title: Dynorphin B is an agonist of nuclear opioid receptors coupling nuclear protein kinase C activation to the transcription of cardiogenic genes in GTR1 embryonic stem cells.
abstract: The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B-like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in Bmax for a kappa opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic kappa opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage-promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes.
authors.affiliation: Department of Biomedical Sciences, Center for Biotechnology Development and Biodiversity Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy. cvent@libero.it
authors.forename: Carlo
authors.initials: C
authors.lastname: Ventura
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Zinellu
authors.forename: Emiliana
authors.initials: E
authors.lastname: Maninchedda
authors.forename: Margherita
authors.initials: M
authors.lastname: Maioli
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004399:Dynorphins / Q000032:analysis / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology; D004625:Embryo, Nonmammalian; D004723:Endorphins / Q000032:analysis / Q000494:pharmacology*; D004745:Enkephalins / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation; D050991:GATA4 Transcription Factor; D018507:Gene Expression Regulation, Developmental; D006321:Heart / Q000196:embryology; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011957:Receptors, Opioid / Q000819:agonists*; D015398:Signal Transduction; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015533:Transcriptional Activation; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; D004723:Endorphins; D004745:Enkephalins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D011498:Protein Precursors; D012333:RNA, Messenger; D011957:Receptors, Opioid; D014157:Transcription Factors; D029867:Xenopus Proteins; D004399:Dynorphins; C036200:rimorphin; C033660:preproenkephalin; D011493:Protein Kinase C