RESUMEN

The hormone oxytocin (OT) has pleiotropic activities both in the central nervous system as well as in peripheral tissues, including uterotonic effects on the myometrium during parturition. OT effects are mediated by a single transmembrane receptor, belonging to the GPCR (G protein-coupled receptor) superfamily and coupled primarily to Gq- and Gi-containing heterotrimeric G proteins. Upon receptor stimulation, one well-studied downstream effect is activation of the ERK1/2 MAP (mitogen-activated protein) kinase, and studies have shown that induction of COX-2 by OT in the myometrium required ERK1/2 activity. Many studies investigating the role of ERK1/2 in myometrial tissue were based on the use of chemical inhibitors that, to varying degrees, also inhibited ERK5/MAPK7. Here we report that OT activates ERK5 in a human myometrial cell line in a dose- and time-dependent manner through the activation of Gi/o heterotrimers. Using complementary approaches, we demonstrate that OT-induced COX-2 induction and the concomitant release of PGF2α into the media are primarily ERK5-dependent and to a much lesser extent ERK1/2-dependent. Moreover, in contrast to ERK1/2 activation, ERK5 activation is downstream of Gi/o activation. Here, we also found that ERK5 impacted both basal and to a lesser extent, OT-mediated myometrial cell contraction in vitro. Finally, tracking both ERK1/2 and ERK5 activity during different stages of gestation in rat myometrium, we showed that they followed distinct patterns starting at the onset of labor corresponding to the highest COX-2 expression levels. Overall, our results reveal an important, hitherto unrecognized role for ERK5 in myometrial cell contraction involving induction of COX-2. This novel pathway is likely to play an important role in supporting uterine contractions during parturition.

Asunto(s)

Miometrio , Oxitocina , Animales , Femenino , Proteína Quinasa 7 Activada por Mitógenos , Miometrio/metabolismo , Oxitocina/metabolismo , Embarazo , Ratas , Receptores de Oxitocina/metabolismo , Transducción de Señal , Contracción Uterina

Asunto(s)

Prolactina , Investigadores , Canadá , Historia del Siglo XX , Historia del Siglo XXI , Humanos

RESUMEN

High-throughput molecular biology studies, such as microarray assays of gene expression, two-hybrid experiments for detecting protein interactions, or ChIP-Seq experiments for transcription factor binding, often result in an "interesting" set of genes - say, genes that are co-expressed or bound by the same factor. One way of understanding the biological meaning of such a set is to consider what processes or functions, as defined in an ontology, are over-represented (enriched) or under-represented (depleted) among genes in the set. Usually, the significance of enrichment or depletion scores is based on simple statistical models and on the membership of genes in different classifications. We consider the more general problem of computing p-values for arbitrary integer additive statistics, or weighted membership functions. Such membership functions can be used to represent, for example, prior knowledge on the role of certain genes or classifications, differential importance of different classifications or genes to the experimenter, hierarchical relationships between classifications, or different degrees of interestingness or evidence for specific genes. We describe a generic dynamic programming algorithm that can compute exact p-values for arbitrary integer additive statistics. We also describe several optimizations for important special cases, which can provide orders-of-magnitude speed up in the computations. We apply our methods to datasets describing oxidative phosphorylation and parturition and compare p-values based on computations of several different statistics for measuring enrichment. We find major differences between p-values resulting from these statistics, and that some statistics recover "gold standard" annotations of the data better than others. Our work establishes a theoretical and algorithmic basis for far richer notions of enrichment or depletion of gene sets with respect to gene ontologies than has previously been available.

RESUMEN

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.

Asunto(s)

Hormona Liberadora de Corticotropina/fisiología , Multimerización de Proteína , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de Vasopresinas/metabolismo , Vasopresinas/fisiología , Glándulas Suprarrenales/citología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafines/metabolismo , Hormona Liberadora de Corticotropina/farmacología , AMP Cíclico/metabolismo , Activación Enzimática , Estrenos/farmacología , Células HEK293 , Humanos , Hidrocarburos Halogenados/farmacología , Indoles/farmacología , Inositol Polifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Cultivo Primario de Células , Unión Proteica , Pirrolidinas/farmacología , Pirrolidinonas/farmacología , Receptor Cross-Talk , Receptores de Hormona Liberadora de Corticotropina/agonistas , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Vasopresinas/agonistas , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/metabolismo , Sistemas de Mensajero Secundario , Tiazinas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Vasopresinas/farmacología

RESUMEN

The oxytocin receptor (OTR) and the ß(2)-adrenergic receptor (ß(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and ß(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/ß(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and ß(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the ß(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third ß(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, ß(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and ß(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and ß(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.

Asunto(s)

Activación Enzimática , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Multimerización de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Oxitocina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Regulación Alostérica , Atenolol/farmacología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Humanos , Inmunoprecipitación , Isoproterenol/farmacología , Sistema de Señalización de MAP Quinasas , Miometrio/citología , Oxitocina/farmacología , Oxitocina/fisiología , Fosforilación , Propranolol/farmacología , Unión Proteica , Receptores de Oxitocina/agonistas , Receptores de Oxitocina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Timolol/farmacología , Vasotocina/análogos & derivados , Vasotocina/farmacología

RESUMEN

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled ß(2)-adrenergic receptor (ß(2)AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and ß(2)AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and ß(2)AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in ß(2)AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the ß(2)AR alone or co-transfected with the OTR. Using this approach, we found that ß(2)AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that ß(2)AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel ß(2)AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors.

Asunto(s)

Activación Enzimática , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miometrio/citología , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Oxitocina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Androstadienos/farmacología , Carbazoles/farmacología , Línea Celular , Receptores ErbB/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica , Humanos , Indoles , Isoproterenol/farmacología , Cinética , Sistema de Señalización de MAP Quinasas , Maleimidas , Oxitocina/farmacología , Oxitocina/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Receptores de Oxitocina/agonistas , Proteínas Recombinantes/metabolismo , Wortmanina

RESUMEN

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein-coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo-pituitary-adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca²(+) in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

Asunto(s)

Encéfalo/metabolismo , Trastornos Mentales/tratamiento farmacológico , Oxitocina/metabolismo , Afecto/fisiología , Analgésicos/uso terapéutico , Animales , Diabetes Mellitus/tratamiento farmacológico , Humanos , Neoplasias/tratamiento farmacológico , Osteoartritis/tratamiento farmacológico , Receptores de Oxitocina/agonistas , Receptores de Oxitocina/antagonistas & inhibidores , Receptores de Oxitocina/metabolismo , Disfunciones Sexuales Fisiológicas/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Conducta Social

RESUMEN

The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.

Asunto(s)

Dinoprost/metabolismo , Parto/efectos de los fármacos , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/tratamiento farmacológico , Péptidos/síntesis química , Péptidos/uso terapéutico , Embarazo , Proteína Quinasa C/metabolismo

RESUMEN

The great diversity of the expression sites and proposed function of the oxytocin (OXT) receptor (OXTR) is paralleled by a diversity of its signalling pathways, many of which have still remained unexplored. We have used different approaches to discover novel pathways. By means of a phosphoproteomics approach, we have detected several distinct OXT-induced changes in tyrosine as well as threonine phosphorylation states of intracellular protein in myometrial cells. The most prominent change involved dephosphorylation of a 95-kDa phosphothreonine moiety. By N-terminal amino acid microsequence analysis, this moiety was shown to correspond to eukaryotic translation factor eEF2. This protein is a key regulator of protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. These findings define a novel mechanism by which OXT assumes a so far unrecognized trophic function. We next elucidated the intracellular pathway(s) involved. We found that this effect is not mediated by any of the known pathways known to induce eEF2 dephosphorylation (mTOR, ERK1/2 or p38) but by protein kinase C. Consistent with this idea, we also found that direct stimulation of protein kinase C with a phorbol ester induced eEF2 dephosphorylation in myometrial cells. Using phosphoERK antibodies, we discovered by Western blotting that OXT induced phosphorylation of a higher molecular weight ERK-related protein. We were able to show that this band corresponded to "big MAP kinase1" or ERK5. ERK5 is part of a distinct MAPK cascade and promotes expression of the myosin light chain gene and plays an obligatory role in muscle cell development and differentiation. The role of ERK5 in myometrium has remained unexplored, but it is likely to represent an important novel pathway mediating OXT's effects on smooth muscle function. Further elucidation of these novel signalling pathways will have significant relevance for the development of novel pathway-specific OXTR agonists and antagonists.

Asunto(s)

Receptores de Oxitocina/fisiología , Transducción de Señal/fisiología , Animales , Quinasa del Factor 2 de Elongación/metabolismo , Femenino , Humanos , Lactancia , Leche/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Contracción Muscular/fisiología , Miometrio/fisiología , Fosforilación , Prolactina/metabolismo , Conducta Sexual , Conducta Sexual Animal , Conducta Social , Contracción Uterina/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

OBJECTIVE: The objective of the study was to investigate changes in the expression of angiogenesis-related genes during the common terminal pathway of parturition including spontaneous labor at term, as well as preterm labor (PTL), induced by either bacteria or ovariectomy. STUDY DESIGN: Preterm pregnant mice (14.5 days of gestation) were treated with the following: (1) intrauterine injection of media; (2) intrauterine injection of heat-inactivated Escherichia coli; (3) ovariectomy; and (4) sham operation. Tissues from mice at term (19.5 days of gestation) were collected at term not in labor, term in labor, and 12 hours postpartum. Angiogenesis-related gene expression levels were quantitated by the measurement of specific mRNAs in uterine tissue by RT-qPCR and analyzed by repeated-measures analysis of variance. RESULTS: The following results were found: (1) microarray analysis of the uterine transcriptome indicated an enrichment for the gene ontology category of angiogenesis in bacteria-induced PTL samples (P < or = .093); (2) several genes related to angiogenesis demonstrated significantly increased expression in samples in either term spontaneous labor or preterm labor; and (3) qRT-PCR measurements demonstrated that spontaneous term labor and preterm labor induced by either bacteria or ovariectomy all substantially increased the expression of multiple angiogenesis-related genes (P < or = .0003; Angpt2, Ctgf, Cyr61, Dscr1, Pgf, Serpine1, Thbs1, and Wisp 1). CONCLUSION: Spontaneous labor at term, as well as pathologically induced preterm labor, all result in greatly increased expression of angiogenesis-related genes in the uterus.

Asunto(s)

Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Neovascularización Fisiológica/fisiología , Parto/fisiología , Útero/metabolismo , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas , Animales , Proteínas CCN de Señalización Intercelular , Factor de Crecimiento del Tejido Conjuntivo , Proteína 61 Rica en Cisteína , Femenino , Proteínas Inmediatas-Precoces/fisiología , Ratones , Modelos Animales , Neovascularización Fisiológica/genética , Trabajo de Parto Prematuro/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas/fisiología , Ovariectomía , Parto/genética , Parto/metabolismo , Periodo Posparto/fisiología , Embarazo , Proteínas Proto-Oncogénicas , Trombospondina 1/fisiología , Regulación hacia Arriba/fisiología

RESUMEN

The hypothalamic post-translational processing of oxytocin (OT)-neurophysin precursor involves the formation of C-terminally extended OT forms (OT-X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT-X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor-mediated cardiomyogenic action of OT-Gly-Lys-Arg (OT-GKR). To obtain further insight into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing the OT-GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT-GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT-GKR/EGFP-expressing embryoid bodies and elevated expression of GATA-4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT-GKR on ES cell-derived cardiomyogenesis. Furthermore, patch-clamp experiments demonstrated induction of ventricular phenotypes in OT-GKR/EGFP-transfected and in OT-GKR-treated cardiomyocytes. Increased connexin 43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.

Asunto(s)

Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Corazón/embriología , Miocitos Cardíacos/citología , Oxitocina/metabolismo , Animales , Western Blotting , Células Cultivadas , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

The nonapeptide oxytocin (OT) mediates a wide spectrum of biological action, many of them related to reproduction. Recently, we have shown that OT exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eukaryotic elongation factor 2 (eEF2). The present study was designed to elucidate the mechanisms underlying this novel action of OT in the well-characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mammalian target of rapamycin (mTOR), and the MAPKs ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in OT-mediated eEF2 dephosphorylation. Because the OT receptor is a G protein-coupled receptor linked to Galphaq, we tested the possibility that this OT action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of OT on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this OT action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester phorbol 12-myristate 13-acetate induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of OT on [(35)S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis.

Asunto(s)

Factor 2 Eucariótico de Iniciación/metabolismo , Miometrio/efectos de los fármacos , Oxitocina/farmacología , Proteína Quinasa C/fisiología , Células Cultivadas , Femenino , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Miometrio/metabolismo , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

We analyzed the response of uterine smooth muscle cells to interleukin-1beta (IL-1beta). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1beta. In total, we identified 198 known genes whose mRNA levels are significantly modulated (> 2.0-fold change) following IL-1beta exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1beta includes transcription factors and inflammatory response genes such as nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFkappaB), pentraxin-related gene (PTX3), and tumor necrosis factor alpha-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1beta elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.

Asunto(s)

Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/farmacología , Miocitos del Músculo Liso/inmunología , Miometrio/inmunología , Trabajo de Parto Prematuro/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Northern Blotting/métodos , Línea Celular , Femenino , Humanos , Immunoblotting/métodos , Contracción Miocárdica , Miocitos del Músculo Liso/efectos de los fármacos , Miometrio/efectos de los fármacos , Embarazo , Estimulación Química

RESUMEN

One of the classical biological actions mediated by the posterior pituitary hormone oxytocin (OT) is contraction of the uterus at parturition. Moreover, premature activation of the OT system is thought to contribute to preterm labor, a major clinical problem in obstetrical practice. However, the molecular mechanisms linking activation of the OT receptor (OTR) to myometrial contractions are not fully understood. Here, we describe an in vitro system that should serve as a useful tool to study this question at a cellular level. The system consists of a collagen lattice contraction assay and two different human myometrial cell lines: a cell clone from a telomerase-immortalized human myometrial cell population (hTERT-C3) as well as a cell line derived from a primary culture of human myometrial cells (M11). Using this approach, we observed that 1 nM OT promoted an almost maximal effect on cell contraction in both cell lines tested. Furthermore, this dose-dependent, OT-induced contraction was antagonized by the specific OTR antagonist d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)(9)]OVT as well as the clinically used antagonist atosiban. This cell line-based contraction assay enables the application of molecular tools aimed at suppressing or overexpressing specific genes. It is also amenable to high-throughput testing approaches. Therefore, this system represents a powerful and improved experimental model that should facilitate the study of the molecular signal transduction pathways involved in the uterotonic actions of OT.

Asunto(s)

Miometrio/efectos de los fármacos , Oxitocina/farmacología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Modelos Biológicos , Miometrio/citología , Oxitocina/agonistas , Unión Proteica , Receptores de Oxitocina/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Contracción Uterina/efectos de los fármacos , Vasotocina/análogos & derivados , Vasotocina/farmacología

RESUMEN

OBJECTIVE: The purpose of this study was to identify changes in gene expression that are associated with preterm labor induced by either bacteria or ovariectomy. STUDY DESIGN: Pregnant mice (14.5 days of gestation) were allocated to: (1) intrauterine injection of heat-inactivated Escherichia coli; (2) media alone; (3) ovariectomy; or (4) sham operation. The uterine transcriptome was studied with photolithographic, very short oligonucleotide-based microarrays, and arachidonate metabolism genes were assayed with quantitative reverse transcriptase-polymerase chain reaction. Significance was determined by analysis of variance. RESULTS: Microarray-based gene expression changes in the arachidonate metabolism pathway are associated globally with bacteria-induced preterm labor (P < or = .0031) and ovariectomy-induced preterm labor (P < or = .00036). Quantitative real-time reverse transcriptase-polymerase chain reaction measurements demonstrated that bacteria-induced preterm labor substantially increased the expression of genes involved in prostaglandin synthesis. In contrast, ovariectomy-induced preterm labor increased the expression of genes involved in lipoxin, leukotriene, and hydroxyeicosatetraenoic acid synthesis. CONCLUSION: Bacteria-induced and ovariectomy-induced preterm labor each express a different balance of genes that are required for the synthesis of prostaglandins, lipoxins, leukotrienes, and hydroxyeicosatetraenoic acids.

Asunto(s)

Ácido Araquidónico/metabolismo , Trabajo de Parto Prematuro/genética , Animales , Ácidos Carboxílicos/metabolismo , Análisis Discriminante , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/complicaciones , Femenino , Leucotrienos/metabolismo , Ratones , Trabajo de Parto Prematuro/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovariectomía , Embarazo , Análisis de Componente Principal , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (K(d)) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.

Asunto(s)

Miometrio/efectos de los fármacos , Miometrio/metabolismo , Oxitocina/farmacología , Factor 2 de Elongación Peptídica/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Células CHO , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Femenino , Expresión Génica , Humanos , Punto Isoeléctrico , Factor 2 de Elongación Peptídica/química , Fragmentos de Péptidos/química , Fosforilación , Fosfotreonina/inmunología , Ratas , Receptores de Oxitocina/genética , Análisis de Secuencia de Proteína , Treonina/metabolismo

RESUMEN

Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, beta-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and beta-arrestin, overexpression of GRK2 and beta-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and beta-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for beta-arrestin-mediated OTR desensitization.

Asunto(s)

Arrestinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Oxitocina/fisiología , Receptores de Oxitocina/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Transferencia de Energía , Femenino , Humanos , Mutación , Fosforilación , Unión Proteica , Receptores de Oxitocina/agonistas , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Quinasas de Receptores Adrenérgicos beta , beta-Arrestinas

RESUMEN

Novel sites of oxytocin receptor expression have recently been detected, including breast cancer cells, bone cells, myoblasts, cardiomyocytes and endothelial cells. These discoveries have greatly expanded the possible spectrum of oxytocin action beyond its classic role as an inducer of uterine contractions and milk ejection. Additional advances in the understanding of oxytocin receptor structure-function relationships, receptor trafficking and novel receptor-linked signaling cascades have made this receptor an attractive model for the study of G-protein-linked receptor function. Finally, the tocolytic efficiency of the oxytocin receptor antagonist atosiban, recently approved for clinical use in Europe, has opened new avenues for the prevention and treatment of preterm labor.

Asunto(s)

Receptores de Oxitocina/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Ligandos , Receptores de Oxitocina/biosíntesis , Receptores de Oxitocina/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

A fuller understanding of the process of parturition is needed in view of the current lack of efficient treatment for preterm labor. Using DNA microarrays, we have analyzed patterns of uterine gene expression at d 0 and 20 of pregnancy, at term in labor or not in labor, and at 1 d post partum. Of the 8740 genes analyzed, 562 genes undergoing significant changes were grouped into 5 distinct clusters, each containing many genes not previously known to be involved with uterine functions. Cluster 1 genes were up-regulated at labor and encompassed immune defense and immediate early response genes, including transcription factors NGFI-B/nurr77 and estrogen-responsive gene 1. Cluster 3 genes were acutely suppressed at labor and included extracellular matrix products and genes related to hormonal signaling, implying novel intrauterine mechanisms regulating intracellular cyclic GMP and local steroid hormone concentrations. At labor, more genes were suppressed than activated, indicating that, for the process of labor induction, gene suppression is at least equally important as the more extensively studied processes of gene activation. The study also points to the existence of novel uterine signaling pathways, including Wnt/frizzled and receptor activator of nuclear factor-kappaB (RANK) and its ligand, as well as the involvement of novel signaling molecules such as estrogen-responsive gene 1, decay-accelerating factor 1, and ebnerin. The present results provide the basis for further studies that will enlarge our knowledge of the mechanisms underlying labor and parturition under physiological and pathophysiological conditions.

Asunto(s)

Análisis de Secuencia por Matrices de Oligonucleótidos , Parto/genética , Útero/fisiología , Animales , Northern Blotting , Análisis por Conglomerados , Femenino , Genoma , Modelos Animales , Embarazo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa