Mansson C, Masironi B, Akerberg S, Wang H, Ekman-Ordeberg G, Sahlin L 2004 Factors involved in the inflammatory events of cervical ripening in humans. Reprod Biol Endocrinol 2:74[CrossRef][Medline]
  36. Belt AR, Baldassare JJ, Molnár M, Romero R, Hertelendy F 1999 The nuclear transcription factor NF-{kappa}B mediates interleukin-1ß-induced expression of cyclooxygenase-2 in human myometrial cells. Am J Obstet Gyneacol 181:359–366
  37. Soloff MS, Cook Jr DL, Jeng Y-J, Anderson GD 2004 In situ analysis of interleukin-1 induced transcription of cox-2 and IL-8 in cultured human myometrial cells. Endocrinology 145:1248–1254[Abstract/Free Full Text]
  38. Schottelius AJG, Mayo MW, Sartor RB, Baldwin AS 1999 Interleukin-10 signaling blocks inhibitor of {kappa}B kinase activity and nuclear factor {kappa}B DNA binding. J Biol Chem 274:31868–31874[Abstract/Free Full Text]
  39. Sato TA, Keelan JA, Mitchell MD 2003 Critical paracrine interactions between TNF-{alpha} and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production. J Immunol 170:158–166[Abstract/Free Full Text]
  40. Sadowsky DW, Novy MJ, Witkin SS, Gravett MG 2003 Dexamethasone or interleukin-10 blocks interleukin-1ß-induced uterine contractions in pregnant rhesus monkeys. Am J Obstet Gynecol 188:252–263[CrossRef][Medline]
  41. Dubnicoff T, Valentine SA, Chen G, Shi T, Lengyel JA, Paroush Z, Courney AJ 1997 Conversion of Dorsal from an activator to a repressor by the global corepressor Groucho. Genes Dev 11:2952–2957[Abstract/Free Full Text]
  42. Chapman NR and Perkins ND 2000 Inhibition of the RelA(p65) NF-{kappa}B subunit by Egr-1. J Biol Chem 275:4719–4725[Abstract/Free Full Text]
  43. Chapman NR, Webster GA, Gillespie PJ, Wilson BJ, Crouch DH, Perkins ND 2002 A novel form of the RelA nuclear factor-{kappa}B subunit is induced by and forms a complex with the proto-oncogene c-Myc. Biochem J 366:459–469[CrossRef][Medline]
  44. Ashburner BP, Westerhide SD, Baldwin AS 2001 The p65 (RelA) subunit of NF-{kappa}B interacts with the histone deacetylase (HDAC) corepressors HDAC-1 and HDAC-2 to negatively regulate gene expression. Mol Cell Biol 21:7065–7077[Abstract/Free Full Text]
  45. Rocha S, Martin AM, Meek DW, Perkins ND 2003 p53 represses cyclin D1 transcription through down regulation of Bcl-3 and inducing increased association of the p52 NF-{kappa}B subunit with histone deacetylase-1. Mol Cell Biol 23:4713–4727[Abstract/Free Full Text]
  46. Campbell KJ, Rocha S, Perkins ND 2004 Active repression of antiapoptotic gene expression by RelA(p65) NF-{kappa}B. Mol Cell 13:858–865
  47. Zhang W and Kone B 2002 NF-{kappa}B inhibits transcription of the H+-K+-ATPase-{alpha}2 subunit gene: role of histone deacetylases. Am J Physiol Renal Physiol 283:F904–F911
  48. Webster GA and Perkins ND 1999 Transcriptional cross talk between NF-{kappa}B and p53. Mol Cell Biol 19:3485–3495[Abstract/Free Full Text]
  49. Wadgaonkar R, Phelps KM, Haque Z, Williams AJ, Silverman ES, Collins T 1999 CREB-binding protein is a nuclear integrator of nuclear factor-{kappa}B and p53 signaling. J Biol Chem 274:1879–1882[Abstract/Free Full Text]
  50. Phaneuf S, Europe-Finner GN, Varney M, Mackenzie IE, Watson SP, López-Bernal A 1993 Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins. J Endocrinol 136:497–509[Abstract]
  51. Rodriguez MS, Wright J, Thompson J, Thomas D, Baleux F, Virelizier JL, Hay RT, Arenzana-Seisdedos F 1996 Identification of lysine residues required for signal-induced ubiquitination and degradation of I{kappa}B{alpha} in vivo. Oncogene 12:2425–2435[Medline]
  52. Perkins ND, Edwards NL, Duckett CS, Agranoff AB, Schmid RM, Nabel GJ 1993 A cooperative interaction between NF-{kappa}B and Sp1 is required for HIV-1 enhancer activation. EMBO J 12:3551–3558[Medline]
  53. Perkins ND, Agranoff AB, Pascal E, Nabel GJ 1994 An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol Cell Biol 14:6570–6583[Abstract/Free Full Text]
  54. Kwok RP, Lundbland JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP is a co-activator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  55. Saccani S, Pantano S, Natoli G 2003 Modulation of NF-{kappa}B activity by exchange of dimers. Mol Cell 11:1563–1574[CrossRef][Medline]
  56. Keelan JA, Blumenstein M, Helliwell RJA, Sato TA, Marvin KW, Mitchell MD 2003 Cytokines, prostaglandins and parturition—a review. Placenta 24(Suppl A):S33–S46
  57. Friese K 2003 The role of infection in preterm labour. BJOG 110(Suppl 20):52–54
  58. Kozasa T, Itoh, H, Tsukamoto T, Kaziro Y 1988 Isolation and characterization of the human Gs{alpha} gene. Proc Natl Acad Sci USA 85:2081–2085[Abstract/Free Full Text]
  59. Chen FE and Ghosh G 1999 Regulation of DNA binding by Rel/NF-{kappa}B transcription factors: structural views. Oncogene 18:6845–6852[CrossRef][Medline]
  60. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  61. Horvai AE, Xu L, Korzus E, Brard G, Kalafus D, Mullen TM, Rose DW, Rosenfeld MG, Glass CK 1997 Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300. Proc Natl Acad Sci USA 94:1074–1079[Abstract/Free Full Text]
  62. Waltner-Law M, Daniels MC, Sutherland C, Granner DK 2000 NF-{kappa}B inhibits glucocorticoid and cAMP-mediated expression of the phosphoenolpyruvate carboxykinase gene. J Biol Chem 275:31847–31856[Abstract/Free Full Text]
  63. Guberman AS, Scassa ME, Giono LE, Varone CL, Cánepa ET 2003 Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator. J Biol Chem 278:2317–2326[Abstract/Free Full Text]
  64. Young A, Thomson AJ, Ledingham M, Jordan F, Greer IA, Norman JE 2002 Immunolocalisation of proinflammatory cytokines in myometrium, cervix and fetal membranes during human parturition at term. Biol Reprod 66:445–449[Abstract/Free Full Text]
  65. Condon JC, Jeyasuria P, Faust JM, Mendelson CR 2004 Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. Proc Natl Acad Sci USA 101:4978–4983[Abstract/Free Full Text]
  66. Singer CA, Salinthone S, Baker KJ, Gerthoffer WT 2004 Synthesis of immune modulators by smooth muscles. BioEssays 26:646–655[CrossRef][Medline]
  67. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  68. Silverman ES, Du J, Williams AJ, Wadgaonkar R, Drazen JM, Collins T 1998 cAMP-response-element-binding-protein-binding-protein (CBP) and p300 are transcriptional co-activators of the early growth response factor-1 (Egr-1). Biochem J 336:183–189
  69. Goodman RH and Smolik S 2000 CBP/p300 in cell growth, transformation and development. Genes Dev 14:1553–1577[Free Full Text]
  70. Long AA, Chapman NR, Europe-Finner GN, Robson SC 2005 Expression and interactions of the transcriptional co-regulators CBP/p300 in the human myometrium during pregnancy. J Soc Gynecol Invest 12:92–97
  71. Yamamoto Y, Verma UN, Prajapati S, Kwak YT, Gaynor RB 2003 Histone H3 phosphorylation by IKK-{alpha} is critical for cytokine-induced gene expression. Nature 423:655–659[CrossRef][Medline]
  72. Smith CL, Oñate SA, Tsai MJ, O’Malley BW 1996 CREB binding protein acts synergistically with steroid receptor co-activator-1 to enhance steroid receptor-dependent transcription. Proc Natl Acad Sci USA 93:8884–8888[Abstract/Free Full Text]
  73. Condon JC, Jeyasuria P, Faust JM, Wilson JW, Mendelson CR 2003 A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition. Proc Natl Acad Sci USA 100:9518–9523