Oxytocin activates NF-κB-mediated inflammatory pathways in human gestational tissues

https://doi.org/10.1016/j.mce.2014.11.008Get rights and content

Highlights

  • In human gestational tissues OT activates NF-κB via the canonical pathway.

  • OT increases expression of NF-κB mediated inflammatory labour-associated genes.

  • Cross-talk exists between the activation of MAPKs and NF-κB signalling cascades in human amnion.

Abstract

Human labour, both at term and preterm, is preceded by NF-κB-mediated inflammatory activation within the uterus, leading to myometrial activation, fetal membrane remodelling and cervical ripening. The stimuli triggering inflammatory activation in normal human parturition are not fully understood. We show that the neurohypophyseal peptide, oxytocin (OT), activates NF-κB and stimulates downstream inflammatory pathways in human gestational tissues. OT stimulation (1 pM–100 nM) specifically via its receptor (OTR) in human myometrial and amnion primary cells led to MAPK and NF-κB activation within 15 min and maximal p65-subunit nuclear translocation within 30 min. Both in human myometrium and amnion, OT-induced activation of the canonical NF-κB pathway upregulated key inflammatory labour-associated genes including IL-8, CCL5, IL-6 and COX-2. IKKβ inhibition (TPCA1; 10 µM) suppressed OT-induced NF-κB-p65 phosphorylation, whereas p65-siRNA knockdown reduced basal and OT-induced COX-2 levels in myometrium and amnion. In both gestational tissues, MEK1/2 (U0126; 10 µM) or p38 inhibition (SB203580; 10 µM) suppressed OT-induced COX-2 expression, but OT-induced p65-phosphorylation was only inhibited in amnion, suggesting OT activation of NF-κB in amnion is MAPK-dependent. Our data provide new insight into the OT/OTR system in human parturition and suggest that its therapeutic modulation could be a strategy for regulating both contractile and inflammatory pathways in the clinical context of term/preterm labour.

Introduction

Oxytocin is widely recognised as playing a major role in parturition by promoting myometrial contractility. Currently, oxytocin is the most potent uterotonin available and is extensively used in the clinical management of dysfunctional labour (Wei et al., 2013). However, myometrial contractions represent a late event in the cascade leading to labour and are preceded by cervical ripening and fetal membrane activation and remodelling. The onset of human labour resembles an inflammatory reaction and a substantial body of evidence suggests that both preterm and term labour are linked with increased NF-κB activity within the uterus (Allport et al, 2001, Chapman et al, 2004, Condon et al, 2006). NF-κB activation occurs in both human myometrium (Khanjani et al., 2011) and the fetal membranes (Lim et al., 2012) prior to the onset of labour and is associated with the upregulation of pro-labour genes including cyclooxygenase type 2 (COX-2) and the oxytocin receptor (OTR) (Terzidou et al., 2011). Consistent with this, inhibition of NF-κB activity inhibits LPS-induced preterm labour in mice (Condon et al, 2004, Pirianov et al, 2009) and IL-1β induced uterine contractions in Rhesus monkeys (Sadowsky et al., 2003). The role of the peptide hormone oxytocin (OT) in the modulation of inflammatory pathways preceding labour has been largely overlooked. Just prior to the onset of human labour, uterine sensitivity to OT increases markedly via upregulation of the OTR (Fang et al, 1996, Fuchs et al, 1982, Soloff et al, 1979). OT is an important regulator of PG production in the endometrium, amnion and decidua in several species, including humans (Fuchs et al, 1981, Hinko, Soloff, 1992, Jeng et al, 2000, Lee et al, 2012, Milne, Jabbour, 2003, Moore et al, 1988, Terzidou et al, 2011, Zhang et al, 2011). This OT effect on PGs release in the human amnion has been shown to be through up regulation of COX-2 (the rate limited step of PG biosynthesis) and was suggested to be protein kinase C (PKC)-dependent (Moore et al, 1991, Wouters et al, 2014).

Amnion is a major site of prostaglandin production in human pregnancy and its activation is critical for cervical ripening and the stimulation of myometrial contractions. Just prior to labour, there is an increase in inflammatory cytokine release from the amnion (Keelan et al, 2003, Satoh et al, 1979) as well as increased PG synthesis, particularly PGE2 (Bennett et al, 1992, Olson, 2003). Collectively these changes promote cervical ripening, lower uterine segment remodelling and initiation of myometrial contractions (Fletcher et al, 1993, Keirse, 1993, Keirse et al, 1983, McLaren et al, 2000, Olson, 2003). In murine parturition surfactant protein A (SP-A) produced by the maturing fetal lung has been suggested to represent the stimulus for a cascade of inflammatory signalling pathways leading to labour onset (Condon et al., 2004). However, in human pregnancy the endocrine or mechanical stimuli triggering inflammatory activation are obscure.

NF-κB is a complex of heterogeneous dimers of various subunits from the Rel/NF-κB family. The Rel/NF-κB family consists of RelA (p65), RelB, c-Rel, p100/p52 and p105/p50, which share a Rel homology domain (RHD). The inactivated NF-κB subunits are present in the cytoplasm as homo- or heterodimers and they are tightly regulated by an inhibitory protein from IκB family, which prevents nuclear translocation of NF-κB. The canonical NF-κB pathway is typically triggered by pro-inflammatory stimuli such as cytokines and lipopolysaccharides (LPS). Following ligand-specific receptor activation, downstream signalling activates IκB kinase (IKK) complex, composed of IKKα, IKKβ, and NF-κB essential modulator (NEMO), which is responsible for phosphorylating IκBα (Traenckner et al., 1995). Phosphorylated IκBα then undergoes tertiary structure changes, which expose motifs recognised by SCF ubiquitin ligases and becomes a target for ubiquination (Yaron et al., 1998). This results in degradation of IκBα by 26S proteosome and frees NF-κB dimers (typically p50/p65) to enter the nucleus. The phosphorylation of the p65 subunit is important for initiating transcription (Sasaki et al, 2005, Vermeulen et al, 2002) and facilitates binding to the promoter region of various contraction associated proteins, including OTR (Fuchs et al, 1982, Terzidou, 2006), PGF receptor (Olson et al., 2003) and COX-2 (Belt et al, 1999, Slater et al, 1995, Soloff et al, 2004) and proinflammatory cytokines IL-6 (Libermann and Baltimore, 1990) and IL-8 (Khanjani et al., 2012).

We have previously demonstrated that OT upregulates the expression of COX-2, which itself is NF-κB dependent, suggesting that OT may activate NF-κB (Moore et al, 1991, Terzidou et al, 2011). In this study, we determined the effects of OT on NF-κB-mediated inflammatory pathways in human gestational tissues. We show that in both human myometrial and amnion cells OT stimulation of its receptor drives the sequential activation of specific MAPKs and NF-κB leading to the production of inflammatory pro-labour cytokines and prostaglandins. Our results demonstrate a previously unrecognised role for oxytocin in modulating NF-κB-mediated inflammatory pathways thereby promoting the labouring phenotype. These findings not only provide novel insight into how the OT/OTR system contributes to normal human parturition but also highlights its potential impact in therapeutic treatments using oxytocin or OTR antagonists.

Section snippets

Cell preparation and culture

All samples were collected with informed consent. Approval was granted by the local ethics committee (Placenta; RREC 2002-6283 and Myometrium; RREC 1997-5089). Fetal membranes and myometrial biopsies were obtained from women undergoing elective caesarean section at term (38+0–39+6 weeks of pregnancy), prior to the onset of labour. The patients did not have pre-existing medical conditions and had not received uterotonics. Those with pre-eclampsia or multiple pregnancies were not included in this

OT activates NF-κB in amnion and myometrium

NF-κB activation by inflammatory stimuli such as IL-1β typically occurs via the canonical pathway whereby p65 homodimers or p50-p65 heterodimers translocate to the nucleus and drive transcriptional activity. Non-canonical activation can also occur and involves nuclear translocation of RelB-p52 heterodimers (Lindstrom, 2005). To examine the effect of OT on NF-κB activation, human primary myometrial cells (n = 6) and amnion (n = 12) were treated with OT and nuclear translocation of the NF-κB p65,

Discussion

Our study shows that OT increases the expression of COX-2 and other inflammatory mediators known to be associated with the onset of labour in both the myometrium and amnion via activation of NF-κB and MAPKs. In myometrium, OT activates the established canonical pathway involving p65/p50 heterodimers with no cross talk between NF-κB and MAPKs. In amnion, however, OT signalling is markedly different. NF-κB activation involves only p65 nuclear translocation and this is dependent on crosstalk with

Acknowledgements

GRANT SUPPORT: This work was supported by an Action Medical Research Project Grant (SP4454), Genesis Research Trust (P14758) and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. The views expressed are those of the author(s) and not necessarily those of Imperial College, the NHS, the NIHR or the Department of Health.

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