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  • A number of studies have demonstrated that oxidative injury

    2018-11-14

    A number of studies have demonstrated that oxidative injury in RPE cultures can be mitigated by compounds that exhibit antioxidant properties, including resveratrol (Chan et al., 2015). In contrast, we observed that piceatannol, a naturally occurring hydroxylated analog of resveratrol, promotes a sustained increase of intracellular Ca2+ that can cause apoptosis (Berridge et al., 2000) and reduces the survival of ARPE-19 cells. These data are consistent with a study showing that resveratrol and some of its analogs exert cytotoxic effects on normal nontransformed glutathione peroxidase (She et al., 2003). Additionally, piceatannol has been previously associated with opposite effects on cell growth depending on its concentration, i.e., low doses (nM range) induce cell growth whereas high doses (μM range) attenuate cell growth (Vo et al., 2010). Resveratrol and its analogs are substrate-specific activators of yeast and human sirtuins (Villalba and Alcain, 2012). Among the seven members of the mammalian sirtuin family, SIRT2 has been detected in adult retina (Geng et al., 2011), and disruption of its signaling has been proposed as an endogenous therapeutic target to preserve the integrity of aging cells against exogenous stressors such as ROS (Cheng et al., 2003). SIRT2 inhibition by AGK2 results in decreased ROS levels (Nie et al., 2014), which coincides with PRL decreasing intracellular levels of ROS in ARPE-19 cells. Consistent with studies showing that oxidative stress up-regulates SIRT2 (Nie et al., 2014), we found that both H2O2 exposure and advancing age cause the elevation of SIRT2 mRNA levels in ARPE-19 cells and mouse retinas, respectively. The fact that AGK2 decreases H2O2-induced apoptosis in ARPE-19 cells indicates that in these cells, SIRT2 mediates oxidative stress-induced apoptosis, as previously reported (Nie et al., 2014). Also, we show that PRL prevents the induction of SIRT2 expression under pro-oxidant conditions in ARPE-19 cells and that SIRT2 expression tends to be elevated in retinas from prlr−/− mice with advancing age compared to age-matched wild-type animals. Most conspicuously, SIRT2 expression was elevated in prlr−/− retinas from young mice. Considering that SIRT2 inactivates the PRL receptor through its deacetylation (Ma et al., 2010), our data suggest that a negative feed-back loop between SIRT2 and PRL signaling may exist. Together with the fact that PRL prevents the reduction in ARPE-19 cell survival induced by piceatannol, these data lead us to propose that PRL, by reducing ROS levels, maintains SIRT2 at levels that contribute to RPE survival. Conversely, under oxidizing conditions, increased levels of SIRT2 blunt PRL signaling, leading to RPE cell death. The possibility that PRL may directly regulate SIRT2 needs further studies. Although PRL signaling, SIRT2, and TRPM2 channels are currently viewed as being independent, we suggest that these pathways are more intricately connected than previously appreciated. We report here that TRPM2 is expressed in ARPE-19 cells. This result complements a previous study showing that all TRP channel genes are expressed in the retina (Gilliam and Wensel, 2011). We also show that H2O2 activates TRPM2 leading to Ca2+ influx in a SIRT2-dependent manner since AGK2 blocks the H2O2-induced Ca2+ increase in ARPE-19 cells. In parallel, silencing TRPM2 inhibited the piceatannol-induced increase in intracellular Ca2+, suggesting that SIRT2 increases intracellular Ca2+ by activating TRPM2. Thus, we assume that TRPM2 channels are activated by H2O2 in a SIRT2-dependent manner in ARPE-19 cells. Undoubtedly, the TRPM2-mediated Ca2+ rise is a dominant source of the Ca2+ required to reduce ARPE-19 cell viability under pro-oxidant conditions (Fig. 5f). Our observations are consistent with previous studies demonstrating that the TRPM2 Ca2+ current links oxidative stress-induced SIRT2 activation to oxidative stress-induced cell death (Faouzi and Penner, 2014). The novelty here consists in demonstrating that PRL inhibits the oxidant-induced SIRT2-dependent activation of TRPM2 channels. PRL alone does not modify Ca2+ levels, and there is no evidence that PRL activates TRPM2. Instead, PRL seems to block TRPM2 once this latter is activated. We emphasize that this effect cannot be attributed to the ability of PRL to reduce SIRT2 transcription, because the time course of the two events is different (seconds/minutes versus 24h). Reduced levels of intracellular ROS may explain the short-term effect of PRL on TRPM2 as well as the restorative effect of PRL at 6h, though further studies using different techniques and dyes are required to address this issue. Interestingly, TRPM2 current density was shown to increase in cultured pyramidal neurons over time in vitro. This increase in current density was prevented by treatment with N-acetyl cysteine and conversely, depletion of GSH augmented TRPM2 currents (Lee et al., 2010).