Nuclear factor kappa B activation occurs in the amnion prior to labour onset and modulates the expression of numerous labour associated genes

Abstract

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 (NFκB). In this study we characterised the level of NFκB 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 NFκB activity while other women exhibit high levels of NFκB activity (n = 12). This activation process does not appear to involve classical pathways of NFκB 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 non-activated 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, embryonic development and tissue development.

Conclusions/significance: Our results indicate that assessment of amnion NFκB activation is critical for accurate sample classification and subsequent interpretation of data. Collectively, our data suggest amnion activation is largely an inflammatory event that occurs in the amnion epithelial layer as a prelude to the onset of labour.

Figure 1. Characterisation of amnion NFκB activity by western blotting.

Primary, pre-labour amnion epithelial cells were derived from 12 women undergoing caesarean section. Protein levels of activated NFκB were examined in each sample by immunoblotting for p65 in nuclear extracts, which were then normalised to β-actin (A). Following densitometric analysis, samples were categorised into three groups based upon their level of NFκB activation – low, medium and high (B). The low NFκB activation group consisted of three samples, the medium of seven samples and the high activation group consisted of two samples. Gel to gel variation was accounted for by loading equal volume of a control sample (X) on each gel. ANOVA revealed significant differences between protein levels of nuclear p65 in the three classified groups (C). Levels of nuclear p65 were also shown to correlate with levels of nuclear phosphorylated p65 in a linear fashion (R² = 0.6205).

Figure 2. Correlations between key components of the canonical, non-canonical and atypical NFκB signalling pathways.

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.

Figure 3. Interactions between p65 and Rel-B exist in pre-labour, human amnion.

(A) 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) 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.

Figure 4. Characterisation of amnion activation for microarray analysis.

Prior to microarray analysis, levels of COX-2 mRNA (A) and COX-2 protein were assessed in 6 amnion samples by western blotting (B) and subsequent densitometric assessment of immuno-reactive bands (C). Three samples characteristically displayed high levels of both COX-2 gene and protein consistent with highly activated amnion. Three samples displaying low levels of COX-2 were also selected as non-activated samples. The six characterised samples were subsequently used for microarray analysis to examine genes involved in amnion activation.

Figure 5. Validation of microarray data.

(A) Gene data derived from the microarray analysis was firstly examined using unsupervised principal components analysis (PCA). Inspection of the PCA score plot revealed clear first component separation of activated (yellow) and low-activated (red) amnion samples. (B) Similarly, hierarchical clustering demonstrated grouping within both the activated and non-activated amnion groups. Collectively these results indicate that gene expression profiles of activated samples are similar to each other, yet distinct to non-activated samples. OTR (C) and IL-8 (D) mRNA levels were then assessed in 6 amnion samples post microarray analysis. Consistent with the microarray data, three samples displayed low levels of OTR and IL-8 mRNA whilst the other three samples displayed characteristically high levels indicative of amnion activation.

Figure 6. Ingenuity Pathway Analysis (IPA) of gene pathways driving amnion activation- network map 1.

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).

Figure 7. IPA of gene pathways driving amnion activation- network map 2.

Network two incorporated 17 focus genes (upregulated >2.5-fold) involved in cell cycle, embryonic development and tissue development. ACTB (actin, beta, cell motility), AMFR (autocrine motility factor receptor, ubiquitin cycle), ASNS (asparagine synthetase, asparagines biosynthetic pathway), Caspase (caspase recruitment domain family, member 10, activation of NFκB inducing kinase), COTL1 (coactosin-like 1 (Dictyostelium), carbohydrate metabolic process), DUSP14 (dual specificity phosphatase 14, protein amino acid dephosphorylation), ENC1 (ectodermal-neural cortex (with BTB-like domain, actin binding), FOXC1 (forkhead box C1, transcription factor activity), GADD45B (growth arrest and DNA-damage-inducible, beta, activation of MAPKK activity), GSPT1 (G1 to S phase transition 1, transition of mitotic cell cycle, translation), JUB (jub, ajuba homolog (Xenopus laevis), zinc ion binding), LDLR (low density lipoprotein receptor (familial hypercholesterolemia), lipid metabolic process), MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian), regulation of transcription), PLAUR (plasminogen activator, urokinase receptor, cell motility), SFN (stratifin, negative regulation of protein kinase activity), SOX9 (SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal), cell fate specification), TNFSF4 (tumor necrosis factor (ligand) superfamily, member 4 (tax-transcriptionally activated glycoprotein 1, 34 kDa), immune response, cytokine activity).