Claire A. Hudson

Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1)

Force generation in smooth muscle is driven by phosphorylation of myosin light chains (MYL), which is regulated by the equilibrium between the activities of myosin light chain kinase (MYLK) and myosin phosphatase (MYLP). MYLK is activated by Ca2+-calmodulin whereas MYLP is inhibited by phosphorylation of its myosin-binding subunit (MYPT1) by Ca2+-independent mechanisms, leading to generation of increased MYL phosphorylation and force for a given intracellular Ca2+ concentration, a phenomenon known as ‘calcium-sensitization’. The regulation of MYPT1 phosphorylation in human myometrium, which shows increasing phasic contractility at the onset of labour, has yet to be fully investigated. Here, we explore phosphorylation of MYPT1 at Thr696 and Thr853, alongside phosphorylation of MYL, in fresh human myometrial tissue and cultured myometrial cells. We report that pMYPT1 (Thr853) levels are dependent on the activity of Rho-associated kinase (ROCK), determined using the ROCK inhibitor g-H-1152 and siRNA-mediated knockdown of ROCK1/2, and are highly correlated to ppMYL (Thr18/Ser19) levels. Pharmacological inhibition of ROCK was associated with a decrease in oxytocin (OXT)-stimulated contractility of myometrial strips in vitro. Moreover, we have measured pMYPT1 and pMYL levels between and during spontaneous and OXT-stimulated phasic contractions by rapidly freezing contracting muscle, and demonstrate for the first time functional coupling between increases in pMYPT1 (Thr853), ppMYL (Thr18/Ser19) and phasic contractility that is ROCK-dependent. The combined approach of measuring contractility and phosphorylation has demonstrated that the phosphorylation of MYPT1 (Thr853) changes dynamically with each contraction and has elucidated a defined role for ROCK in regulating myometrial contractility through MYLP, providing new insights into uterine physiology which will stimulate further research into treatments for preterm labour.

Regulation of PIKfyve phosphorylation by insulin and osmotic stress.

PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.

Central role for protein kinase C in oxytocin and epidermal growth factor stimulated cyclooxygenase 2 expression in human myometrial cells

BackgroundProstaglandins are important mediators of uterine contractility and cervical ripening during labour. Cyclooxygenase-2 (COX-2), also known as prostaglandin-endoperoxide synthase 2, is a rate limiting enzyme involved in the conversion of arachidonic acid into prostaglandins at parturition. In this paper, the pathways underlying agonist-induced cyclooxygenase-2 expression in human myometrial cells were studied.ResultsMyometrial cells were stimulated with different agonists: oxytocin (OXT), epidermal growth factor (EGF), interleukin-1β (IL1β), and phorbol-12-myristate-13-acetate (PMA) alone and in the presence of specific signalling pathway inhibitors. The nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathway was inhibited by means of the IKK-2 inhibitor TPCA-1. Signalling through extracellular signal-regulated kinases (ERK) was inhibited using the MEK1/2 inhibitor PD-184352. Bisindolylmaleimide-I was used to inhibit protein kinase C (PKC) signalling. COX-2 expression and ERK phosphorylation were measured using immunoblotting.OXT induced COX-2 expression by activating PKC and ERK. EGF increased COX-2 expression via stimulation of PKC, ERK and NFKB. As expected, the pro-inflammatory cytokine IL1β induced COX-2 expression by activating PKC- and NFKB-dependent pathways. Stimulation of PKC directly with PMA provoked strong COX-2 expression.ConclusionsPKC plays a central role in OXT and EGF induced COX-2 expression in human myometrial cells. However, other pathways, notably ERK and NFKB are also involved to an extent which depends on the type of agonist used.

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

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 (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. 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 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 INTERESTS This work was supported by an Action Medical Research grant (SP4612). The authors have no competing interests to declare.

Bariatric surgery in morbidly obese insulin resistant humans normalises insulin signalling but not insulin-stimulated glucose disposal

Aims Weight-loss after bariatric surgery improves insulin sensitivity, but the underlying molecular mechanism is not clear. To ascertain the effect of bariatric surgery on insulin signalling, we examined glucose disposal and Akt activation in morbidly obese volunteers before and after Roux-en-Y gastric bypass surgery (RYGB), and compared this to lean volunteers. Materials and Methods The hyperinsulinaemic euglycaemic clamp, at five infusion rates, was used to determine glucose disposal rates (GDR) in eight morbidly obese (body mass index, BMI=47.3±2.2 kg/m2) patients, before and after RYGB, and in eight lean volunteers (BMI=20.7±0.7 kg/m2). Biopsies of brachioradialis muscle, taken at fasting and insulin concentrations that induced half-maximal (GDR50) and maximal (GDR100) GDR in each subject, were used to examine the phosphorylation of Akt-Thr308, Akt-473, and pras40, in vivo biomarkers for Akt activity. Results Pre-operatively, insulin-stimulated GDR was lower in the obese compared to the lean individuals (P<0.001). Weight-loss of 29.9±4 kg after surgery significantly improved GDR50 (P=0.004) but not GDR100 (P=0.3). These subjects still remained significantly more insulin resistant than the lean individuals (p<0.001). Weight loss increased insulin-stimulated skeletal muscle Akt-Thr308 and Akt-Ser473 phosphorylation, P=0.02 and P=0.03 respectively (MANCOVA), and Akt activity towards the substrate PRAS40 (P=0.003, MANCOVA), and in contrast to GDR, were fully normalised after the surgery (obese vs lean, P=0.6, P=0.35, P=0.46, respectively). Conclusions Our data show that although Akt activity substantially improved after surgery, it did not lead to a full restoration of insulin-stimulated glucose disposal. This suggests that a major defect downstream of, or parallel to, Akt signalling remains after significant weight-loss.

Smoking alters hydroxyprostaglandin dehydrogenase expression in fetal membranes

INTRODUCTION The way in which tobacco smoking increases the risk of preterm labor remains uncertain. Altered prostaglandin metabolism is one potential mechanism. METHODS Proteins in fetal membrane samples (amniochoriodecidua) from 20 women were relatively quantified using Tandem Mass Tagging nano-liquid chromatography mass spectrometry. RESULTS Prostaglandin synthases and two enzymes involved in prostaglandin degradation, hydroxyprostaglandin dehydrogenase (HPGD) and CBR1, were detected by the mass spectrometer. The expression of HPGD was significantly lower in smokers relative to non-smokers (0.43 fold, p = 0.016). There was no effect of labor, inflammatory status or gestational age on the HPGD levels. DISCUSSION We describe for the first time an association between maternal smoking and HPGD expression. We propose that reduced expression of HPGD is one mechanism through which smoking may contribute to preterm labor. Lower levels of this enzyme, key to metabolising prostaglandins, may result in higher levels of prostaglandins and therefore precipitate labor prematurely.