the onset of labour is associated with changes in gene expression in the myometrium consistent with activation of inflammatory mediators and leukocyte chemotaxis [ 1 , 2 ] .
the uterine expression of the chemokine il8 increases dramatically with the onset of term and preterm labour [ 3 , 4 ] and is thought to promote cervical remodelling [ 5 , 6 ] and myometrial contractility by promoting neutrophil infiltration and activation [ 4 , 7 ] .
myometrial il8 expression is increased by stretch and the inflammatory cytokines in a mapk- and nfb - dependent manner [ 8 , 9 ] .
consistent with these observations , il8 expression has been shown to be critically dependent on a region in its promoter , spanning nucleotides 1 to 133 , which contains binding sites for the transcription factors ap-1 , cebp , and nfb [ 1013 ] .
the binding sites are in close proximity to each other and to the coding region of the gene , forming a transcriptional enhanceosome ( figure 1(a ) ) .
many researchers have investigated the role and interaction of these transcription factors in regulating the il8 gene and have shown different mechanisms of regulation depending on the cell type [ 1321 ] .
studies in reproductive tissues show that nfb is important in il1b - driven il8 expression in amnion and myometrium , ap-1 is involved in the stretch - induced expression of il8 , oxtr , and cox2 in human myometrial and amnion cells [ 9 , 22 , 23 ] , and cebp has been shown to bind to two different regions of the il8 promoter and interact with nfb to regulate the il8 expression .
the marked inflammatory infiltration of the myometrium and cervix associated with human labour is driven by increased expression of il8 and other chemokines .
the transcription factors , nfb , cebp , and ap-1 , have all been implicated in il8 expression in various tissues / cell types , but which actually regulate myometrial il8 expression remains unclear . in this study
, we have used il1b stimulation to mimic labour in order to define the relative importance of nfb , cebp , and ap-1 in myometrial il8 expression .
myometrial cells were extracted from biopsies taken at the time of elective caesarean section at term with the ethics committee approval and patient consent .
tissue was minced and digested for 45  min in dmem with 1  mg / ml collagenase type ia and ix ( sigma - aldrich corp . , st .
cells were centrifuged at 400  g for 10  min and grown in dmem with 10% fetal calf serum , l - glutamine , and penicillin - streptomycin ( 37c and 5% co2 ) .
cells were serum starved for 16  h before treatment with 1  ng / ml il1b ( r&d systems , inc . , minneapolis , mn , usa ) .
cells were used at passage three for all experiments . to obtain whole cell lysates ,
cells were lysed for 20  min on ice in radioimmunoprecipitation assay buffer ( 1% np-40 , 1% triton , 1% sodium deoxycholate , 0.1% sds , 150  mm nacl , 10  mm tris , ph 8.0 , and 2  mm naf ) .
protein concentrations were determined using detergent - compatible protein assay reagents ( bio - rad laboratories ) .
protein samples ( 50  g ) were denatured by boiling for 5  min and run on a 10% sds - page for 60  min at 140  v , followed by a transfer to a hybond ecl nitrocellulose membrane ( amersham biosciensces ) .
the membrane was blocked in 5% milk protein solution over night or for 1 hour , washed and hybridized with the primary antibody for 1 hour at room temperature in a fresh blocking buffer ( 1  pbs , 1% milk protein and 0.1% tween-20 ) containing antibodies for rela and cebp ( santa cruz biochemicals , sc-8008 and sc-7962 , resp . ) or beta actin ( abcam , ab6276 ) .
immunoreactivity was then visualised using a chemiluminescent substrate for hrp ( ecl plus ; amersham biosciensces ) .
the promoter region of the il8 gene ( 135/+46  bp ) was amplified by polymerase chain reaction , and the fragment was ligated into the luciferase reporter plasmid pgl2-basic ( promega , southampton , uk ) to give the wild - type construct .
the mutation of individual base pairs in plasmid dna was achieved using the quikchange site - directed mutagenesis kit ( stratagene , uk ) according to manufacturer 's instructions .
transient transfections were performed using fugene 6 transfection reagent ( roche , indianapolis , in , usa ) .
cytomegalovirus - renilla vector ( 1/10th of reporter ) was used to control for transfection efficiency and cell number .
luciferase reporter vectors containing either the wild - type il8 promoter or the mnfb ( mutation of nfb ) or the mcebp ( mutation of cebp ) or the map-1 ( mutation of ap-1 ) were transfected at 0.2  g / well .
cells were cultured for a total of 48  h ( including 24  h of il1b stimulation for half the experiment ) followed by harvesting and analysis with a dual firefly / renilla luciferase assay ( luclite , packard bell , and coelenterazine cn biosciences ) .
the cells were lysed , and chromatin was sheared into 200 to 1000 bp fragments by sonication .
antibodies recognizing the c - terminal of rela , cebp ( santa cruz biochemicals , sc-8008 and sc-7962 resp . ) , and acetyl - histone h4 ( upstate cell signalling solutions , 06 - 866 ) were used ( 1  :  1000 dilution ) for immunoprecipitation , and the chromatin fragments containing the crosslinked protein were purified by immunoabsorption and elution from protein a / g beads .
the crosslinks were reversed , and the dna was purified using a qiaquick nucleotide removal kit ( qiagen inc . , valencia , ca , usa ) .
pre - pcr cycle was 10  min at 95c followed by 35 cycles of 95c for 1  min , 5660c for 1  min , and 72c for 1  min followed by final extension at 72c for 10  min .
on - targetplus smart pool human nfbp65 , cebp , cebp , c - fos , and c - jun sirnas ( dharmacon ) were used .
siglo ( dharmacon ) was used as a positive control , giving a high transfection efficiency of approximately 90% , and on - targetplus nontargeting pool ( dharmacon ) was used as a negative control .
the sirnas were transfected using dharmafect 2 ( dharmacon ) transfection reagent at a final concentration of 100  nm according to manufacturer 's instructions .
tube one contained the sirna diluted in phenol - red - free dmem , and tube two contained the transfection reagent diluted in phenol - red - free dmem .
each tube was incubated at room temperature for 5  min before mixing the contents of the two tubes .
prewarmed dmem containing 10% fbs and 2  mm l - glutamine , but no antibiotics were then added to the mixture .
the culture media was removed from the cells , and the cells were fed with the sirna mixture .
the media was changed to dmem containing 10% fbs , 2  mm l - glutamine , and 100 u / ml penicillin - streptomycin 24 hours after the transfection .
the sirna transfections were performed at different time courses ( 12 , 24 , 48 , and 72  h and 5 , 6 , and 7 days ) .
the changes were of the same pattern at all time points , but maximal response was observed at 5 days . the cell viability was monitored closely before harvesting the cells for appropriate analysis . at the end of incubation assays , 1  ml of medium was collected and immediately frozen at 80c for analysis by il8 elisa kit ( biosource , khc0081 ) .
the il8 elisa had a sensitivity of 5  pg / ml ; the inter- and intra assay variations were 7.8% and 5.3% , respectively .
protein concentrations were determined using detergent - compatible protein assay reagents ( bio - rad laboratories ) .
statistical analysis was performed by anova with the level of significance defined as p < 0.05 .
chip studies showed that il1b induces the binding of nfbp65 , cebp and ap-1 to the endogenous il8 promoter . il1b stimulated the recruitment of nfbp65 , cebp , and - , cfos , and cjun to their respective cis - elements in the transcriptional enhanceosome at 6  hrs ( figure 1(b ) ) .
this was associated with concomitant acetylation of h4 at that region of the promoter . a sequence extending from 135 to + 46 which contains the transcription enhanceosome was used as a wild - type control .
site - directed mutagenesis was performed to make three further constructs with mutations for each of the nfb , cebp , or ap-1 sites individually ( figure 2 ) .
basal luciferase activity following transient transfection of the wild - type construct was significantly increased by 2-fold upon il1b stimulation . in primary human myometrial cells ,
mutation of the nfb site reduced the unstimulated reporter activity to 10% of that of the wild type and obliterated the response to il1b .
mutation of the cebp site had no effect on basal promoter activity but did abolish the response to il1b .
mutation of the ap-1 site decreased basal il8 promoter activity although a response to il1b stimulation was retained ( figure 2 ) .
knockdown mediated by sirna against nfbp65 , cebp , c - jun , and c - fos was performed with protein expression studies showing highly - specific and better than 95% gene silencing ( figure 3(a ) ) .
the effect of sirna knockdowns against nfbp65 , cebp , c - jun , and c - fos was measured on il8 protein production and its response to il1b stimulation ( for 24  h ) using elisa . silencing of each individual transcription factor had no effect on basal il8 protein release into the culture medium , measured by elisa .
knockdown of cebp increased the response to il1b by 1.5-fold whilst c - jun and c - fos knockdown reduced the response by half ( figure 3(b ) ) . since cebp may also bind to the il8 promoter ( see figure 1 ) , we repeated the experiment using sirna knockdown of cebp which also increased the response to il1b by 1.5-fold as with knockdown of cebp ( figure 3(c ) ) .
nfb , cebp , and ap-1 are important transcription factor families that are involved in immune and inflammatory functions as well as in cell growth and differentiation .
nfb , cebp , and ap-1 are each expressed in human myometrium , and their expression is increased in response to the proinflammatory cytokine , il1b .
labour - associated genes such as pghs-2 , otr , il8 , il6 , and tnf- have been shown to be regulated by either one or a combination of theses transcription factors [ 14 , 16 , 20 , 21 , 2429 ] .
targeting activators of inflammation has been proposed as a strategy for prevention or delay of preterm birth and to improve outcome in preterm neonates .
have demonstrated a central role for nfb in the onset of term labour in the mouse , and that inhibition of nfb can delay parturition .
for example , sulfasalazine is a synthetic anti - inflammatory drug comprising 5-aminosalicylic acid ( 5-asa ) , linked to sulfapyridine , which is routinely used for the treatment of inflammatory bowel disease and rheumatoid arthritis .
this has been shown to inhibit labour - associated gene expression in cell culture models [ 31 , 32 ] , although there are currently no animal or clinical studies . in a mouse model of inflammation - induced preterm labour the cyclopentenone prostaglandin j2 , which acts via inhibition of nfb , both delays preterm birth and improves pup survival .
glucocorticoids such as dexamethasone or prednisolone have well - established anti - inflammatory and immunosuppressive activities through their inhibitory effects on ap-1 and nfb pathways   although there is no evidence that these drugs will delay or prevent preterm birth .
progesterone , which , when used clinically , can reduce the risk of preterm labour , has been shown to repress il1b - induced expression of nfb regulated genes in human amnion and myometrium [ 35 , 36 ] . in this study , we examined the role of nfb , cebp , and ap-1 in regulation of il8 expression in human myometrium and used il1b to mimic the effects of inflammation seen with human labour .
we used chip to show that il1b induces the binding of all three transcription factors to the il8 promoter .
that il1b increases both nfbp65 and cebp binding to the endogenous il8 promoter in myometrial cells .
for example in a study in human conjunctiva epithelial cells chip showed no binding of nfb , cebp , or ap-1 alone , but when immunoprecipitation was performed for both nfb and cebp simultaneously , chip showed binding of both to the il8 promoter .
conversely treatment of human airway smooth muscle cells with tryptase , which mimics inflammation , induced the binding of all three transcription factors to the promoter . since we found that , in human myometrial cells all three transcription factors bound to the il8 promoter , we considered two approaches to identify the relative importance of each transcription factor in il8 expression .
first we used site - directed mutagenesis to alter the binding sites for ap1 , cebp , and nfb .
we found that mutating both the nfb and ap-1 sites markedly reduced both basal and stimulated il8 promoter activity .
however , mutating the cebp site did not change the basal promoter activity but did completely inhibit the il1b - induced increase in promoter activity .
these data suggest that the nfb and ap-1 binding sites are essential for both basal and il1b - stimulated il8 expression .
in contrast , it appeared that cebp binding sites are not important for basal promoter activity but are essential for the il1b - induced increase in promoter activity . using a similar approach in human amnion and cervical epithelial cells , elliott et al .
found similar results for the nfb binding site but found no effect of mutating the cebp or ap-1 binding sites on the il8 promoter activity .
however , site - directed mutagenesis of dna binding sites gives limited information about the role and importance of transcription factors , particularly where , as in the il8 promoter , those sites are close to each other or overlap .
therefore in an alternative approach we used the more novel technique of sirna against ap-1 , cebp , and nfb to knock down the expression of their endogenous proteins .
the data generated by sirna knockdown is probably the most reliable in terms of the role of individual transcription factors since it is highly specific .
in addition , the readout is activity of the endogenous promoter , not a transfected construct .
in contrast to the finding from the site - directed mutation studies , basal il8 levels were not reduced by knockdown of any of the transcription factors . this may be because although we achieved 90% knockdown , this was not complete
. however , knockdown of p65 did completely inhibit the il1b - stimulated increase in il8 levels .
the effects of c - jun and c - fos knockdown were less marked but still inhibitory .
cebp knockdown did not inhibit the il1b - induced increase in il8 expression and actually tended to increase it .
in some cell types cebp has been shown to inhibit the action of nfb in regulation of il8 .
certainly our data does not point to a central positive role for cebp in driving il8 expression .
our data show that , in human myometrial cells , nfb is essential for il8 expression ; ap-1 plays a less important but still stimulatory role , while the role of cebp is less clear .
the site - directed mutagenesis studies suggested a positive role in il8 expression ; however , the nfb and cebp binding sites are close together , and it remains possible that mutation of the cebp site may have interfered with nfb binding or function .
the probably more reliable data from sirna would suggest that pharmacological inhibition of cebp might enhance il8 expression and may therefore not be beneficial .
the dominant role played by nfb in il8 expression confirms that it is an attractive therapeutic target for the prevention of preterm labour .
these data show that nfb is essential for il8 myometrial expression , ap-1 plays a less important but still stimulatory role , while the role of cebp is less clear .
these results provide further support for the notion that nfb represents an attractive therapeutic target for the prevention of preterm labour .
 the uterine expression of the chemokine il8 increases dramatically with the onset of labour both at term and preterm . 
 the il8 promoter contains binding sites for the transcription factors nuclear factor - kappa b ( nfb ) , activator protein-1 ( ap-1 ) , and ccaat / enhancer - binding protein ( cebp ) . in this study we investigated the roles of these transcription factors in il1b regulation of the il8 gene in human myometrium . using chromatin immune precipitation ( chip ) assay , we showed that each of nfb , cebp , and ap-1 binds to the il8 promoter upon il1b stimulation . to examine the relative importance of each site in il8 gene expression , site - directed mutagenesis of each of these sites 
 was performed . 
 we found that the nfb site was essential for basal and il1b - stimulated gene expression . 
 mutation of the ap-1 site reduced both basal and il1b - stimulated expression but to a lesser extent . 
 mutation of the cebp site had no effect upon basal expression but eliminated the il1b response . 
 small interfering rna ( sirna ) silencing of nfb abolished the il8 response to il1b significantly ; sirna against ap-1 reduced it to a lesser extent whilst knockdown of cebp enhanced the response . 
 our data confirms a central and essential role for nfb in regulation of il8 in human myometrium . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion