Fig 2: PRL maintains human RPE cell survival by inhibiting the oxidant-induced SIRT2-dependent induction of TRPM2-mediated intracellular Ca2+ increase. (a) RT-PCR and (b) Western-blotting of TRPM2 in ARPE-19 cell lysates. (a) TBP was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) ARPE-19 cells were untreated (Ctl), subjected to lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 (siRNA) or scramble sequence (Scr.). TRPM2 siRNA efficiently reduced TRPM2 expression as observed by immunoblotting using an anti-TRPM2 antibody that labeled a protein at the expected molecular weight for TRPM2 (171 kDa). ß-Actin served as loading control. Extracts from three independent ARPE-19 cell cultures in each condition were analyzed (N = 3). (c, d) Measurement of the change in intracellular Ca2+ measured by the change in fluo-8 fluorescence (? fluorescence) in ARPE19 cells exposed to (c) H2O2 (100 µM) or (d) piceatannol (10 µM) while TRPM2 was blocked by siRNA against TRPM2 or PRL was applied (hPRL, 100 pmol/l, 15-min pretreatment). Scramble sequence (siRNA ctl) was used as a negative control for siRNA against TRPM2. Treatments with H2O2 or piceatannol began at time = 0 s (n = 170–190 cells; N = 3 independent replicates). (e) Ca2+-dependent fluorescence change in ARPE19 cells 180 s after application of SIRT2 inhibitor AGK2 (10 µM) combined or not with H2O2 (100 µM) (n = 160–190 cells; N = 3 independent replicates). (f) Effect of TRPM2 inhibition on survival of ARPE-19 subjected to a 24-h H2O2 insult (100 µM) by MTT assay. ARPE-19 cells were untreated (Ctl), treated with lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 or the scramble sequence (Scr.) 24 h prior initiating the MTT assay. In (c–e), signals were normalized by subtracting the Fluo-8 ? fluorescence to the one in untreated conditions. All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test.

Fig 3: Gene expression analysis by qRT-PCR in the frontal cortex (FC) and hippocampus (HIP). A) TRPV1; B) TRPV2; C) TRPC1; D) TRPC6; E) TRPM2. CHOW, rats fed with standard diet; DIO, rats fed with high-fat diet. Data are expressed as folds comparing DIO with CHOW rats used as control. Data are the mean ± SEM. *p<0.05 vs CHOW rats.

Fig 4: Schematic depiction of the antiapoptotic conditions including the PRL signaling pathway in young RPE versus the proapoptotic conditions in aging RPE. Young RPE cells express PRL and its receptor (PRLR) that (1) signal for the synthesis of reduced glutathione (GSH) that transforms two molecules of hydrogen peroxide (H2O2) into water and (2) induces the transcription of catalase, which also processes H2O2 into water; both of these processes limit ROS levels. This antioxidant pathway is more prominent than the pro-oxidant pathway comprised by the TRPM2-mediated intracellular Ca2+ rise induced by the metabolite 2'O-acetyl-ADP-ribose (O-Ac-ADPR) that results from the deacetylation (removal of Ac group) of various substrates by SIRT2. The viability of RPE is therefore not jeopardized. In contrast, in aged RPE cells, the PRL signaling pathway is blunted. Therefore, the pro-oxidant pathway predominates over the antioxidant one. High levels of ROS, illustrated by increased H2O2, promote the up-regulation of SIRT2 which, in turn, activates TRPM2 by producing O-Ac-ADPR. The subsequent sustained rise in intracellular Ca2+ levels can depolarize mitochondria (Berridge et al., 2000), which causes apoptosis. RPE from PRLR null (-/-) mice emphasizes these features.

Fig 5: Expression of TRPM2 and TRPM8 did not differ between IBS-D patients and HCs. (a, b) No difference in the expression of TRPM8 was found between IBS-D and HC groups. (c, d) No difference in the expression of TRPM2 was found between IBS-D and HC groups. Scale bar, 50 µM.