Up-regulation Of PTEN By Peroxynitrite Contributes To Cytokine-induced Apoptosis In Pancreatic β-cells

Both type 1 and type 2 diabetes are characterized by a significant deficit inβ-cell mass, mainly caused by apoptosis, which appears to play an important role in the development of insulin deficiency and the onset and/or progression of the disease. In type 1 diabetes, proinflammatory cytokines such as IL-1β, TNF-αand IFN-γ, secreted by activated macrophages and T cells, are the major mediators ofβ-cell dysfunction and death. Moreover, an increasing number of recent studies have suggested that the inflammatory mediators are also implicated in the development of type 2 diabetes. Together, these data strongly imply that blocking cytokine signaling may be a potential way for inhibiting pancreaticβ-cell loss.Phosphatase and tensin homolog (PTEN), originally identified as a tumor suppressor gene frequently mutated in a large percentage of human cancers, is the central negative regulator of the PI3K-Akt signal transduction cascade. A reduced PTEN function determines a marked activation of Akt survival pathways, leading to hyperplasia and inhibition of apoptosis and thereby contributing to tumor formation. Recent studies have made it clear that PTEN does much more than suppressing tumors. In major insulin target organs, principally the liver, muscle, and fat, PTEN deficiency affects glucose metabolism and protects mice from developing diabetes. Importantly, pancreas-specific PTEN deletion leads to resistance to streptozotocin (STZ)-induced diabetes and decreasedβ-cell apoptosis, but the underlying mechanism remains unclear. Cytokine-mediated cytotoxicity is presumed to be one major mechanism of STZ and there is growing evidence suggesting that cytokines causeβ-cell loss by inducing apoptosis, so the central hypothesis guiding this study is that PTEN may play a role in cytokine-inducedβ-cell apoptosis.The generation of high levels of reactive oxygen/nitrogen species has been reported to play a key role in cytokine-inducedβ-cell apoptosis. Peroxynitrite (ONOO-), produced by the reaction of nitric oxide (NO) with superoxide, is a highly reactive radical species with increased toxic potential compared with either NO or superoxide alone. Scavenger of ONOO- can prevent diabetes development in NOD mice and significantly decrease the destruction ofβ-cells incubated with the combination of cytokines. Expression of calbindin-D28k can protectβTC3 cells against cytokine-mediated apoptosis by inhibiting ONOO- formation. These findings suggest that ONOO- is a key mediator ofβ-cell apoptosis induced by cytokines.Furthermore, several studies have demonstrated that oxidative stress is an important regulator of PTEN expression/activation. Especially, it has been reported that ONOO- exerts neuroprotection by inhibiting PTEN, hence activating the anti-apoptotic PI3K-Akt pathway in primary neurons. While, data from other laboratory using human umbilical vein endothelial cells have shown that hyperglycemia triggers apoptosis by inhibiting Akt signaling via ONOO--mediated PTEN activation. In view of this, in the present study, the role of PTEN in cytokine-induced apoptosis was investigated and the question whether ONOO- formation modulates PTEN expression in was explored. Experimental contents as follows:Part 1 PTEN is involved in the cytokine-inducedβcell apoptosisObjective: To explore whether PTEN is involved in theβTC6-cells apoptosis induced by cytokines. Methods: Set up the in vitro model:βTC6-cells apoptosis induced by cytokines. The role of PTEN in cytokine-induced apoptosis was investigated and the change of Akt was observed. Results: Exposure to the cytokine mixture of IL-1β(2 ng/ml), TNFα(5 ng/ml) and IFNγ(25 ng/ml) for 24 h resulted in significantly decreased viability of mouse pancreaticβTC-6 cells, as determined by MTT assay. Moreover, the results from Hoechst staining and Caspase-3 activity assay confirmed that apoptosis was the primary mode of death caused by the cytokine mixture. The enhanced PTEN and p-PTEN expression and the inhibited Akt activation were observed. Conclusion: In pancreaticβ-cells, PTEN is a component of a functional signaling pathway activated by cytokines. Moreover, the increased protein stability through phosphorylation may be a key mechanism for the upregulated level of PTEN expression after cytokines treatment.Part 2 The effect of PTEN knowckdown on cytokine-inducedβcell apoptosisObjective: To investigate the effect of PTEN knockdown onβTC6-cells apoptosis induced by cytokines. Methods: MTT assay was used to detect the cell viability, Hoechst staining and Caspase-3 activity were employed to observe the cell apoptosis, and Western Blotting was adopted to observe the change of Akt expression/activation. Results: PTEN siRNA specifically suppressed basal expression of PTEN and p-PTEN. In parallel, PTEN siRNA abolished cytokine-enhanced PTEN expression and PTEN phosphorylation. Transfection with PTEN-specific siRNA protectedβTC-6 cells from cytokine-mediated apoptosis and reversed Akt suppression. Conclusion: Cytokine-inducedβ-cell apoptosis and Akt inactivation are PTEN-dependent.Part 3 ONOO- mediates PTEN upregulation induced by cytokinesObjective: To explore whether ONOO- formation modulates PTEN expression in the apoptosis ofβ-cells induced by cytokines. Methods: ONOO- formation was detected by ELISA. After preincubation ofβTC-6 cells with NAC (an O2·- scavenger), L-NMMA (a NOS inhibitor), and UA (an ONOO- scavenger) and then treatment with cytokines. The production of ONOO-, the number of apoptotic cells and the expression of PTEN/Akt were observed. To further confirm the role of ONOO- in the signaling pathway,βTC-6 cells transfected with or without PTEN siRNA were directly treated with exogenous ONOO-. In addition, the oxidation of PTEN was observed. Results: Incubation ofβTC-6 cells with cytokine mixture (IL-1β, TNF-αand IFN-γ) significantly promoted the ONOO- formation. Preventing ONOO- formation by administrating NAC/L-NMMA or scavenging ONOO- directly by UA abolished cytokine-induced PTEN enhancement, Akt inactivation and cell apoptosis. Exogenous ONOO- induced apoptosis ofβTC-6 cells. Furthermore, similar to cytokines, exogenous ONOO- also increased PTEN expression and PTEN phorsphoylation, while inhibited Akt activation. Cells transfected with PTEN siRNA resisted ONOO--induced apoptosis and Akt inhibition, but cytokines treatment did not result in PTEN oxidation. Conclusion: ONOO- cause PTEN up-regulation in cytokine-induced apoptosis ofβTC-6 cells, but treatment with cytokines did not cause PTEN oxidation.