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Query: UNIPROT:P01178 (oxytocin)
 15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endocrine factors involved in the transcriptional regulation of the oxytocin (OT) gene were investigated in heterologous expression systems. Plasmids having a 5'-flanking region of the rat OT gene (-363/+16) or the human OT gene (-382/+41) cloned in front of the firefly luciferase gene were co-transfected with an expression vector for the rat thyroid hormone receptor alpha in P19 embryonal carcinoma (EC) cells. Thyroid hormone (T3) stimulated the activity of the rat and human OT promoters about 10-fold. In MCF-7 breast tumor cells transfected with the human OT promoter-luciferase fusion gene, T3 stimulation through endogenous thyroid hormone receptors was about 5-fold. Co-transfection experiments in P19EC cells using 5' deletion mutants of the rat OT gene showed that thyroid hormone responsiveness was located in two regions, one located between nucleotides -195 and -172, the other between nucleotides -172 and -148. Each region accounted for about 3-fold T3 stimulation. Gel retardation analysis using extracts from HeLa cells over-producing the c-erbA/TR alpha protein showed specific binding to the -172/-148 element, while no binding occurred on the -195/-172 element. The -172/-148 element which contains the imperfect estrogen response element, GGTGACCTTGACC, has inverted as well as direct repeats of the TGACC motif. Mutagenesis of TGACC motifs separately reduced thyroid hormone responsiveness by about 50%. However, simultaneous mutation of two TGACC motifs abolished the responsiveness to T3 completely. There was no cooperativity between the activated thyroid hormone and estrogen receptors in transfected MCF-7 cells nor in thyroid hormone receptor and estrogen receptor co-transfected P19EC cells. Negative interactions between these two receptors were observed and gel retardation assays showed interaction between the two receptors proteins. It was shown in an in vivo experiment that treatment of rats with thyroid hormone increased hypothalamic OT mRNA levels, the pituitary OT content, as well as OT levels in blood. The results reveal thyroid hormone as a physiological regulator of OT gene expression, which stimulates OT promoter activity directly through interaction with a thyroid hormone-response element in the OT gene.
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PMID:Thyroid hormone regulates the oxytocin gene. 137 Dec 78

DNA sequences in the 5'-flanking region of rat and bovine oxytocin genes were examined for their capacity to confer estrogen responsiveness to their homologous promoters. In contrast to the 5'-flanking region of the rat oxytocin gene, upstream promoter sequences up to 3200 bp of the bovine gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene which were transfected in estrogen receptor expressing MCF-7 cells did not respond to estrogen. Testing 5'-deletion mutants of the rat upstream region linked to the luciferase gene in P19 embryocarcinoma cells co-transfected with an estrogen receptor expression plasmid showed that two regions each associated with approximately 15-fold stimulation of promoter activity were located between nucleotides -172 and -149 and between -148 and +16 in the rat gene. The former region contains the imperfect palindrome GGTGACCTTGACC which differs in one nucleotide from the estrogen response element (ERE) consensus. It is concluded that the corresponding motive CATAACCTTGACC of the bovine gene is not a functional ERE. Thus, the estrogen responsiveness of oxytocin genes is species-dependent.
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PMID:Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters. 199 97

The genes for the alpha subunit of inhibin and for the nonapeptide hormone oxytocin are both expressed in the granulosa cells of the ruminant follicle as well as in the Sertoli cells of the ruminant testis. Northern hybridization of mRNA from both ovary and testis indicate that in both gonads the expression of the two genes is inversely regulated. In the luteinizing granulosa cells, in vitro as in vivo, the alpha-inhibin gene is down-regulated when the oxytocin gene is up-regulated. In the Sertoli cells of the bull and sheep testis, the situation is similar, with the alpha-inhibin gene being up-regulated in the prepubertal gonad and down-regulated concomitantly with an up-regulation of the oxytocin gene in early puberty. The gene for the bovine alpha-inhibin subunit was cloned and characterized. Assessment of transcriptional initiation by primer extension and ribonuclease protection assays showed that several different sites were used in both granulosa cells and testis. Transient transfection of primary bovine granulosa cells with alpha-inhibin/luciferase gene constructs indicated that a major promoter element resided in the region -178 to -245 respective to the methionine start codon of translation, a region that contains a cAMP response element. The ability of forskolin to up-regulate the transcription of transfected gene constructs also depended on the integrity of this region. In contrast, transfection of TM4 cells led to transcriptional initiation from an unusual site in the alpha-inhibin gene and to a lack of forskolin regulation. Comparison of the alpha-inhibin and oxytocin genes indicates that although both can be up-regulated by FSH or by forskolin within the same cells, different mechanisms of signal transduction are involved to explain the temporal differences in expression. Together the results indicate that a differentiation step occurring in Sertoli cells at early puberty and in granulosa cells at luteinization involves comparable regulation of genes through the sequential action of different cAMP-linked transcription factors.
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PMID:Structure of the alpha-inhibin gene and its regulation in the ruminant gonad: inverse relationship to oxytocin gene expression. 814 57

By Northern blot analysis and in situ hybridization, we have determined that, at term, the rat uterine epithelium represents a major site of oxytocin (OT) gene expression. OT mRNA levels increase > 150-fold during pregnancy and, at term, exceed hypothalamic OT mRNA by a factor of 70. By cryoultramicroscopy, OT immunoreactivity was localized to transport vesicles in the apical compartment of uterine epithelial cells. Estrogens (E) act as a strong inducer of uterine OT gene expression in vivo, and this effect is potentiated 7-fold by concomitant progesterone (P) administration. We have also cloned the rat OT receptor (OTR) gene and developed a polymerase chain reaction (PCR)-based assay to measure OTR mRNA. Whereas OTR mRNA is strongly induced by E, P does not potentiate but slightly attenuates the E-induced rise. However, E-induced OT binding is completely reversed by concomitant P administration, suggesting an additional post-transcriptional effect of P. The mechanisms of E-induction of the uterine OT gene remain unclear, inasmuch as the OTR gene promoter does not contain a classical estrogen response element (ERE). Moreover, transfection analysis of a 3.1 kb OTR gene promoter fragment linked to a luciferase reporter gene indicates that promoter activity is induced 5-fold by calcium ionophore A23187 but not by E.
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PMID:Gonadal steroid regulation of oxytocin and oxytocin receptor gene expression. 871 94

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.
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PMID:Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor. 909 98

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.
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PMID:Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis. 1007 81

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including the initiation of labor and milk ejection. OTR messenger RNA levels are highly regulated, reaching the greatest concentration in the uterus at the end of gestation, and in the mammary gland during lactation. Factors directly effecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by characterizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region between -85 and -65 that was required for both basal and serum-induced expression in a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OTRs. This DNA region contains an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif. The specific Ets factor binding to the OTR promoter was identified, by electrophoretic mobility immunoshift assays, to be GABP alpha/beta. Co-transfection of a -85 OTR/luciferase construct with vectors expressing GABP alpha and GABP beta1 had only a modest effect on expression, but cotransfection with GABP alpha/beta- with c-Fos/c-Jun-expressing plasmids resulted in an increase of almost 10-fold in luciferase activity. Mutation of either the GABP- or CRE-like binding sites obliterated the induction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We showed the requirement for GABP alpha/beta and c-Fos/c-Jun in endogenous OTR gene expression, using oligonucleotide GABP and AP-1 binding decoys to inhibit serum-induced increases in 125I-labeled OT antagonist binding to Hs578T cells. Our work is the first characterization of the proximal promoter region of the human OTR gene, and it sets the stage for studying regulation of OTR expression in breast cells.
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PMID:Identification of a GABP alpha/beta binding site involved in the induction of oxytocin receptor gene expression in human breast cells, potentiation by c-Fos/c-Jun. 1021 80

Human placental leucine aminopeptidase (P-LAP) plays a major role in the clearance of oxytocin, which is a key hormone in regulating labour pain. To explore the transcriptional regulation of P-LAP gene expression in placenta, we performed systematic studies using human choriocarcinoma cells, BeWo and JEG-3, as a model of placental trophoblastic cells. Transient transfection and luciferase assays using various 5'-deleted P-LAP-luciferase constructs showed that the region from -297 to +49 of the transcription start site was responsible for promoter activity in these cells. Footprinting analysis with nuclear extracts from both cell lines demonstrated at least four sites for nucleoprotein interactions in this region (FP1 to FP4). Site-directed deletion of FP1-4 in luciferase assays indicated the significance of the FP3 region (-214 to -183) for high promoter activity in the cells. Electrophoretic mobility shift assays to identify the proteins interacting with DNA at FP3 revealed three retarded bands, one of which was generated by activator protein-2 (AP-2) binding. Our findings suggest that AP-2 may be one of the important factors regulating P-LAP gene expression in human placenta.
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PMID:Transcriptional regulation of human placental leucine aminopeptidase/oxytocinase gene. 1151 97

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively. Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.
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PMID:Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer. 1466 7

Studies using estrogen receptor alpha (ERalpha) knock-out mice indicate that ERalpha masculinizes male behavior. Recent studies of ERalpha and male prosocial behavior have shown an inverse relationship between ERalpha expression in regions of the brain that regulate social behavior, including the medial amygdala (MeA), and the expression of male prosocial behavior. These studies have lead to the hypothesis that low levels of ERalpha are necessary to "permit" the expression of high levels of male prosocial behavior. To test this, viral vectors were used to enhance ERalpha in male prairie voles (Microtus ochrogaster), which display high levels of prosocial behavior and low levels of MeA ERalpha. Adult male prairie voles were transfected with ERalpha in the MeA (MeA-ERalpha) or the caudate-putamen (ERalpha control) or luciferase (MeA-site-specific control), and 3 weeks later tested for spontaneous alloparental behavior and partner preference. Enhancing ERalpha in the MeA altered/reduced male prosocial behavior. Only one-third of MeA-ERalpha males, compared with all control males, were alloparental. MeA-ERalpha males also displayed a significant preference for a novel female. This is a critical finding because the manipulations of neuropeptides, oxytocin and vasopressin, can inhibit the formation of a partner preference, but do not lead to the formation of a preference for a novel female. The results support the hypothesis that low levels of ERalpha are necessary for high levels of male prosocial behavior, and provide the first direct evidence that site-specific ERalpha expression plays a critical role in the expression of male prosocial behavior.
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PMID:Estrogen receptors in the medial amygdala inhibit the expression of male prosocial behavior. 1912 78


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