title: Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells.
abstract: The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca(2+) in the extracellular medium, induced a slow and progressive increase of [Ca(2+)](c) toward a stable level. Melatonin did not inhibit the typical Ca(2+) response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca(2+) in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl(3), to inhibit Ca(2+) entry, we could not detect any change in [Ca(2+)](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca(2+). When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca(2+) in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca(2+)]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca(2+). Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1 nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100 µM) or PMA (1 µM). On the contrary, pretreatment with Ro31-8220 (3 µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca(2+) signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells.
authors.affiliation: Cell Physiology Research Group (FICEL), Department of Physiology, University of Extremadura, Caceres, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Santofimia-Castaño
authors.affiliation: Facultade de Agronomia & Medicina Veterinaria, Universidade de Brasilia, 70900-100, Brasilia DF, Brazil.
authors.forename: Deborah
authors.initials: D
authors.lastname: Clea Ruy
authors.affiliation: Cell Physiology Research Group (FICEL), Department of Physiology, University of Extremadura, Caceres, Spain.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Garcia-Sanchez
authors.affiliation: Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC, Salamanca, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Jimenez-Blasco
authors.affiliation: Cell Physiology Research Group (FICEL), Department of Physiology, University of Extremadura, Caceres, Spain; Department of Gastroenterology, San Pedro de Alcantara Hospital, E-10003 Caceres, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Fernandez-Bermejo
authors.affiliation: Institute of Functional Biology and Genomics (IBFG), University of Salamanca-CSIC, Salamanca, Spain.
authors.forename: Juan P
authors.initials: JP
authors.lastname: Bolaños
authors.affiliation: Cell Physiology Research Group (FICEL), Department of Physiology, University of Extremadura, Caceres, Spain.
authors.forename: Gines M
authors.initials: GM
authors.lastname: Salido
authors.affiliation: Cell Physiology Research Group (FICEL), Department of Physiology, University of Extremadura, Caceres, Spain. Electronic address: agmateos@unex.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Gonzalez
mesh_terms: D061354:Acinar Cells / Q000378:metabolism; D000818:Animals; D061745:Antioxidant Response Elements / Q000235:genetics; D000975:Antioxidants / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D008550:Melatonin / Q000008:administration & dosage*; D051379:Mice; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D051547:Heme Oxygenase-1; D016660:NAD(P)H Dehydrogenase (Quinone); C000597536:Noq1 protein, mouse; D011493:Protein Kinase C; C560749:GCLM protein, mouse; D005721:Glutamate-Cysteine Ligase; D008550:Melatonin; D002118:Calcium
keywords: Antioxidant response; Calcium; Melatonin; Nrf2; Pancreas; Protein kinase C