Inflammatory cytokines such as interleukin-1β (IL-1β) and TNF-α are thought to be involved in the initiation of labour (Brockelhurst, 1999). Preterm labour accounts for one-third of premature births, and one of the leading causes is intra-uterine infection as evidenced by increased levels of IL-1β and TNF-α. IL-1β increases expression of the enzyme cyclo-oxygenase-2 (COX-2), which catalyses the limiting step in the production of prostaglandins PGE₂ and PGF_2α, involved in muscle contraction in preterm and term labour. The mechanisms underlying IL-1β-induced increases in prostaglandins via COX-2 are not fully elucidated. IL-1β activates the three major mitogen-activated protein kinase (MAPK) pathways, and there is evidence implicating p38 MAPK and jun kinase (JNK) in cytokine-induced COX-2 expression (Guan et al. 1998). The COX-2 promoter contains several regulatory elements including NFκB sites, and it is possible that p38 MAPK may mediate COX-2 transcription via activation of NFκB possibly through involvement of protein kinase C (PKC) pathways.

Expression of COX-2 in human myometrial smooth muscle cells (HMSMCs), obtained from lower segment myometrial biopsies (the study was approved by the ethical committee of GKT School of Biomedical Sciences), was examined including inhibitors of MAP kinases (SB203580, 2 µM, for p38; U0126, 250 nM, for ERK 1/2; and SP600125, 20 µM, for JNK), PKC (GF109203X, 10 µM) and NFκB (MG-132, 42 µM; and PG490, 1 µM). Western blot analyses revealed that IL-1β increased COX-2 expression via signalling pathways involving p38 MAPK, PKC and NFκB. In parallel Northern blots we showed that inhibition of p38 MAPK and PKC reduced COX-2 mRNA levels in cells challenged with IL-1β for 3 h.

To examine NFκB translocation, we used electrophoretic mobility shift assays (EMSA) with antibodies for NFκB subunits and inhibitors of MAPKs and PKC. Using two NFκB COX-2 promoter sites, we have shown that these inhibitors do not affect translocation of p65 and p50 subunits to the nucleus. MG132, a proteosome inhibitor known to prevent degradation of IκB, reduced NFκB translocation, whereas PG490, an inhibitor of nuclear NFκB transactivation was ineffective. These data suggest that p38 MAPK and/or PKC may affect NFκB-mediated COX-2 transcription through a mechanism other than translocation to the nucleus after Iκ kinase degradation, possibly through phosphorylation of NFκB dimers.

This work was supported by The Wellcome Trust.