Human Oxytocin Receptor Research Articles

The design of orally bioavailable 2, 5‐diketopiperazine oxytocin antagonists: from concept to clinical candidate for premature labor

A short, efficient and highly stereoselective synthesis has been developed for a series of 6-indanyl-3-alkyl-7-aryl/heterocyclic-(3R, 6R, 7R)-2, 5-diketopiperazine amides that are potent and selective oxytocin (OT) antagonists. Property-based design using an estimate of human oral absorption enabled focus to be directed to those templates with the greatest chance of delivering high bioavailability in humans. This led to the 2', 4'-difluorophenyl dimethylamide 40, a highly potent (pK(i) =9.2) and selective OT antagonist (>1,000-fold selectivity vs. the human vasopressin receptors V1a, V2, and V1b) with good oral bioavailability (>50%) in the rat and dog. Increased solubility and an improved Cyp450 profile was achieved with a range of 2'-substituted 7-(1',3'-oxazol-4'-yl)-(3R,6R,7R)-2,5-diketopiperazine amides and branching at the α-carbon of the 3-butyl group led to a superior rat pharmacokinetic profile that resulted in the discovery of the 2'-methyl-1',3'-oxazol-4'-yl morpholine amide derivative 74 GSK221149A (Retosiban), which had the best oral exposure and bioavailability in the rat. Retosiban has sub-nanomolar affinity (K(i) =0.65 nM) for the oxytocin receptor with >1400-fold selectivity over the closely related vasopressin receptors. It has good solubility, low protein binding and has a good Cyp450 profile with no significant inhibition IC(50) >100 µM. Retosiban is >15-fold more potent at the human oxytocin receptor than atosiban (a marketed i.v, peptide OT antagonist) and it has been shown to be an effective tocolytic by i.v. and by oral administration in rats, and was selected for progression as a potential clinical candidate for preterm labor.

Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor

Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu 1.35 in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu 1.35 is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu 1.35 by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu 1.35 projects into the receptor's central binding crevice and provides agonist-specific contacts not utilized by antagonists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist–receptor and antagonist–receptor binding modes.

Modulating Oxytocin Activity and Plasma Stability by Disulfide Bond Engineering

Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K(i) = 1.5 nM, and EC₅₀ = 32 nM), selenylsulfide (K(i) = 0.29/0.72 nM, and EC₅₀ = 2.6/154 nM), diselenide (K(i) = 11.8 nM, and EC₅₀ = 18 nM), and ditelluride analogues (K(i) = 7.6 nM, and EC₅₀ = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K(i) = 0.79 nM, and EC₅₀ = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t(½) = 12 h). By contrast, the all-D-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

Association between neuropeptide oxytocin and male infertility.

To investigate the relationship between oxytocin (OT) and male infertility, serum OT baseline concentration and oxytocin receptor (OTR) gene expression in fertile and infertile men were investigated. Twenty obstructive azoospermia patients, twenty five idiopathic asthenozoospermia patients, twenty idiopathic oligozoospermia patients and twenty healthy subjects were taken into consideration. Serum OT baseline concentration was determined by radioimmunoassay. Moreover, serum concentration of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were determined by chemoluminescence to evaluate the correlation with OT. OTR gene promotor and OTR mRNA expressions were determined by polymerase chain reaction and reverse transcriptase-polymerase chain reaction, respectively. OTR protein expression was also performed by Western Blot. Serum OT baseline concentrations in infertile groups were significantly higher than in fertile group (F0.05/2(2,82) = 8.29, p < 0.001). Serum baseline concentration of OT was not correlated with that of LH, FSH and T. There was no significant difference in gene sequences of OTR gene promotor and OTR mRNA when comparing infertile patients with fertile. Human OTR was in the form of oligomers and monomers, and the oligomers were in the majority containing tetramers and hexamers. Monomer expression was significantly higher in idiopathic asthenozoospermia and idiopathic oligozoospermia than that in obstructive azoospermia and control group (F0.05/2(2,82) = 115.50, p < 0.001). There was no significant difference in oligomer expression between different groups, but 20% of idiopathic asthenozoospermia cases showed a decrease. Significantly different OT baseline concentrations and OTR expressions between fertile and infertile men strongly suggest that OT/OTR system is likely to be linked with male infertility, providing new insights into the pathogenesis and treatment of male infertility.

Intracellular Calcium Elevation Induced by Extracellular Application of Cyclic‐ADP‐Ribose or Oxytocin is Temperature‐Sensitive in Rodent NG108‐15 Neuronal Cells with or without Exogenous Expression of Human Oxytocin Receptors

ADP-ribosyl cyclase and/or CD38 are activated after oxytocin receptor stimulation in the hypothalamus and pituitary in adult mice, leading to facilitation of oxytocin secretion. Although cyclic adenosine 5'-diphosphoribose (cADPR) primarily acts as an intracellular second messenger, it has been suggested that extracellular cADPR stimulates intracellular ryanodine receptors after internalisation via the nucleotide-transporting capacity of CD38 in fibroblasts and astrocytes. However, little is known about whether extracellular cADPR activates neurones. To address this question, we used a model neuronal cell line, NG108-15 mouse neuroblastoma x rat glioma hybrid cells possessing CD38 but not oxytocin receptors, and measured cytosolic free calcium concentrations ([Ca(2+)](i)). Extracellular application of cADPR to NG108-15 cells elevated [Ca(2+)](i) at 35 degrees C. The elevation was significantly enhanced when measured at 40 degrees C. The cADPR and heat-induced [Ca(2+)](i) increase were blocked under extracellular Ca(2+)-free conditions and by 2-aminoethoxydiphenyl borate, an antagonist of melastatin-related transient receptor potential channel 2 (TRPM2) cation channels. Reverse transcriptation-polymerase chain reaction analyses indicated that TRPM2 channels were expressed in NG108-15 cells. Application of oxytocin elevated [Ca(2+)](i) in NG108-15 cells transformed to transiently express cloned human oxytocin receptors. The oxytocin-induced [Ca(2+)](i) response was also enhanced by heat. These results indicate that the extracellular application of cADPR, together with heat, activates cation influx downstream of oxytocin receptor signalling in NG108-15 neuronal cells, and suggest the possible involvement of TRPM2 channels in oxytocin release in the mammalian brain.

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors.

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Galpha(q) protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.

Evidence for the involvement of genetic variation in the oxytocin receptor gene (OXTR) in the etiology of autistic disorders on high‐functioning level

An increasing number of animal studies advert to a substantial role of the neuropeptide oxytocin in the regulation of social attachment and affiliation. Furthermore, animal studies showed anxiety and stress-reduced effects of oxytocin. First human studies confirm these findings in animal studies and implicate a crucial role of oxytocin in human social attachment behavior and in social interactions. Thus, the oxytocin system might be involved in the impairment of social interaction and attachment in autism spectrum disorders (ASD). The human oxytocin receptor gene (OXTR) represents a plausible candidate gene for the etiology of ASD. To analyze whether genetic variants in the OXTR gene are associated with ASD we performed family-based single-marker and haplotype association analyses with 22 single nucleotide polymorphisms (SNPs) in the OXTR and its 5' region in 100 families with autistic disorders on high-functioning level (Asperger syndrome (AS), high-functioning autism (HFA), and atypical autism (AA)). Single-marker and haplotype association analyses revealed nominally significant associations of one single SNP and one haplotype with autism, respectively. Furthermore, employing a "reverse phenotyping" approach, patients carrying the haplotype associated with autism showed nominally significant impairments in comparison to noncarriers of the haplotype in items of the Autism Diagnostic Interview-Revised algorithm describing aspects of social interaction and communication. In conclusion, our results implicate that genetic variation in the OXTR gene might be relevant in the etiology of autism on high-functioning level.

In position 7 l- and d-Tic-substituted oxytocin and deamino oxytocin: NMR study and conformational insights

Incorporation of L- or D-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa(1)]OT) resulted in four analogues, [L-Tic(7)]OT (1), [D-Tic(7)]OT (2), [Mpa(1),L-Tic(7)]OT (3) and [Mpa(1),D-Tic(7)]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799-806, 2007). Their NMR study (NOESY, TOCSY, (1)H-(13)C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC(50) value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of D analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys(6)-D-Tic(7) bond is supported by observation of two sets of cross-peaks for (1)H and (13)C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in (1)H-(13)C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.

Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: structure activity study

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of L- or D-beta-(2-thienyl)-alanine [L- or D-Thi], or L- or D-3-Pyridylalanine [L- or D-3-Pal]) were combined with D-tyrosine(OEthyl) [D-Tyr(Et)] or D-1-naphthylalanine [D-1-Nal] in position 2 and beta-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having alpha-aminoisobutyric acid [Aib] or D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (D-Tic) or diethylglycine (Deg) in position 9 and D-Tyr(Et) or D-1-Nal or D-Tic in position 2 and Mpa or Pen (beta beta-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and D-Tyr(Et) or D-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa(1), D-Tyr(Et)(2), Deg(9)]OT (pA(2) = 8.68 +/- 0.26); this analogue was also selective.

Intracellular trafficking of the human oxytocin receptor: evidence of receptor recycling via a Rab4/Rab5 “short cycle”

As in the case of most G protein-coupled receptors, agonist stimulation of human oxytocin receptors (OTRs) leads to desensitization and internalization; however, little is known about the subsequent intracellular OTR trafficking, which is crucial for reestablishing agonist responsiveness. We examined receptor resensitization by first using HEK293T cells stably expressing human OTRs. Upon agonist activation, the receptors were almost completely sequestered inside intracellular compartments that were not labeled by lysosomal markers, thus indicating that the internalized receptors were not sorted to these degrading organelles. Binding and fluorescence assays showed that almost 85% of the receptors had returned to the cell surface after 4 h, by which time cell responsiveness to the agonist was also completely restored, as shown by measuring phospholipase C activation. Similar results were also obtained in the presence of cycloheximide, thus indicating that receptor recycling and not de novo receptor synthesis was responsible for the resensitization. Notably, very similar internalization and recycling kinetics were observed in endogenous OTRs expressed on myometrial cells. We also investigated the role of beta-arrestin2 in OTR recycling as these receptors have been previously classified as slowly or nonrecycling receptors on the basis of their stable association with this interacting protein. Our data suggest that the stable OTR/beta-arrestin2 interaction plays an important role in determining the rate of recycling of human OTRs, but does not determine the fate of endocytosed receptors. Subsequent investigations of receptor recycling pathways showed that OTRs localize in vesicles containing the Rab5 and Rab4 small GTPases (markers of the "short cycle"), whereas there was no colocalization with Rab11 (a marker of the "long cycle") or Rab7 (a marker of vesicles directed to endosomal/lysosomal compartments). Taken together, these data indicate that OTRs are capable of very efficient and complete resensitization due to receptor recycling via the short cycle.

Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.

Oxytocin analogues with amide groups substituted by tetrazole groups in position 4, 5 or 9.

Eleven oxytocin analogues substituted in position 4, 5 or 9 by tetrazole analogues of amino acids were prepared using solid-phase peptide synthesis method and tested for rat uterotonic in vitro and pressor activities, as well as for their affinity to human oxytocin receptor. The tetrazolic group has been used as a bioisosteric substitution of carboxylic, ester or amide groups in structure-activity relationship studies of biologically active compounds. Replacement of the amide groups of Gln(4) and Asn(5) in oxytocin by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities. Oxytocin analogues in which the glycine amide residue in position 9 was substituted by tetrazole analogues of glycine had diminished activities as well. The analysis of differences in rat uterotonic activity and in the affinity to human oxytocin receptors of analogues containing either an acidic 5-substituted tetrazolic group or a neutral 1,5- or 2,5-tetrazole nucleus makes it possible to draw some new conclusions concerning the role of the amide group of amino acids in positions 4, 5 and 9 of oxytocin for its activity. The data suggest that the interaction of the side chain of Gln(4) with the oxytocin receptor is influenced mainly by electronic effects and the hydrogen bonding capacity of the amide group. Steric effects of the side chain are minor. Substitution of Asn(5) by its tetrazole derivative gave an analogue of very low activity. The result suggests that in the interaction between the amide group of Asn(5) and the binding sites of oxytocic receptor hydrogen bonds are of less importance than the spatial requirements for this group.

Modeling the human oxytocin receptor for drug discovery efforts

The oxytocin receptor belongs to class A receptors within the great family of G protein-coupled receptors. The endogenous ligand oxytocin is a nonapeptide hormone that induces the uterine contractions at parturition and is used to induce the labor. The peptide oxytocin and, even more, its non-peptide antagonist, could be valuable tools in tocolysis. The knowledge of the three-dimensional structure of the oxytocin receptor and the determination of the main interaction points between the receptor and the ligands may help to develop selective oxytocin agonists and antagonist. This review summarizes the knowledge about the mapping of the binding domain of the oxytocin receptor and the efforts in the field of molecular modeling studies related to oxytocin receptor–ligand interactions.

Synthesis and biological activity of oxytocin analogues containing conformationally-restricted residues in position 7

We report the solid-phase synthesis and some pharmacological properties of twenty oxytocin (OT) analogues. Basic modifications at position 7 (introduction of α-aminoisobutyric acid [Aib], l- or d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [ l/ d-Tic], l- α- t-butylglycine [Gly(Bu t )] and pipecolic acid [Pip]) were combined with d-Tyr(Et) 2, l/ d-( pEt)Phe 2, d-Tic 2, and Mpa 1 modifications and their various combinations in a total of 14 analogues. Additionally, two analogues having one more modification in position 3, i.e. Gly(Bu t ), and three analogues having glycine in position 9 substituted by d-Tic or Aib, were prepared. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest antioxytocic activity was [Mpa 1, d-Tyr(Et) 2, d-Tic 7, Aib 9]OT having pA 2 = 8.31 ± 0.19; this analogue was also selective.

Cigarette smoke extract enhances oxytocin-induced rhythmic contractions of rat and human preterm myometrium

Although smoking during pregnancy is a major risk factor for preterm delivery, the underlying mechanism by which smoking stimulates uterine contractions is still poorly understood. In the present study, we tried to clarify the effects of smoking on myometrial contractility induced by oxytocin (OT) using cigarette smoke extract (CSE). Myometrial strips, which were taken from the rat on day 16 of pregnancy, and from human preterm and term delivery groups, were incubated overnight with several doses of CSE at 37 degrees C under non-hormonal conditions. The uterine contractile sensitivity and activity (force and frequency) upon exposure to OT were investigated. Furthermore, the expression levels of oxytocin receptor (OTR) mRNA in the myometrial strips were investigated by real-time PCR. Contractile sensitivity to OT in the rat CSE (10(-7) pieces/ml) group was found to be significantly higher than in the control group (P < 0.05). Contractile activity did not differ between the CSE and control groups. The expression levels of rat OTR mRNA in the CSE (10(-7) pieces/ml) group were significantly higher than in the control group (P < 0.01). Similarly, in preterm myometrial strips, the expression levels of human OTR mRNA in the CSE (10(-7) pieces/ml) group were significantly higher than in the control group (P < 0.05). These findings suggest that CSE directly increases the contractile sensitivity of preterm myometrium in response to OT by upregulating the expression of OTR mRNA and thereby increases the risk of preterm delivery in women, who smoke during pregnancy.

Receptor-based QSAR studies of non-peptide human oxytocin receptor antagonists

In the present study, QSAR calculations were performed on the receptor-based alignment of 58 non-peptide human oxytocin receptor antagonists. With the aid of different scoring functions (AutoDock 3.05 built-in and X-Score 1.2) the evolved receptor–ligand complexes were characterized. By means of various datasets it was confirmed that the scoring functions were not capable to predict the biological activity correctly in compounds containing a rigid derivative in the variable region. To improve the p K i prediction 3D-QSAR calculation was performed. The regions related to the biological activity were determined by using cross-validated r 2( q 2)-guided region selection ( q 2-GRS) method. The predictive power of the CoMFA model [ r pred 2 = 0.89 , q 2(LMO, five groups) = 0.695 ± 0.034] allowed prediction of the biological activities of newly synthesized compounds and confirmed the receptor-based alignment.

Effects of cholesterol manipulation on the signaling of the human oxytocin receptor

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-beta-cyclodextrin (MbetaCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MbetaCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MbetaCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.

G-protein coupling specificity of the human oxytocin receptor

G-protein coupling specificity of the human oxytocin receptor

Regulation of the Human Oxytocin Receptor by Nuclear Factor-κB and CCAAT/Enhancer-Binding Protein-β

Increased myometrial sensitivity to oxytocin at term is mediated through increased oxytocin receptor (OTR) expression. OTR promoter contains putative transcription factor-binding sites for activating protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP), and nuclear factor-kappaB (NF-kappaB), which may be activated by IL-1beta, whose concentrations increase with labor. The objective of this study was to examine the effect of IL-1beta on OTR expression and the roles of AP-1, C/EBP, and NF-kappaB in OTR promoter function. IL-1beta induces an increase in OTR mRNA concentrations and OTR ligand binding in myometrial cells, which is maximal at 4 h and decreased after 20 h. IL-1beta activates the transcription factors AP-1 C/EBPbeta, and NF-kappaB. Using computer-based analysis and EMSA studies, we have identified three AP-1, nine C/EBP, and three NF-kappaB DNA-binding sites in the OTR promoter. In transient transfection studies, OTR promoter activity was increased by C/EBPbeta and NF-kappaB, but not by AP-1. C/EBPbeta and NF-kappaB together had a synergistic action in the induction of OTR promoter activity. Site-directed mutagenesis of each individual C/EBP and NF-kappaB site had no effect on the ability of C/EBPbeta, NF-kappaB, or their combination to activate OTR promoter. However, mutation of both NF-kappaB sites inhibited promoter activation by NF-kappaB alone, but not that by the combination of C/EBPbeta and NF-kappaB. Deletion studies showed that a region between -851 and -656 of the OTR confers responsiveness to the combination of C/EBPbeta and NF-kappaB. IL-1beta has a biphasic effect on OTR expression in myometrial cells, and C/EBP and NF-kappaB play synergistic roles in OTR promoter activation.