Characterization of highly proliferative decidual precursor cells during the window of implantation in human endometrium.

Pregnancy depends on the wholesale transformation of the endometrium, a process driven by differentiation of endometrial stromal cells (EnSC) into specialist decidual cells. Upon embryo implantation, decidual cells impart the tissue plasticity needed to accommodate a rapidly growing conceptus and invading placenta, although the underlying mechanisms are unclear. Here we characterize a discrete population of highly proliferative mesenchymal cells (hPMC) in midluteal human endometrium, coinciding with the window of embryo implantation. Single-cell transcriptomics demonstrated that hPMC express genes involved in chemotaxis and vascular transmigration. Although distinct from resident EnSC, hPMC also express genes encoding pivotal decidual transcription factors and markers, most prominently prolactin. We further show that hPMC are enriched around spiral arterioles, scattered throughout the stroma, and occasionally present in glandular and luminal epithelium. The abundance of hPMC correlated with the in vitro colony-forming unit activity of midluteal endometrium and, conversely, clonogenic cells in culture express a gene signature partially conserved in hPMC. Cross-referencing of single-cell RNA-sequencing data sets indicated that hPMC differentiate into a recently discovered decidual subpopulation in early pregnancy. Finally, we demonstrate that recurrent pregnancy loss is associated with hPMC depletion. Collectively, our findings characterize midluteal hPMC as novel decidual precursors that are likely derived from circulating bone marrow-derived mesenchymal stem/stromal cells and integral to decidual plasticity in pregnancy.

Embryo biosensing by uterine natural killer cells determines endometrial fate decisions at implantation.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

Loss of Endometrial Plasticity in Recurrent Pregnancy Loss.

Menstruation drives cyclic activation of endometrial progenitor cells, tissue regeneration, and maturation of stromal cells, which differentiate into specialized decidual cells prior to and during pregnancy. Aberrant responsiveness of human endometrial stromal cells (HESCs) to deciduogenic cues is strongly associated with recurrent pregnancy loss (RPL), suggesting a defect in cellular maturation. MeDIP-seq analysis of HESCs did not reveal gross perturbations in CpG methylation in RPL cultures, although quantitative differences were observed in or near genes that are frequently deregulated in vivo. However, RPL was associated with a marked reduction in methylation of defined CA-rich motifs located throughout the genome but enriched near telomeres. Non-CpG methylation is a hallmark of cellular multipotency. Congruently, we demonstrate that RPL is associated with a deficiency in endometrial clonogenic cell populations. Loss of epigenetic stemness features also correlated with intragenic CpG hypomethylation and reduced expression of HMGB2, coding high mobility group protein 2. We show that knockdown of this sequence-independent chromatin protein in HESCs promotes senescence and impairs decidualization, exemplified by blunted time-dependent secretome changes. Our findings indicate that stem cell deficiency and accelerated stromal senescence limit the differentiation capacity of the endometrium and predispose for pregnancy failure.

The clock protein period 2 synchronizes mitotic expansion and decidual transformation of human endometrial stromal cells.

Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss.

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

Functionally Selective Inhibition of the Oxytocin Receptor by Retosiban in Human Myometrial Smooth Muscle.

Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared with first-generation oxytocin antagonists when used for the prevention of preterm birth. The aim was to determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban compared with the currently used peptide-based compound atosiban. Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Retosiban antagonism of oxytocin action in human myometrium was potent, rapid, and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single-site competitive binding kinetics for epelsiban, retosiban, and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban but not retosiban inhibited forskolin, and calcitonin stimulated 3',5'-cyclic adenosine 5'-mono-phosphate (cAMP) production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated extracellular regulated kinase (ERK)1/2 activity in a time- and concentration-dependent manner. Oxytocin and atosiban stimulated cyclo-oxygenase 2 activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα i G-proteins. Retosiban and epelsiban demonstrate distinct pharmacology when compared with atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.

Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window.

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage.

Impact of sitagliptin on endometrial mesenchymal stem-like progenitor cells: A randomised, double-blind placebo-controlled feasibility trial.

BACKGROUND: Recurrent pregnancy loss (RPL) is associated with the loss of endometrial mesenchymal stem-like progenitor cells (eMSC). DPP4 inhibitors may increase homing and engraftment of bone marrow-derived cells to sites of tissue injury. Here, we evaluated the effect of the DPP4 inhibitor sitagliptin on eMSC in women with RPL, determined the impact on endometrial decidualization, and assessed the feasibility of a full-scale clinical trial. METHODS: A double-blind, randomised, placebo-controlled feasibility trial on women aged 18 to 42 years with a history of 3 or more miscarriages, regular menstrual cycles, and no contraindications to sitagliptin. Thirty-eight subjects were randomised to either 100 mg sitagliptin daily for 3 consecutive cycles or identical placebo capsules. Computer generated, permuted block randomisation was used to allocate treatment packs. Colony forming unit (CFU) assays were used to quantify eMSC in midluteal endometrial biopsies. The primary outcome measure was CFU counts. Secondary outcome measures were endometrial thickness, study acceptability, and first pregnancy outcome within 12 months following the study. Tissue samples were subjected to explorative investigations. FINDINGS: CFU counts following sitagliptin were higher compared to placebo only when adjusted for baseline CFU counts and age (RR: 1.52, 95% CI: 1.32-1.75, P<0.01). The change in CFU count was 1.68 in the sitagliptin group and 1.08 in the placebo group. Trial recruitment, acceptability, and drug compliance were high. There were no serious adverse events. Explorative investigations showed that sitagliptin inhibits the expression of DIO2, a marker gene of senescent decidual cells. INTERPRETATION: Sitagliptin increases eMSCs and decreases decidual senescence. A large-scale clinical trial evaluating the impact of preconception sitagliptin treatment on pregnancy outcome in RPL is feasible and warranted. FUNDING: Tommy's Baby Charity. CLINICAL TRIAL REGISTRATION: EU Clinical Trials Register no. 2016-001120-54.

Paradoxical behavior of neuromedin U in isolated smooth muscle cells and intact tissue.

Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

Lack of receptor-selective effects of either RGS2, RGS3 or RGS4 on muscarinic M3- and gonadotropin-releasing hormone receptor-mediated signalling through G alpha q/11.

Termination of signalling by G-protein-coupled receptors requires inactivation of the G alpha-subunits of heterotrimeric G-proteins and the re-association of G alpha- and G betagamma-subunits. Inactivation of G alpha-subunits is achieved by the hydrolysis of bound GTP by an intrinsic GTPase activity, which is considerably enhanced by GTPase activating proteins. Regulators of G-protein signalling (RGS) proteins are a large family of GTPase activating proteins, many of which have structures indicating roles beyond GTPase activating protein activity and suggesting that the identity of the RGS protein recruited may also be critical to other aspects of signalling. There is some evidence of selective effects of RGS proteins against different G-protein-coupled receptors coupling to the same signalling pathways and growing evidence of physical interactions between RGS proteins and G-protein-coupled receptors. However, it is unclear as to how common such interactions are and the circumstances under which they are functionally relevant. Here we have examined potential selectivity of RGS2, 3 and 4 against signalling mediated by G alpha q/11-coupled muscarinic M3 receptors and gonadotropin-releasing hormone in an immortalised mouse pituitary cell line. Despite major structural differences between these two receptor types and agonist-dependent phosphorylation of the muscarinic M3- but not gonadotropin-releasing hormone receptor, signalling by both receptors was similarly inhibited by expression of either RGS2 or RGS3, whereas RGS4 has little effect. Thus, at least in these circumstances, RGS protein-dependent inhibition of signalling is not influenced by the nature of the G-protein-coupled receptor through which the signalling is mediated.

Reciprocal regulation of ß2-adrenoceptor-activated cAMP response-element binding protein signalling by arrestin2 and arrestin3.

Activation of Gs coupled receptors (e.g. ß2-adrenoreceptor (ß2AR)) expressed within the uterine muscle layer (myometrium), promotes intracellular cAMP generation, inducing muscle relaxation through short-term inhibition of contractile proteins, and longer-term modulation of cellular phenotype to promote quiescence. In the myometrium cAMP-driven modulation of cell phenotype is facilitated by CREB activity, however despite the importance of CREB signalling in the promotion of myometrial quiescence during pregnancy, little is currently known regarding the molecular mechanisms involved. Thus, we have characterised ß-adrenoceptor-stimulated CREB signalling in the immortalised ULTR human myometrial cell line. The non-selective ß-adrenoceptor agonist isoprenaline induced time- and concentration-dependent CREB phosphorylation, which was abolished by the ß2AR selective antagonist ICI118,551. ß2AR-stimulated CREB phosphorylation was mediated through a short-term PKA-dependent phase, and longer-term Src/p38 MAPK-dependent/PKA-independent phase. Since in model cells, arrestin2 can facilitate ß2AR-mediated Src/p38 recruitment, we examined whether CREB signalling was activated through a similar process in myometrial cells. Depletion of arrestin2 attenuated p38 phosphorylation, whilst arrestin3 depletion enhanced and prolonged isoprenaline-stimulated p38 signals, which was reversed following inhibition of Src. Knockdown of arrestin2 led to enhanced short-term (up to 10min), and attenuated longer-term (>10min) isoprenaline-stimulated CREB phosphorylation. Contrastingly, removal of arrestin3 enhanced and prolonged isoprenaline-stimulated CREB phosphorylation, whilst depletion of both arrestins abolished CREB signals at time points >5min. In summary, we have delineated the molecular mechanisms coupling ß2AR activity to CREB signalling in ULTR myometrial cells, revealing a biphasic activation process encompassing short-term PKA-dependent, and prolonged Src/arrestin2/p38-dependent components. Indeed, our data highlight a novel arrestin-mediated modulation of CREB signalling, suggesting a reciprocal relationship between arrestin2 and arrestin3, wherein recruitment of arrestin3 restricts the ability of ß2AR to activate prolonged CREB phosphorylation by precluding recruitment of an arrestin2/Src/p38 complex.

Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium.

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells.

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca(2+) release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.

Confocal microscopy: theory and applications for cellular signaling.

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to interrogate a wide range of structural and functional aspects on both fixed and live cells. Here we describe the basic principles underlying confocal microscopy and provide methodological accounts of how it can be used to study aspects related particularly (but not exclusively) to the expression, activation, and regulation of signaling by G-protein-coupled receptors. Specifically we provide detailed protocols for examining: the cellular expression and distribution of proteins by immunocytochemistry; cytoplasmic and organelle Ca(2+) signaling using fluorescent indicators; second messenger generation using fluorescently tagged biosensors; and ligand/receptor internalization using fluorescently tagged peptide agonists and receptors.

G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling.

AIMS: prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5'-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). METHODS AND RESULTS: mesenteric arteries contracted in response to UTP challenge: When an EC(50) UTP concentration (30 µM, 5 min) was added 5 min before (R(1)) and after (R(2)) the addition of a maximal UTP concentration (R(max): 100 µM, 5 min), R(2) responses were decreased relative to R(1), indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca(2+)-sensitive dyes. A similar protocol (R(1)/R(2) = 10 µM; R(max) = 100 µM, applied for 30 s) revealed markedly reduced R(2) when compared with R(1) responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y(2)-receptor almost completely abolished UTP-stimulated IP(3)/Ca(2+) signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype. CONCLUSION: these new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Arrestins differentially regulate histamine- and oxytocin-evoked phospholipase C and mitogen-activated protein kinase signalling in myometrial cells.

BACKGROUND AND PURPOSE: The uterotonins oxytocin and histamine, mediate contractile signals through specific G protein-coupled receptors, a process which is tightly controlled during gestation to prevent preterm labour. We previously identified G protein-coupled receptor kinase (GRK)2 and GRK6 as respective cardinal negative regulators of histamine H(1) and oxytocin receptor signalling. GRK-mediated phosphorylation promotes arrestin recruitment, not only desensitizing receptors but activating an increasing number of diverse signalling pathways. Here we investigate potential roles that arrestins play in the regulation of myometrial oxytocin/histamine H(1) receptor signalling. EXPERIMENTAL APPROACH: Endogenous arrestins2 and 3 were specifically depleted using RNA-interference in a human myometrial cell line and the consequences of this for G protein-coupled receptor-mediated signalling were assessed using Ca(2+) /inositol 1,4,5-trisphophate imaging and standard mitogen-activated protein kinase (MAPK) assays. KEY RESULTS: Depletion of arrestin3, but not arrestin2 enhanced and prolonged H(1) receptor-stimulated Ca(2+) responses, whilst depletion of either arrestin increased oxytocin receptor responses. Arrestin3 depletion decreased H(1) receptor desensitization, whilst removal of either arrestin isoform was equally effective in preventing oxytocin receptor desensitization. Following arrestin3 depletion oxytocin-induced phospho-extracellular signal-regulated kinase1/2 signals were diminished and histamine-stimulated signals virtually absent, whereas depletion of arrestin2 augmented extracellular signal-regulated kinase1/2 responses to each agonist. Conversely, depletion of arrestin3 enhanced p38 signals to each agonist, whilst arrestin2 suppression increased oxytocin-, but not histamine-induced p38 MAPK responses. CONCLUSIONS AND IMPLICATIONS: Arrestin proteins are key regulators of H(1) and oxytocin receptor desensitization, and play integral roles mediating uterotonin-stimulated MAPK-signalling. These data provide insights into the in situ regulation of these receptor subtypes and may inform pathophysiological functioning in preterm labour.

Characterization of the endocannabinoid system, CB(1) receptor signalling and desensitization in human myometrium.

BACKGROUND AND PURPOSE: The endocannabinoid plays vital roles in several aspects of reproduction, including gametogenesis, fertilization and parturition. However, little is known regarding the presence or role of the endocannabinoid system in myometrial function. Here the presence of the endocannabinoid system and signalling properties of cannabinoid receptors were characterized. EXPERIMENTAL APPROACH: Components of the endocannabinoid system were identified using qRT-PCR, immunohistochemical, immunoblotting and radioligand binding experiments. Cannabinoid receptor signalling pathways were characterized using standard MAPK and second messenger assays. KEY RESULTS: Primary myometrium expresses the endocannabinoid synthesizing enzyme N-acyl-phosphatidyl ethanolamine-specific phospholipase D, endocannabinoid degrading enzyme fatty acid amide hydrolase and cannabinoid CB(1) , but not CB(2) receptors or transient receptor potential vanilloid-type-1 channels. The CB(1) receptor ligand anandamide caused a Gα(i/o) -dependent inhibition of adenylate cyclase reducing intracellular cAMP levels, and Gα(i/o) , phosphoinositide-3-kinase, Src-kinase-dependent ERK activation. CB(1) receptor-generated signals declined following continual anandamide stimulation, possibly due to ligand metabolism since free anandamide concentrations declined during the experiment from 2.5 µM initially, to 500 nM after >30 min. However, identical loss of CB(1) receptor responsiveness occurred in the presence of the metabolically stable derivative methanandamide. Moreover, RNAi-mediated depletion of arrestin3 (a negative regulator of receptor signalling) prevented loss of CB(1) receptor activity, enhancing and prolonging ERK signals. CONCLUSIONS AND IMPLICATIONS: The myometrium has the capacity to synthesize, respond to and degrade endocannabinoids. Furthermore, reduced CB(1) receptor responsiveness occurs as a consequence of receptor desensitization, not agonist depletion and we identify a key role for arrestin3 in this process.

Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

Characterization of anandamide-stimulated cannabinoid receptor signaling in human ULTR myometrial smooth muscle cells.

Accumulating evidence highlights the importance of the endocannabinoid anandamide (AEA) as a key mediator in reproductive physiology. Current data suggest potential roles for AEA in gametogenesis, fertilization, and parturition. AEA exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of AEA signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2, and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling after stimulation with AEA. Radioligand binding analysis revealed a total CB receptor expression of 76 +/- 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. AEA caused Galpha(i/o)-dependent inhibition of adenylate cyclase to reduce intracellular cAMP levels. In addition, AEA caused a 2.5- to 3.5-fold increase in ERK activation, which was ablated by inhibition of Galpha(i/o), phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca(2+)/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings, the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial AEA signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.