NFκB and AP-1 Drive Human Myometrial IL8 Expression

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 (NFκB), 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 NFκB, 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 NFκB 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 NFκB 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 NFκB in regulation of IL8 in human myometrium

Nuclear Factor Kappa B Activation Occurs in the Amnion Prior to Labour Onset and Modulates the Expression of Numerous Labour Associated Genes

Background: Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFkB). In this study we characterised the level of NFkB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour. Methodology/Principal Findings: We found that a proportion of women exhibit low or moderate NFkB activity while other women exhibit high levels of NFkB activity (n = 12). This activation process does not appear to involve classical pathways of NFkB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised nonactivated amnion (n = 3) samples and compared to activated samples (n = 3). A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold), interleukin 8 (IL-8, 6-fold), IL-1 receptor accessory protein (IL-1RAP, 4.5-fold), thrombospondin 1 (TSP-1, 3-fold) and, unexpectedly, oxytocin receptor (OTR, 24-fold). Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i) cell death, cancer and morphology and ii) cell cycle, embryoni

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

Nuclear factor kappa B and its interactions in human parturition

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Inflammatory genes significantly upregulated in activated amnion.

<p>A list for inflammatory genes upregulated in activated amnion samples compared to non-activated samples was generated. In total, 8 significant inflammatory genes were identified (<i>P</i><0.01).</p

Correlations between key components of the canonical, non-canonical and atypical NFκB signalling pathways.

<p>Protein levels of activated NFκB were examined in each sample by immunoblotting as previously described. Levels of both nuclear p65 and nuclear phospho-p65 were shown to correlate highly with nuclear levels with Rel-B (A and B, R² = 0.8145 and R² = 0.6288 respectively). No correlation was detected between nuclear levels of p65 and p50 (C, R² = 0.06856), nuclear Rel-B and p52 (D, R² = 0.00008) or nuclear p65 and IκBα (E, R² = 0.0077). Collectively these results suggest that neither the canonical, non-canonical nor the atypical signalling pathways are responsible for the observed differences in NFκB activation.</p

Ingenuity Pathway Analysis (IPA) of gene pathways driving amnion activation- network map 1.

<p>Network one comprising of upregulated genes (>2.5-fold) involved predominately in cell death, cancer and morphology. A total of 25 focus genes were used in the construction of the network. ADM (adrenomedullin/progesterone biosynthetic process), CTGF (connective tissue growth factor/angiogenesis), CYR61 (cysteine-rich angiogenic inducer/patterning of blood vessels), EMP2 (epithelial membrane protein 2/cell death), ETS1 (v-ets erythroblastosis virus E26 oncogene homolog 1 (avian)/DNA dependent transcription), ETS2 (DNA dependent transcription), HMGCS1 (transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)/protein complex assembly), IL1RAP (interleukin 1 receptor accessory protein/transmembrane receptor activity involved in the inflammatory response), ITGA6 (integrin, alpha 6/cell adhesion and calcium ion binding), KLF4 (Kruppel-like factor 4 (gut)/DNA dependent transcription and nucleic acid binding), KLF6 (Kruppel-like factor 6/DNA dependent transcription and nucleic acid binding), NET1 (neuroepithelial cell transforming gene 1/regulation of Rho protein signal transduction), NME1 (non-metastatic cells 1, protein (NM23A)/nucleic acid binding), ODC1 (ornithine decarboxylase 1/polyamine biosynthetic process), PIM1 (pim-1 oncogene/protein serine/threonine kinase activity), PSAT1 (phosphoserine aminotransferase 1/phosphoserine transaminase activity), RND3 (Rho family GTPase 3/cell adhesion), SGK (serum/glucocorticoid regulated kinase/serine/threonine kinase activity), SLC7A1 (solute carrier family 7, (cationic amino acid transporter, y+ system) member 11/amino acid transport activity), SPRR1B (small proline-rich protein 1B (cornifin)/epidermis development), TAF5L (TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa/DNA dependent transcription), THBS1 (Thrombospondin 1/cell motility, adhesion in response to inflammation), TUBB3 (tubulin, beta 3/microtubule-based movement), TUBB2A (tubulin, beta 2A/microtubule-based movement), UGT1A6 (UDP glucuronosyltransferase 1 family/xenobiotic metabolic process).</p

Interactions between p65 and Rel-B exist in pre-labour, human amnion.

<p>(<b>A</b>) Whole cell lysate from unstimulated and IL-1β stimulated pre-labour amnion epithelial cells was incubated p65 conjugated beads. The lysate was recaptured under denaturing conditions and Western immunoblotting performed with either anti-p65, anti-phospho-p65 or anti-Rel-B antibodies. Non-stimulated amnion was shown to contain both p65-pp65 and to a greater extent, p65-Rel-B dimers. When stimulated with IL-1β, p65-pp65 dimer levels increased maximally at 30 min and then decreased gradually over 24 h. Dimers of p65-Rel-B were maintained at high levels throughout the time series. (<b>B</b>) Non-radioactive DNA binding assay kit (TRANSAM perbioscience) measuring the binding of Rel-B to the NFκB consensus binding sequence in response to IL-1β showed an increase from the unstimulated state at 30 min before dropping slightly 1 h. Binding peaked at 4 h before again subsiding at 24 h.</p