Molecular Human Reproduction Vol.7, No.8 pp. 787–790, 2001
Nuclear factor-kappa B is essential for up-regulation of
interleukin-8 expression in human amnion and cervical
epithelial cells
C.L.Elliott1,3, V.C.Allport2, J.A.Z.Loudon2, G.D.Wu2 and P.R.Bennett2
1Institute
of Obstetrics and Gynaecology, Imperial College School of Medicine, Queen Charlotte’s Hospital, Goldhawk Road, London
W6 0XG, UK and 2Division of Gastroenterology, Department of Internal Medicine, University of Pennsylvania School of Medicine,
Philadelphia, PA 19104-6144, USA.
3To
whom correspondence should be addressed. E-mail: [email protected]
Interleukin-8 (IL-8) is a cytokine which recruits and activates neutrophils into tissue stroma. It is present in uterine
tissues and its concentration increases in the third trimester and with labour. The promoter region of the IL-8 gene
contains binding sites for the transcription factors, nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1) and
CCAAT/enhancer-binding protein (C/EBP). These are in close proximity to each other and to the coding region of
the gene. This study used site-directed mutagenesis of each of these sites to examine the relative importance of each
site in IL-8 gene expression in a cervical cell line and in amnion cells obtained before and after labour. We found
that the NF-κB site was essential for basal and IL-1β-stimulated gene expression in all cell types. Neither of the
other binding sites was consistently essential for gene expression but may have an additive role in promoter activity.
We conclude that the NF-κB binding site is essential for up-regulation of IL-8 gene expression in these uterine cell
types. An increase in IL-8 expression has been shown to occur in the uterus in association with parturition and
NF-κB binding to the promoter may be of importance at this time.
Key words: amnion/cervix/interleukin-8/NF-kB/parturition
Introduction
Interleukin-8 (IL-8) is a chemokine of the CXC type that is a
potent attractor and activator of neutrophils (Baggiolini et al.,
1989). It is present within the uterus in cervix (Barclay et al.,
1993), placenta (Elliott et al., 1998), myometrium (Elliott
et al., 2000), amnion and choriodecidua (Kelly et al., 1992).
Its production by fetal membranes, placenta, myometrium and
lower segment fibroblasts has been shown to increase in the
late third trimester or with labour at term (Winkler et al.,
1998, 1999). We have shown that IL-8 in amnion is increased
in the third trimester and after labour at term and that
its expression in the choriodecidua shows parallel increases
(unpublished data). An influx of neutrophils has been shown
to occur into the uterine cervix and myometrium (Junqueira
et al., 1980; Thomson, 1999) in association with labour and
it is thought that IL-8 plays a key role in this neutrophil
recruitment. The cervical changes of effacement and dilatation
that occur with labour are due to the release of metalloproteinases, such as MMP-8 (neutrophil elastase), from
activated neutrophils (Osmers et al., 1992). IL-8 thus plays a
key role in the events of parturition.
The promoter region of the IL-8 gene contains functional
© European Society of Human Reproduction and Embryology
binding sites for the transcription factors (NF-κB), activator
protein (AP-1) and CCAAT/enhancer-binding protein (C/EBP)
in close proximity within 130 bp of the coding region (Figure 1).
Located upsteam of these sites are two glucocorticoid response
elements (GRE) (Mukaida et al., 1989). In other cell types it
has been shown that the NF-κB site is essential for transcription
(Stein et al., 1993; Kunsch et al., 1994; Wu et al., 1997).
The role for the AP-1 and C/EBP sites varies between the
tissues studied.
The aim of this study was to determine the role of the transcription binding sites for NF-κB, AP-1 and C/EBP in regulation
of basal and stimulated IL-8 gene expression in primary amnion
cells obtained before and after labour at term and in the human
cervical epithelial cell line HOG-1 (Gleeson et al., 1990).
Materials and methods
IL-8–luciferase reporter constructs
The constructs used were as previously described (Wu, 1997). In
brief, the promoter region of the IL-8 gene (–135/⫹46bp) was
amplified by polymerase chain reaction and the fragment ligated into
the luciferase reporter plasmid pGL2-Basic (Promega, Southampton,
UK) to give the wild type construct (wt)LUC. Three further constructs
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C.L.Elliott et al.
Figure 1. Representation of the interleukin-8 (IL-8) promoter
region and the mutations used in these studies, as indicated by the
underlined nucleotides. mAP-1 ⫽ activator protein-1 mutation;
mNF-κB ⫽ nuclear factor-kappa B mutation; mC/EBP ⫽ CCAAT/
enhancer-binding protein mutation.
were developed with site-directed mutations at the AP-1, NF-κB and
C/EBP binding sites to produce (mAP-1)LUC, (mNF-κB)LUC and
(mC/EBP)LUC respectively (Figure 1).
Amnion cell cultures and transient transfections
Amnion was obtained from elective Caesarean section (L–) before
labour at term or after spontaneous vaginal delivery at term (L⫹).
Ethical approval for the collection of the tissues was obtained from
the Hammersmith Hospital LREC. Term was defined as 37–42
completed weeks of pregnancy. There was no clinical evidence of
infection or meconium staining in any of the samples obtained.
Elective Caesarean section was performed for maternal indications,
such as breech presentation or previous Caesarean or uterine surgery
or maternal request. There was no uterine activity prior to surgery.
Amnion was digested and cell cultures were established as previously
described (Bennett, 1987). Cells were grown to 70–80% confluence
and maintained serum-free prior to transfection. Transfection conditions were optimized for charge ratio in each cell type. 1 µg plasmid
DNA was transfected into each well using a liposome-mediated
method with Tfx-50 reagent (Promega, Southampton, UK). Cells
were cultured overnight and then either left unstimulated or stimulated
with IL-1β (1 ng/ml) for 6 h. Cytoplasmic extract of the cells was
prepared and analysed for luciferase activity with a commercially
available kit (Promega, Southampton, UK).
HOG-1 cell culture and transient transfections
Human cervical epithelial cells (HOG-1) (Gleeson et al., 1990) were
cultured to 70–80% confluence. Charcoal-stripped serum was added
to the medium used prior to transfection. Transient transfection with
each construct was performed as described above except that 0.5 µg
plasmid DNA was used in each well.
Reporting of results and statistics
Three replicates were studied for each cell type and three samples
of each were used. Results are expressed as percentage of the
unstimulated wild-type promoter activity. Significance was determined
using an ANOVA test with Fisher’s protected least significant difference (PLSD) post-hoc analysis and significance was determined at
the 95% level.
Figure 2. Functional effect of site-directed mutations altering
transcription factor binding sites in the interleukin-8 (IL-8)
promoter on promoter activity in the cervical epithelial cell line
HOG-1. Analysis of variance showed a significant effect of each
mutation and of IL-1β stimulation on reporter activity. Individual
post-hoc analysis P values are shown. mAP-1 ⫽ activator protein-1
mutation; mNF-κB ⫽ nuclear factor-kappa B mutation; mC/EBP ⫽
CCAAT/enhancer-binding protein mutation; ns ⫽ non-stimulated.
of the wild type construct resulted in luciferase activity in
each of the cell types studied. This was significantly increased
by stimulation with IL-1β.
HOG-1 cells
In these immortalized cervical epithelial cells, mutation of the
NF-κB site significantly reduced the unstimulated reporter
expression (P ⬍ 0.0001) and prevented the increase in
expression by IL-1β. Mutation of the C/EBP and the AP-1
sites also significantly repressed constitutive promoter activity
(P ⬍ 0.0001). Mutation of the AP-1 site did not prevent
significant stimulation of promoter expression by IL-1β. Cells
transfected with the C/EBP mutated construct showed a slight
increase in reporter expression after IL-1β stimulation but the
difference was not significant (Figure 2).
Pre-labour amnion cells
Primary cell cultures transiently transfected with the mutated
NF-κB construct also demonstrated reduced basal reporter
activity as compared with those transfected with the wild
type construct (P ⬍ 0.005). This mutation prevented further
stimulation in promoter activity by IL-1β. In these cells
mutation of the C/EBP or the AP-1 sites did not alter
unstimulated activity, while small increases in promoter activity
(significant for the AP-1 mutation but not for the C/EBP
mutation) occurred after IL-1β stimulation (Figure 3).
Results
Post-labour amnion cells
Transient transfection of a promoterless ‘empty vector’
generated negligible luciferase activity. Transient transfection
Transient transfection of these primary cells with any of
the three mutated constructs did not significantly alter the
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NF-κB is essential for IL-8 expression in uterine cells
Figure 3. Functional effect of site-directed mutations altering transcription factor binding sites in the interleukin-8 (IL-8) promoter on
promoter activity in pre-labour term primary amniocyte cultures. Analysis of variance showed a significant effect of the nuclear factor-kappa
B mutation (mNF-κB) and of IL-1β stimulation on reporter activity, whereas the activator protein-1 (mAP-1) or CCAAT/enhancer-binding
protein (mC/EBP) mutations did not have significant effects. Individual post-hoc analysis P values are shown. ns ⫽ non-stimulated.
unstimulated reporter expression. Increased reporter expression
from the wild type promoter was seen following IL-1β stimulation. IL-1β caused an increase in the expression from the
C/EBP and AP-1 mutant constructs but the differences were
not significant. No increase in reporter activity was observed
following IL-1β stimulation of the NF-κB mutant construct
(Figure 4).
Discussion
We and others have previously shown that an increase in IL-8
production occurs in uterine tissues in association with the
onset and progression of labour in women. This study examined
the mechanism by which IL-8 expression may be regulated in
uterine tissues. The region of the IL-8 promoter used in these
studies was truncated at –135 bp. This truncated region contains
binding sites for NF-κB, AP-1 and C/EBP, and was able to
drive reporter gene expression in both unstimulated and IL-1β
stimulated cells. The binding sites in this short region therefore
appear to be sufficient for regulation of gene expression.
Mutation of the NF-κB binding site produced a significant
reduction in gene expression in unstimulated and stimulated
conditions. This has been described in several other cell types.
In pulmonary epithelial cells, the NF-κB site was shown to
be essential for stimulation of IL-8 production in response to
mycobacteria and mediated by IL-1 (Wickremasinghe et al.,
1999). In the colon carcinoma cell line Caco-2, mutation of
the NF-κB binding site did not alter basal promoter activity
but prevented stimulation with IL-1β (Wu et al., 1997). Our
data suggest that NF-κB plays a central role in both basal
and IL-1β-stimulated expression of IL-8 in both the fetal
membranes and the cervix.
NF-κB is a hetero- or homodimer composed of two members
of the Rel gene family which includes RelA (p65), c-Rel,
Figure 4. Functional effect of site-directed mutations altering
transcription factor binding sites in the interleukin-8 promoter
(IL-8) on promoter activity in post-labour term primary amniocyte
culture. Analysis of variance showed a significant effect of the
nuclear factor-kappa B mutation (mNF-κB) and of IL-1β
stimulation upon reporter activity, but not the activator protein-1
(mAP-1) or CCAAT/enhancer-binding protein (mC/EBP) mutations.
Individual post-hoc analysis P values are shown. ns ⫽ nonstimulated.
NF-kB1 (p50), Rel B and NF-kB2 (Baldwin, 1996). In lymphocyte and fibrosarcoma cell lines it has been found that binding
of Rel A or c-Rel to the IL-8 promoter is sufficient to activate
transcription, with Rel A binding to the NF-κB binding
site appearing to have the most consistent effect on IL-8
transcription (Kunsch and Rosen, 1993).
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C.L.Elliott et al.
The results of our study suggest a difference in regulation
of basal IL-8 gene expression between pre- and post-labour
amnion cells. In pre-labour cells, as in HOG-1 cells, mutation
of the NF-kB binding site resulted in a reduction in basal
activity of the promoter. In post-labour cells, however, this
mutation did not alter unstimulated activity, although it did
prevent up-regulation of expression by IL-1β. It is possible
that, in post-labour cells, several alternate transcription factors
may be available to drive basal IL-8 expression so that mutation
of only one transcription factor binding site does not suppress
basal promoter activity. Alternatively, it could be that after the
onset of labour there is a change in the NF-κB subunits
available which form more weakly active dimers. Stimulation
with IL-1β may then increase the availability of strongly
binding dimers in the tissue. It is also possible that there may
be changes in the activity of repressor transcription factors
such as Oct-1 (Wu et al., 1997). After the onset of labour
there may also be increases in the activity of other transcription
factors which act on sites other than the NF-κB binding site.
Our data suggest that in amnion cells intact C/EBP and
AP-1 binding sites are not critical for either basal or stimulated
IL-8 gene expression. In contrast, in the immortalized epithelial
cell line HOG-1, mutation of the AP-1 or C/EBP sites
significantly reduced basal promoter activity. The close proximity of the C/EBP and the NF-κB binding sites enables an
interaction between these two transcription factors, allowing
synergistic activation of the promoter, as has been shown to
occur in non-uterine cell types (Stein and Baldwin, 1993;
Kunsch et al., 1994; Roebuck, 1999). At term, there may be
an increased availability of NF-κB in the amnion cells, reducing
the requirement for an intact C/EBP or AP-1 binding site for
constitutive activity. This could be due to alterations in the
cell environment with increased inflammatory stimulation.
Mutation of the AP-1 and C/EBP sites still allowed some
stimulation by IL-1β in amnion cells, though the increases
were not generally significant. The proposed increase in
availability of NF-κB in amnion cells at term, while reducing
the need for other transcription factors to promote basal
activity, may not be sufficient to allow significant stimulated
activity in the absence of an intact AP-1 or C/EBP site.
We have recently described the importance of an intact
NF-κB binding site for stimulation of COX-2 gene expression
in amnion cells (Allport et al., 2000). Labour is proposed to
occur due to a cascade of inflammatory mediators. We have
now shown that NF-κB is an essential for transcriptional upregulation of two of the genes whose increase is associated
with labour, COX-2 and IL-8. NF-kB therefore appears to play
a central role in the onset of human parturition.
References
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HOG-1 cells were a kind gift from Dr John White. This work was
funded by Tommy’s Campaign.
Received on March 5, 2001; accepted on May 16, 2001
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