Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The microphthalmia (mi) locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). We also found that mi/mi CMCs did not express a receptor (MC1R) for alpha-melanocyte-stimulating hormone. The overexpression of the wild-type (+/+) MITF but not mi-MITF normalized the expression of the MC1R in mi/mi CMCs, indicating the involvement of +-MITF in the MC1R gene expression. Next, we analyzed the promoter region of the MC1R gene by the transient cotransfection assay. The luciferase construct under the control of the MC1R promoter and the cDNA-encoding +-MITF or mi-MITF were cotransfected into NIH/3T3 fibroblasts. The cotransfection of +-MITF but not mi-MITF increased the luciferase activity. There were five CANNTG motifs recognized by bHLH-Zip-type transcription factors in the cloned promoter region. We found +-MITF bound two of five CANNTG motifs, and both motifs were essential for the transactivation of the MC1R gene by +-MITF. These results indicated that +-MITF directly transactivated the MC1R gene through these two motifs.
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PMID:Involvement of mi-transcription factor in expression of alpha-melanocyte-stimulating hormone receptor in cultured mast cells of mice. 1062 32

The processing of pro-opiomelanocortin (POMC) to generate bioactive ACTH in the anterior pituitary is mediated by prohormone convertase 1 (PC1). Leukemia inhibitory factor (LIF) and interleukin 6 (IL-6), two cytokines sharing the common gp130 receptor subunit and functioning through activation of the intracellular JAK/STAT pathway, induce POMC synthesis and ACTH release. We investigated the effects of LIF and IL-6 on PC1 expression and its subsequent processing of POMC. A significant time-dependent up-regulation of both PC1 protein and mRNA by LIF and IL-6 was seen in mouse corticotroph AtT-20 cells. IL-6 or LIF increased the synthesis of ACTH-related products with a concomitant increase in bioactive 5 and 13 kDa ACTH indicating coordinated regulation of substrate and processing enzyme. AtT-20 cells transiently transfected with a human PC1-promoter-luciferase reporter construct and treated with LIF or IL-6 showed significantly increased luciferase activity. Additionally, lipopolysaccharide (LPS) administration to rats resulted in an increase in both pituitary PC1 and POMC mRNA. These findings suggest that the ACTH increase induced by LIF and IL-6 is due to both increased POMC synthesis as well as increased POMC processing by up-regulation of PC1. These two coordinately regulated processing events probably exert central roles in the pathophysiological response to some stresses, such as inflammatory stress.
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PMID:Regulation of prohormone convertase 1 (PC1) by gp130-related cytokines. 1063 Apr 14

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5'UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5'upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3' end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5'UTR, similar to that described in the rat oxytocin receptor gene. 5'RACE and RNase protection analysis mapped two major putative transcription start points at -830 and -861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP-1 and AP-2 sites and a cluster of Sp1 sites upstream of the AP-2 sites. A luciferase construct containing a 2.1-kb of putative promoter, and part of the 5'UTR including the first intron, showed promoter activity when transfected into COS-7, CHO and PC12 cell lines but not in AtT-20 cells. A similar construct without the intron and distal 5'UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5'flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.
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PMID:Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene. 1079 83

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nm. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8-13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of alpha-melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis approximately 3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.
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PMID:Leptin regulates prothyrotropin-releasing hormone biosynthesis. Evidence for direct and indirect pathways. 1096 95

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.
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PMID:Development of a facile method for high throughput screening with reporter gene assays. 1108 Jun 97

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, alpha-MSH-ND was the most efficient alpha-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of alpha-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor-ligand complex by NMR, [Gln6]alpha-MSH-ND and [Lys6]alpha-MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30-50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor-ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.
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PMID:Differential regulation of cAMP-mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors. 1116 97

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.
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PMID:Inhibition of corticotropin releasing hormone type-1 receptor translation by an upstream AUG triplet in the 5' untranslated region. 1117 43

The pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) mediates broad anti-inflammatory and immunomodulatory effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of alpha-MSH, alpha-MSH(1-10), and alpha-MSH(11-13) on NO production and nuclear factor-kappaB (NF-kappaB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-gamma (LPS/IFN-gamma), all three peptides inhibited NO production with an order of potency alpha-MSH > or = alpha-MSH(11-13) > alpha-MSH(1-10). All three MSH peptides inhibited NF-kappaB nuclear translocation with the maximal effect of alpha-MSH and alpha-MSH(11-13) being seen in the range 1 nM-1 microM, and that of alpha-MSH(1-10) at 1 microM. By use of (125)I-(Nle(4),D-Phe(7))alpha-MSH(NDP-MSH) radioligand binding, MC(1) receptor-binding sites were demonstrated on RAW 264.7 cells. alpha-MSH and alpha-MSH(1-10) competed with the (125)I-NDP-MSH binding at these MC(1) receptor-binding sites, but alpha-MSH(11-13) even in concentrations up to 1 mM did not. Moreover, alpha-MSH and alpha-MSH(1-10) caused powerful stimulation of cyclic 3',5'-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas alpha-MSH(11-13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as alpha-MSH and alpha-MSH(1-10), but did not modify the translocation of NF-kappaB. Whereas the protein kinase A inhibitor H89 did not modify the effect of alpha-MSH on NF-kappaB translocation, H89 caused a partial inhibition of the inhibitory effect of alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin on NO production. In addition alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin also inhibited the activity of an NF-kappaB-dependent luciferase reporter and these effects were partially counteracted by H89. We suggest that melanocortin peptides act via dual mechanisms of action: one cAMP-independent and causing inhibition of NF-kappaB translocation and the other dependent on MC(1) receptor/cAMP activation.
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PMID:Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action. 1123 5

Previous studies have demonstrated that angiotensin-II (A-II) increases the human adrenocorticotropin receptor (hMC2R) gene expression in adrenal cells. In the present study, we have characterized two activator protein-1 (AP-1)-binding sites involved in the A-II stimulation of hMC2R gene transcription. Vectors containing different fragments of the hMC2R gene promoter inserted upstream of the luciferase gene, have been constructed. After transfection of H295R cells with these constructs and treatment of the cells by A-II during 48 h, maximal stimulation of the luciferase activity was obtained using the construct p(-263/+22)luc. Using progressively deleted constructs, three regions responsible for the A-II-stimulated transcription of hMC2R have been delineated. Inside these regions, two sequences displayed some homology with an AP-1 binding element (AP-108 and AP-203). Mutation of either AP-108 or AP-203 site induced a decrease of A-II-stimulated luciferase activity by 40% and 25%, respectively. Gel-shift analysis showed protein binding to these sites which was increased by an A-II treatment (maximum obtained after 3 h). Moreover, A-II could rapidly increase mRNA levels of several factors belonging to the Fos and Jun families which may be components of the AP-1 complex.
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PMID:Activator protein-1 is necessary for angiotensin-II stimulation of human adrenocorticotropin receptor gene transcription. 1124

Although opioid peptides are involved in the regulation of the hypothalamic-pituitary-adrenal axis, their role in pro-opiomelanocortin (POMC) gene expression at the pituitary level is not known. We therefore examined the effects of opioid receptor agonists, including recently discovered endogenous opioid peptides, on POMC gene expression using the AtT20PL cell line, a subclone of AtT20 in which the rat POMC 5'-promoter-luciferase fusion gene was stably incorporated. The endogenous mu-opioid receptor agonists endomorphin 1 and 2 had no effect on either basal or corticotropin-stimulating-hormone-induced POMC expression. This was also the case with the delta-agonist BUBUC, the kappa-agonist U50488H and the orphan receptor agonist orphanin FQ. In contrast, the synthetic mu-agonist loperamide significantly inhibited basal and yet enhanced cAMP-induced POMC expression. The inhibitory effect of loperamide was mimicked by the calmodulin antagonist W7 and antagonized by the calcium channel blocker nifedipine, whereas neither the inhibitory nor the enhancing effect of loperamide was influenced by the opioid antagonist naloxone. These results suggest that the synthetic mu-agonist loperamide has a modulatory effect on the 5'-promoter activity of the POMC gene. This effect does not seem to be mediated through the classical mu-opioid receptor but rather in part through a calcium/calmodulin-related mechanism.
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PMID:Effects of loperamide and other opioid-related substances on the transcriptional regulation of the rat pro-opiomelanocortin gene in AtT20 cells. 1147 16


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