Phlda3 is a novel stress-responsive gene in beta cells that regulates cytokine and oxidative stress-induced apoptosis
(2016) 52nd Annual Meeting of the European Association for the Study of Diabetes — Location: Munich, Germany (12.September.2016)
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- et. al.
- Abstract
- Background and aims: Beta cell death is implicated in the loss of functional beta cell mass in type 1 and type 2 diabetes, but the mechanisms are not clear. Pleckstrin homology-like, domain family A, member 3 (Phlda3) has been identified as a tumor suppressor in pancreatic neuroendocrine tumors. However, its role in beta cells has not been investigated. Here we investigated: 1) whether Phlda3 expression is regulated by stress in beta cells, and 2) the role of Phlda3 expression in beta cell pathophysiology under stress conditions. Materials and methods: Phlda3 expression was assessed in islets from diabetic db/db mice and age-matched lean control db/+ mice. MIN6 cells were exposed for 24h to various stress stimuli including cytokines [TNFa (100 U/ml) + IFNg (250 U/ml) + IL1b (100 U/ml)], thapsigargin (300 nM, 1 µM), tunicamycin (1-5 µg/ml), H2O2 (200 µM), ribose (50 mM) and modulators of Ca2+ influx diazoxide (250 µM) and nifedipine (10 µM) . Phlda3 was inhibited using siRNA. Gene expression was assessed by real-time RT-PCR. Apoptosis was measured by DNA fragmentation ELISA. Results: The mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic db/db mice by ~4,6-fold (p<0,001). In vitro, exposure of MIN6 cells to cytokines upregulated Phlda3 mRNA levels by ~4-fold (p<0,01) in parallel with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Interestingly, thapsigargin treatment, but not tunicamycin treatment, significantly upregulated Phlda3 mRNA levels despite marked upregulation of ER stress genes by both agents. This implicates perturbation of Ca2+ homeostasis in the regulation of Phlda3 expression. Diazoxide and nifedipine treatment reduced Phlda3 mRNA levels by ~50% (p<0,05). On the other hand, exposure of MIN6 cells to ribose, which induces oxidative stress, strongly upregulated Phlda3 mRNA levels by ~13-fold (p<0,001). Interestingly, siRNA-mediated knockdown of Phlda3 potentiated cytokine-induced apoptosis by ~2,6-fold (p<0,05). This effect was associated with ~1,5-fold further upregulation of iNos mRNA levels (p<0,01) and downregulation of antioxidant genes Gpx1 (p<0,01) and Srxn1 (p<0,05) and adaptive UPR genes Xbp1 (p<0,001), Hspa5 (p<0,05) and Fkbp11 (p<0,01), whereas beta-cell enriched genes Glut2, Pc and Mafa as well as Ddit3 were unchanged. These findings suggest that the inhibition of Phlda3 potentiates the effects of cytokines to induce nitrosative stress and depletes defenses against oxidative and ER stress. Finally, Phlda3 knockdown also potentiated H2O2 and ribose-induced apoptosis (p<0,05). Conclusion: We have identified Phlda3 as a novel stress-response gene in beta cells that plays an important protective role under stress conditions. Phlda3 may regulate the nitrosative, oxidative and ER stress response in beta cells via the maintenance of antioxidant and adaptive UPR gene expression and repression of iNos. The induction of Phlda3 may promote beta cell survival under stress conditions in diabetes.
- Affiliations
Garvan Institute Of Medical Research
Citations
Bensellam, M., Laybutt, R., & et al. (2016). Phlda3 is a novel stress-responsive gene in beta cells that regulates cytokine and oxidative stress-induced apoptosis. Diabetologia : clinical and experimental diabetes and metabolism, 59, S208. https://hdl.handle.net/2078.5/218598 (Original work published 2016)