title: Steroid receptor co-activator interacting protein (SIP) mediates EGF-stimulated expression of the prostaglandin synthase COX2 and prostaglandin release in human myometrium.
abstract: 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.
  
  
  
  SIP is phosphorylated in myometrial cells in response to epidermal growth factor (EGF)-stimulation and is required for EGF-stimulated increases in COX2 expression, PGE2 and PGF2α release, and expression of interleukins (IL) 6 and IL8.
  
  
  
  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.
  
  
  
  Mass spectrometry was used to identify SIP from cultured 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 expression were 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.
  
  
  
  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.
  
  
  
  While we describe a new role for myometrial SIP, we are yet to determine whether SIP phosphorylation is required for its effects on regulating COX2 expression and PG release. Our data are from in vitro studies using fresh tissue and cultured myometrial cells therefore may not fully reflect the conditions in vivo.
  
  
  
  Our group has previously described increases in myometrial COX2 expression with labour at term and preterm. EGF levels rise in the amniotic fluid near term suggesting it may participate in paracrine signalling events, altering gene expression in the myometrium. Our novel data describe a role for SIP in regulating EGF-stimulated expression of myometrial COX2 and PG release. Moreover, our profile of SIP-dependent TF activation provides a platform for further investigations into additional roles for SIP in uterine function. These findings may facilitate the development of new, targeted drugs for the management of labour.
  
  
  
  Not applicable.
  
  
  
  This work was supported by an Action Medical Research grant (SP4612). The authors have no competing interests to declare.
authors.affiliation: School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK claire.hudson@bristol.ac.uk.
authors.forename: Claire A
authors.initials: CA
authors.lastname: Hudson
authors.affiliation: School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK.
authors.forename: Craig A
authors.initials: CA
authors.lastname: McArdle
authors.affiliation: School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK Department of Obstetrics and Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, UK.
authors.forename: Andrés
authors.initials: A
authors.lastname: López Bernal
mesh_terms: D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015237:Dinoprost / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D006801:Humans; D009215:Myometrium / Q000187:drug effects* / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C000613889:Kank2 protein, human; D011453:Prostaglandins; D025521:Tumor Suppressor Proteins; D004815:Epidermal Growth Factor; D015237:Dinoprost; D051546:Cyclooxygenase 2; D015232:Dinoprostone
keywords: KANK2; cyclooxygenase-2; epidermal growth factor; gene expression; human myometrium; parturition; prostaglandin-endoperoxide synthase 2; steroid receptor co-activator-interacting protein