Steroid receptor co-activator interacting protein (SIP) mediates EGF-stimulated expression of the prostaglandin synthase COX2 and prostaglandin release in human myometrium

Abstract

Study hypothesis: Steroid receptor coactivator interacting protein (SIP/KANK2) is involved in regulating the expression of the prostaglandin (PG)-endoperoxide synthase 2 (PTGS2; also known as cyclo-oxygenase 2, COX2) and PG release in human myometrium. studyfinding: SIP is phosphorylated in myometrial cells in response to epidermal growth factor (EGF)-stimulation and is required for EGFstimulated increases in COX2 expression, PGE2 and PGF2a release, and expression of interleukins (IL) 6 and IL8. what is known already: Human parturition involves inflammatory and non-inflammatory pathways and requires activation of the intrauterine PG cascade. A key mediator of uterine PG production is the highly inducible enzyme COX2. Regulation of COX2 expression is complex, and novel factors involved in its induction may play an important role during labour. The expression and function of SIP in uterine tissues has never been investigated. study design, samples/materials, methods: Mass spectrometrywas used to identify SIP fromcultured primary myometrial cells, and its expression in fresh placenta, fetal membranes, decidua and myometrium from pregnant and non-pregnant women was determined by western blotting. SIP expression in myometrial cells was reduced using small interfering RNA (siRNA), and COX2 expression was stimulated with EGF. COX2, IL6 and IL8 mRNA and COX2 protein expressionwere measured using quantitative RT-PCR (RT-qPCR) and western blotting respectively, and release of PGE2 and PGF2α by enzyme immunoassay. The time course and dose response of SIP phosphorylation in response to EGF were determined, and phosphorylation was measured in the presence of the mitogen-activated protein kinase kinase 1(MEK1) inhibitor PD-184352. Fresh myometrial tissue was used to confirm effects of EGF and MEK1 inhibition on SIP phosphorylation and COX2 expression. A profile of transcription factor (TF) activity after SIP knockdown was carried out using a commercially available array. main results and the role of chance: We have demonstrated expression of SIP in human myometrium. siRNA-mediated knockdown of SIP resulted in decreased EGF-stimulated COX2 protein expression (P < 0.001), and decreased release of PGE2 (P < 0.001) and PGF2α (P < 0.01). EGF stimulation resulted in rapid and transient phosphorylation of SIP, which was blocked by pharmacological inhibition of the MEK1/ERK (extracellular signal-regulated kinase) signalling pathway with PD-184352 (P < 0.001). Moreover inhibition of ERK signalling significantly decreased EGF-stimulated COX2 expression (P < 0.001). EGF phosphorylated SIP and increased COX2 expression in a MEK1/ERK-dependent manner in freshly isolated pregnant myometrium. Our data have uncovered a pathway mediating EGF-stimulated COX2 expression that is ERK and SIP dependent, providing a novel function for SIP in the pregnant uterus. Furthermore, EGF stimulated the expression of IL6 and IL8 mRNA in a SIP-dependent manner (both P < 0.05), and SIP expression was positively associated with activation of serum response factor (SRF) and YY1 TF (P < 0.001 and P < 0.05, respectively), suggesting additional important roles for myometrial SIP.