Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nr12-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca2+ 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 mu M to 1 mM), in the presence of Ca2+ in the extracellular medium, induced a slow and progressive increase of [Ca2+](c), toward a stable level. Melatonin did not inhibit the typical Ca2+ response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca2+ in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl3, to inhibit Ca2+ entry, we could not detect any change in [Ca2+](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca2+. When the cells were incubated with the PKC activator PMA (1 mu M) in the presence of Ca2+ in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 pM. However, in the presence of Ro31-8220 (3 mu M), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca2+](c). Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKC alpha activation, either in the presence or in the absence of external Ca2+. Melatonin induced the phosphorylation arid nuclear translocation of the transcription factor Nrf2, and evoked a concentration dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidorecluctase 1, catalytic subunit of glutamate-cysteine ligase, arid home oxygenase-1. Incubation of MitoSOX Red loaded pancreatic acinar cells in the presence of 1 Ai 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 mu M) or PMA (1 riM). On the contrary, pretreatment with Ro31-8220 (3 mu M) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphoiylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca2+ signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells. (C) 2015 Elsevier Inc. All rights reserved.