co-activator interacting protein (SIP) mediates EGF-stimulated expression of the prostaglandin synthase COX2 and prostaglandin release in human myometrium. Molecular Human Reproduction, 22(7), 512-525. DOI: 10.1093/molehr/gaw031

17 Study hypothesis: Steroid receptor coactivator interacting protein (SIP/KANK2) is involved in 18 regulating the expression of the prostaglandin (PG)-endoperoxide synthase 2 (PTGS2; also known as 19 cyclo-oxygenase 2, COX2) and PG release in human myometrium. 20 Study finding: SIP is phosphorylated in myometrial cells in response to epidermal growth factor 21 (EGF)-stimulation and is required for EGF-stimulated increases in COX2 expression, PGE2 and 22 PGF2α release, and expression of interleukins (IL) 6 and IL8. 23 What is known already: Human parturition involves inflammatory and non-inflammatory pathways 24 and requires activation of the intrauterine PG cascade. A key mediator of uterine PG production is the 25 highly inducible enzyme COX2. Regulation of COX2 expression is complex, and novel factors 26 involved in its induction may play an important role during labour. The expression and function of 27 SIP in uterine tissues has never been investigated. 28 Study design, samples/materials, methods: Mass spectrometry was used to identify SIP from 29 cultured primary myometrial cells, and its expression in fresh placenta, fetal membranes, decidua and 30 myometrium from pregnant and non-pregnant women was determined by western blotting. SIP 31 expression in myometrial cells was reduced using small interfering RNA (siRNA), and COX2 32 expression was stimulated with EGF. COX2, IL6 and IL8 mRNA and COX2 protein expression were 33 measured using quantitative RT-PCR (RT-qPCR) and western blotting respectively, and release of 34 PGE2 and PGF2α by enzyme immunoassay. The time course and dose response of SIP phosphorylation 35 in response to EGF were determined, and phosphorylation was measured in the presence of the 36 mitogen-activated protein kinase kinase 1(MEK1) inhibitor PD-184352. Fresh myometrial tissue was 37 used to confirm effects of EGF and MEK1 inhibition on SIP phosphorylation and COX2 expression. 38 A profile of transcription factor (TF) activity after SIP knockdown was carried out using a 39 commercially available array. 40 Main results and the role of chance: We have demonstrated expression of SIP in human 41 myometrium. siRNA-mediated knockdown of SIP resulted in decreased EGF-stimulated COX2 42