1. Steroid receptor co-activator interacting protein (SIP) mediates EGF-stimulated expression of the prostaglandin synthase COX2 and prostaglandin release in human myometrium
- Author
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Craig A. McArdle, Andrés López Bernal, and Claire A. Hudson
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,Embryology ,medicine.medical_specialty ,Biology ,Dinoprost ,Dinoprostone ,prostaglandin-endoperoxide synthase 2 ,03 medical and health sciences ,0302 clinical medicine ,Epidermal growth factor ,Internal medicine ,Gene expression ,Serum response factor ,Genetics ,medicine ,Humans ,Protein kinase A ,parturition ,Molecular Biology ,Gene knockdown ,030219 obstetrics & reproductive medicine ,Epidermal Growth Factor ,Kinase ,Tumor Suppressor Proteins ,Myometrium ,Obstetrics and Gynecology ,steroid receptor co-activator-interacting protein ,Cell Biology ,Cell biology ,epidermal growth factor ,030104 developmental biology ,Endocrinology ,Reproductive Medicine ,Cyclooxygenase 2 ,cyclooxygenase-2 ,Prostaglandins ,gene expression ,Female ,KANK2 ,Apoptosis Regulatory Proteins ,Carrier Proteins ,human myometrium ,Developmental Biology - 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. Study finding 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. 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 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. 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 (P2 (P2α (PPPIL6 and IL8 mRNA in a SIP-dependent manner (both PPP Limitations, reasons for caution 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 at passage 7 therefore may not fully reflect the conditions in vivo. Wider implications of the findings 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. Large scale data: Not applicable. Study funding and competing interest(s) This work was supported by an Action Medical Research grant (SP4612). The authors have no competing interests to declare.
- Published
- 2016