Expression of the GTP-Binding Protein (Gs) Is Repressed by the Nuclear Factor B RelA Subunit in Human Myometrium
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《内分泌学杂志》
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
Academic Unit of Reproductive and Developmental Medicine (N.R.C., D.O.C.A.), Sheffield Teaching Hospitals NHS Trust, Sheffield S10 2SF, United Kingdom
Abstract
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 B (NF-B) family of transcription factors. Recently, we have provided evidence that the RelA NF-B subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Gs/cAMP/protein kinase A pathway. TNF is a potent activator of NF-B, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNF was observed to repress expression of Gs while, at the same time, stimulating NF-B activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNF was seen to repress endogenous Gs mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Gs promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Gs expression. Together, these data suggest that, in human myometrium, repression of the Gs gene by NF-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
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, Gs (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 Gs expression is regulated in any system including the myometrium. Recently, our group reported that the Gs promoter was regulated by Sp-like transcription factors requiring phosphorylation by PKA (12). The precise nature of these factors and those that down-regulate Gs levels at term, however, have yet to be defined (12).
Elevation of cAMP and activation of PKA has been shown to inhibit nuclear factor B (NF-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-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, which serves as a potent activator of NF-B, has been shown to reduce cAMP levels in cardiac myocytes (20) and up-regulate expression of Gi and Gq in human airway smooth muscle (21). More recently, it has been demonstrated that NF-B is required to stimulate expression of the Gi2 gene (22).
NF-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-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-B1 (p105/p50), and NF-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-B is retained within the cytoplasm in an inactive form, bound to its inhibitor protein, IB, of which there are several isoforms (23, 24). Activation of NF-B is ultimately facilitated by the ubiquitination and degradation of IB with the concomitant release of particular NF-B subunits, for example, RelA:p50 heterodimers, which translocate to the nucleus where they modulate gene expression (23, 24).
A role for NF-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-B-regulated genes examined include COX-2, IL-1, IL-8, and phospholipase-A2 (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), Furthermore, inhibitors of NF-B activity, including IL-10 (38), can suppress both lipopolysaccharide (LPS)-induced preterm labor in mice and rats (39) and IL-1-induced uterine contractions in Rhesus monkeys (40), providing further, albeit circumstantial, evidence for NF-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-B-mediated gene activation.
Significantly, however, although NF-B will generally activate transcription, the Drosophila NF-B homolog, Dorsal, has also been documented to repress gene expression in the presence of corepressor proteins such as Groucho (41). Furthermore, mammalian NF-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-B and cAMP/PKA pathways within myometrium, there are no data examining whether NF-B may actually regulate Gs expression. Consequently, we tested the hypothesis that NF-B represses Gs expression while concomitantly inducing the up-regulation of the proinflammatory cascade associated with parturition.
Materials and Methods
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 Gs-luciferase reporter (Gs-Luc) has been described previously (12). The 3x-B-ConA-luciferase (3x-B-ConA-Luc) and enh-ConA-luciferase (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--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 or 2 μg/ml bacterial LPS. For experiments using exogenous RelA, 250 ng RSV-RelA or 250 ng RSV--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 Gs was then determined using RT-PCR analyses with the following primers: Gs sense, 5'-ATGGGCTGCCTCGGGAACAGTAAGACC-3' and Gs 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 (Gs) 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-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-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 B sites within the IB promoter (sense, 5'-GACGACCCCAATTCAAATCG-3' and antisense, 5'-TCAGGCTCGGGGAATTTCC-3') or the putative B site within the Gs 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 (IB promoter) and approximately 360 bp (Gs 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 -32P-labeled double-stranded HIV 3' long terminal repeat B site (5'-GATCCGCTGGGGACTTTCCAGGC G-3'; B site in bold) or the putative Gs B site (5'-GTACCAGGGACCCCCTCC-3'; putative 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 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
TNF and LPS repress expression of the Gs promoter while inducing NF-B activity
A number of proinflammatory cytokines, including TNF, are associated with the onset of both normal and preterm birth (56). When primary myometrial myocytes were transfected with the Gs-Luc reporter and stimulated with TNF for 24 h, a significant reduction in luciferase activity was observed, indicating that TNF could repress the Gs promoter (Fig. 1A). Importantly, in parallel experiments where primary myometrial cells were transfected with a NF-B-responsive reporter (3x-B-ConA-Luc), TNF stimulation was seen to strongly activate NF-B activity (Fig. 1B); removal of the B sites (B-ConA-Luc) abrogated this response to TNF (Fig. 1C). To confirm that TNF was activating NF-B, nuclear extracts were prepared from primary cultures of myometrial myocytes exposed to TNF and subject to Western analysis. Immunoblots were then probed for the RelA NF-B subunit because this subunit is rapidly activated by TNF (24). As expected, TNF induced a strong nuclear localization of RelA (Fig. 1D).
Approximately 40% of cases of preterm labor have an underlying bacterial infection (57). Consequently, we tested the notion that LPS, through activation of NF-B, may also repress Gs expression. When primary myometrial myocytes were stimulated with LPS for 24 h, Gs-Luc activity was greatly reduced (Fig. 2A) while NF-B activity was induced (Fig. 2B). Again, removal of the B elements abolished the LPS response (Fig. 2C). We confirmed that LPS was able to induce increased nuclear localization of RelA (Fig. 2D).
The RelA NF-B subunit specifically represses Gs expression
It is well documented that both TNF and LPS are potent inducers of NF-B activity in many cell types (24) including myometrial myocytes (Figs. 1 and 2). Therefore, we addressed the question of whether expression of exogenous RelA NF-B could also repress basal Gs promoter activity. When primary myometrial myocytes were transfected with the RelA NF-B subunit, Gs-Luc activity was seen to be repressed (Fig. 3A) while activating NF-B-responsive controls (Fig. 3B). No significant effect was seen on the B-ConA-luc control (Fig. 3C), thus reflecting the effect seen with TNF and LPS and reinforcing the notion that RelA NF-B can repress Gs expression. No repression of Gs 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. 3D). Interestingly, down-regulation of Gs could also be induced by RelA in HEK 293 cells, suggesting this effect is a general mechanism working in other cell types (Fig. 3E). Furthermore, a nonspecific transcriptional activator, Gal4-VP16, could not repress Gs-luc activity (data not shown).
TNF represses expression of the endogenous Gs gene but RelA NF-B subunits do not bind to a putative B element in the Gs promoter
We have shown that TNF and LPS can repress the Gs gene when it is placed in a reporter plasmid (Figs. 1 and 2). However, such data does not consider the natural Gs 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 for 24 h, total RNA was isolated, and then the level of endogenous Gs expression was assessed using RT-PCR. Significantly, we were able to confirm, in vivo, that TNF consistently repressed endogenous Gs mRNA expression by 50% (Fig. 4A).
The data presented thus far illustrate that TNF and LPS can repress Gs expression through induction of the RelA NF-B subunit. However, it was unclear from these studies how RelA was mediating this repression. A search of the Gs promoter using the AliBaba2.1 web-based software (Ref.58 and www.gene-regulation.com/cgi-bin/pub/programs/alibaba2/) illustrated an imperfect B element centralized over position –545 with the sequence 5'-AGGGACCCCC-3'. This may permit binding of NF-B to the promoter and facilitate recruitment of corepressor proteins, including HDACs (44, 45, 46, 47), to the Gs promoter causing its repression. Importantly, the consensus NF-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-B could bind to this putative B site in the Gs promoter. No binding to this site was observed compared with that seen with the positive control (3' B site from the HIV-long terminal repeat; Fig. 4B).
The EMSA data illustrated in Fig. 4B highlighted that RelA did not bind to a putative B element located in the Gs 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 for 24 h and the ChIP assay was performed as detailed in Materials and Methods. However, the Gs promoter could not be recovered by immunoprecipitating with an anti-RelA serum (Fig. 4C, top panel). We confirmed that the assay was functioning through the ability of TNF to induce RelA DNA-binding to a known NF-B-responsive promoter, namely IB (Fig. 4C, lower panel).
CBP relieves RelA-induced repression of the Gs 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-B, can be regulated (48, 49, 60, 61, 62, 63). Consequently, we tested the hypothesis that the RelA-induced repression of Gs was occurring through the ability of RelA to sequester the coactivator, CBP, from the Gs-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 Gs 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. 5A). Western blot analysis confirmed that exogenous expression of CBP did not have a negative effect on the expression of RelA (Fig. 5B). Finally, we were also able to confirm that CBP could relieve RelA-induced Gs repression in cultures of primary myometrial cells (Fig. 5C). Again, CBP had no negative effects on the expression of RelA (Fig. 5D).
Discussion
TNF, LPS, and RelA repress Gs expression
We have examined the effect of proinflammatory mediators on the expression of the Gs promoter in primary myometrial cells. Our findings illustrate, for the first time, that TNF and LPS can repress the expression of the Gs gene. Significantly, this effect was mediated by the RelA NF-B subunit in both human myometrial cultures and HEK 293 cells, suggesting a conserved cellular mechanism regulates Gs expression. Furthermore, repression could not be induced when the RHD of RelA was replaced with the Gal4 DNA-binding region (Gal4-RelA; Fig. 3D). 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 Gs uses competition for limiting amounts of cellular coactivators (see below). Finally, a control transcription factor, Gal4-VP16, could not repress Gs-luc activity (data not shown).
Therefore, these data suggest a novel mode of action for NF-B whereby it also functions to repress certain proquiescent genes within the myometrium. As such, our data would imply that NF-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 on other G protein subunits
The source of proinflammatory mediators within the myometrium is unclear. There is evidence to suggest that TNF is derived from myometrial macrophages in response to LPS (64), whereas in the mouse uterus, IL-1 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 itself serves as a potent activator of NF-B and has been shown previously to reduce cAMP levels in cardiac myocytes (20), up-regulate expression of Gi and Gq in human airway smooth muscle (21), and activate expression of Gi2 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 Gs levels as judged by RT-PCR. Importantly, earlier studies have demonstrated that there is marked reduction in both Gs protein and intracellular cAMP from laboring myometrium (7, 8) and this would be consistent with our current observations.
Mechanism of RelA-induced Gs repression
In the present study, we have demonstrated that the RelA NF-B subunit can repress Gs in both human myometrial cells and HEK 293 cells. This repression does not involve direct binding of RelA to the Gs 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 Gs 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 Gs 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 Gs promoter through binding one or more of these factors. Interestingly, we observed that when CBP alone was expressed in myometrial myocytes it readily activated Gs expression (data not shown), although it remains to be defined whether this was a promoter-specific effect.
Functional significance of RelA-induced Gs 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. 6, where CBP is associated with factors that are needed to activate Gs expression. However, before induction of parturition, when levels of CBP are again limiting (72), the immediate activation of RelA NF-B would facilitate removal of CBP from the Gs 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-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).
In summary, this study illustrates that proinflammatory agents, including both LPS and TNF, serve to repress Gs expression in human primary myometrial cells. This repression is mediated by the RelA NF-B subunit and also occurs in other cell types. Significantly, RelA does not bind directly to the Gs 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 Gs 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.
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-B, nuclear factor B; PKA, protein kinase A; RHD, Rel homology domain.
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Academic Unit of Reproductive and Developmental Medicine (N.R.C., D.O.C.A.), Sheffield Teaching Hospitals NHS Trust, Sheffield S10 2SF, United Kingdom
Abstract
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 B (NF-B) family of transcription factors. Recently, we have provided evidence that the RelA NF-B subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Gs/cAMP/protein kinase A pathway. TNF is a potent activator of NF-B, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNF was observed to repress expression of Gs while, at the same time, stimulating NF-B activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNF was seen to repress endogenous Gs mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Gs promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Gs expression. Together, these data suggest that, in human myometrium, repression of the Gs gene by NF-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
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, Gs (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 Gs expression is regulated in any system including the myometrium. Recently, our group reported that the Gs promoter was regulated by Sp-like transcription factors requiring phosphorylation by PKA (12). The precise nature of these factors and those that down-regulate Gs levels at term, however, have yet to be defined (12).
Elevation of cAMP and activation of PKA has been shown to inhibit nuclear factor B (NF-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-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, which serves as a potent activator of NF-B, has been shown to reduce cAMP levels in cardiac myocytes (20) and up-regulate expression of Gi and Gq in human airway smooth muscle (21). More recently, it has been demonstrated that NF-B is required to stimulate expression of the Gi2 gene (22).
NF-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-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-B1 (p105/p50), and NF-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-B is retained within the cytoplasm in an inactive form, bound to its inhibitor protein, IB, of which there are several isoforms (23, 24). Activation of NF-B is ultimately facilitated by the ubiquitination and degradation of IB with the concomitant release of particular NF-B subunits, for example, RelA:p50 heterodimers, which translocate to the nucleus where they modulate gene expression (23, 24).
A role for NF-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-B-regulated genes examined include COX-2, IL-1, IL-8, and phospholipase-A2 (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), Furthermore, inhibitors of NF-B activity, including IL-10 (38), can suppress both lipopolysaccharide (LPS)-induced preterm labor in mice and rats (39) and IL-1-induced uterine contractions in Rhesus monkeys (40), providing further, albeit circumstantial, evidence for NF-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-B-mediated gene activation.
Significantly, however, although NF-B will generally activate transcription, the Drosophila NF-B homolog, Dorsal, has also been documented to repress gene expression in the presence of corepressor proteins such as Groucho (41). Furthermore, mammalian NF-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-B and cAMP/PKA pathways within myometrium, there are no data examining whether NF-B may actually regulate Gs expression. Consequently, we tested the hypothesis that NF-B represses Gs expression while concomitantly inducing the up-regulation of the proinflammatory cascade associated with parturition.
Materials and Methods
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 Gs-luciferase reporter (Gs-Luc) has been described previously (12). The 3x-B-ConA-luciferase (3x-B-ConA-Luc) and enh-ConA-luciferase (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--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 or 2 μg/ml bacterial LPS. For experiments using exogenous RelA, 250 ng RSV-RelA or 250 ng RSV--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 Gs was then determined using RT-PCR analyses with the following primers: Gs sense, 5'-ATGGGCTGCCTCGGGAACAGTAAGACC-3' and Gs 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 (Gs) 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-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-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 B sites within the IB promoter (sense, 5'-GACGACCCCAATTCAAATCG-3' and antisense, 5'-TCAGGCTCGGGGAATTTCC-3') or the putative B site within the Gs 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 (IB promoter) and approximately 360 bp (Gs 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 -32P-labeled double-stranded HIV 3' long terminal repeat B site (5'-GATCCGCTGGGGACTTTCCAGGC G-3'; B site in bold) or the putative Gs B site (5'-GTACCAGGGACCCCCTCC-3'; putative 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 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
TNF and LPS repress expression of the Gs promoter while inducing NF-B activity
A number of proinflammatory cytokines, including TNF, are associated with the onset of both normal and preterm birth (56). When primary myometrial myocytes were transfected with the Gs-Luc reporter and stimulated with TNF for 24 h, a significant reduction in luciferase activity was observed, indicating that TNF could repress the Gs promoter (Fig. 1A). Importantly, in parallel experiments where primary myometrial cells were transfected with a NF-B-responsive reporter (3x-B-ConA-Luc), TNF stimulation was seen to strongly activate NF-B activity (Fig. 1B); removal of the B sites (B-ConA-Luc) abrogated this response to TNF (Fig. 1C). To confirm that TNF was activating NF-B, nuclear extracts were prepared from primary cultures of myometrial myocytes exposed to TNF and subject to Western analysis. Immunoblots were then probed for the RelA NF-B subunit because this subunit is rapidly activated by TNF (24). As expected, TNF induced a strong nuclear localization of RelA (Fig. 1D).
Approximately 40% of cases of preterm labor have an underlying bacterial infection (57). Consequently, we tested the notion that LPS, through activation of NF-B, may also repress Gs expression. When primary myometrial myocytes were stimulated with LPS for 24 h, Gs-Luc activity was greatly reduced (Fig. 2A) while NF-B activity was induced (Fig. 2B). Again, removal of the B elements abolished the LPS response (Fig. 2C). We confirmed that LPS was able to induce increased nuclear localization of RelA (Fig. 2D).
The RelA NF-B subunit specifically represses Gs expression
It is well documented that both TNF and LPS are potent inducers of NF-B activity in many cell types (24) including myometrial myocytes (Figs. 1 and 2). Therefore, we addressed the question of whether expression of exogenous RelA NF-B could also repress basal Gs promoter activity. When primary myometrial myocytes were transfected with the RelA NF-B subunit, Gs-Luc activity was seen to be repressed (Fig. 3A) while activating NF-B-responsive controls (Fig. 3B). No significant effect was seen on the B-ConA-luc control (Fig. 3C), thus reflecting the effect seen with TNF and LPS and reinforcing the notion that RelA NF-B can repress Gs expression. No repression of Gs 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. 3D). Interestingly, down-regulation of Gs could also be induced by RelA in HEK 293 cells, suggesting this effect is a general mechanism working in other cell types (Fig. 3E). Furthermore, a nonspecific transcriptional activator, Gal4-VP16, could not repress Gs-luc activity (data not shown).
TNF represses expression of the endogenous Gs gene but RelA NF-B subunits do not bind to a putative B element in the Gs promoter
We have shown that TNF and LPS can repress the Gs gene when it is placed in a reporter plasmid (Figs. 1 and 2). However, such data does not consider the natural Gs 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 for 24 h, total RNA was isolated, and then the level of endogenous Gs expression was assessed using RT-PCR. Significantly, we were able to confirm, in vivo, that TNF consistently repressed endogenous Gs mRNA expression by 50% (Fig. 4A).
The data presented thus far illustrate that TNF and LPS can repress Gs expression through induction of the RelA NF-B subunit. However, it was unclear from these studies how RelA was mediating this repression. A search of the Gs promoter using the AliBaba2.1 web-based software (Ref.58 and www.gene-regulation.com/cgi-bin/pub/programs/alibaba2/) illustrated an imperfect B element centralized over position –545 with the sequence 5'-AGGGACCCCC-3'. This may permit binding of NF-B to the promoter and facilitate recruitment of corepressor proteins, including HDACs (44, 45, 46, 47), to the Gs promoter causing its repression. Importantly, the consensus NF-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-B could bind to this putative B site in the Gs promoter. No binding to this site was observed compared with that seen with the positive control (3' B site from the HIV-long terminal repeat; Fig. 4B).
The EMSA data illustrated in Fig. 4B highlighted that RelA did not bind to a putative B element located in the Gs 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 for 24 h and the ChIP assay was performed as detailed in Materials and Methods. However, the Gs promoter could not be recovered by immunoprecipitating with an anti-RelA serum (Fig. 4C, top panel). We confirmed that the assay was functioning through the ability of TNF to induce RelA DNA-binding to a known NF-B-responsive promoter, namely IB (Fig. 4C, lower panel).
CBP relieves RelA-induced repression of the Gs 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-B, can be regulated (48, 49, 60, 61, 62, 63). Consequently, we tested the hypothesis that the RelA-induced repression of Gs was occurring through the ability of RelA to sequester the coactivator, CBP, from the Gs-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 Gs 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. 5A). Western blot analysis confirmed that exogenous expression of CBP did not have a negative effect on the expression of RelA (Fig. 5B). Finally, we were also able to confirm that CBP could relieve RelA-induced Gs repression in cultures of primary myometrial cells (Fig. 5C). Again, CBP had no negative effects on the expression of RelA (Fig. 5D).
Discussion
TNF, LPS, and RelA repress Gs expression
We have examined the effect of proinflammatory mediators on the expression of the Gs promoter in primary myometrial cells. Our findings illustrate, for the first time, that TNF and LPS can repress the expression of the Gs gene. Significantly, this effect was mediated by the RelA NF-B subunit in both human myometrial cultures and HEK 293 cells, suggesting a conserved cellular mechanism regulates Gs expression. Furthermore, repression could not be induced when the RHD of RelA was replaced with the Gal4 DNA-binding region (Gal4-RelA; Fig. 3D). 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 Gs uses competition for limiting amounts of cellular coactivators (see below). Finally, a control transcription factor, Gal4-VP16, could not repress Gs-luc activity (data not shown).
Therefore, these data suggest a novel mode of action for NF-B whereby it also functions to repress certain proquiescent genes within the myometrium. As such, our data would imply that NF-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 on other G protein subunits
The source of proinflammatory mediators within the myometrium is unclear. There is evidence to suggest that TNF is derived from myometrial macrophages in response to LPS (64), whereas in the mouse uterus, IL-1 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 itself serves as a potent activator of NF-B and has been shown previously to reduce cAMP levels in cardiac myocytes (20), up-regulate expression of Gi and Gq in human airway smooth muscle (21), and activate expression of Gi2 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 Gs levels as judged by RT-PCR. Importantly, earlier studies have demonstrated that there is marked reduction in both Gs protein and intracellular cAMP from laboring myometrium (7, 8) and this would be consistent with our current observations.
Mechanism of RelA-induced Gs repression
In the present study, we have demonstrated that the RelA NF-B subunit can repress Gs in both human myometrial cells and HEK 293 cells. This repression does not involve direct binding of RelA to the Gs 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 Gs 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 Gs 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 Gs promoter through binding one or more of these factors. Interestingly, we observed that when CBP alone was expressed in myometrial myocytes it readily activated Gs expression (data not shown), although it remains to be defined whether this was a promoter-specific effect.
Functional significance of RelA-induced Gs 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. 6, where CBP is associated with factors that are needed to activate Gs expression. However, before induction of parturition, when levels of CBP are again limiting (72), the immediate activation of RelA NF-B would facilitate removal of CBP from the Gs 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-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).
In summary, this study illustrates that proinflammatory agents, including both LPS and TNF, serve to repress Gs expression in human primary myometrial cells. This repression is mediated by the RelA NF-B subunit and also occurs in other cell types. Significantly, RelA does not bind directly to the Gs 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 Gs 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.
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-B, nuclear factor B; PKA, protein kinase A; RHD, Rel homology domain.
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