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For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD+-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of β-cells to nitric oxide.
Nitric oxide plays a central role in regulating the response(s) of pancreatic β-cells to cytokine treatment. Cytokines such as IL-1 (rat) and a combination of IL-1 + IFN-γ (mouse and human) stimulate the expression of the inducible isoform of nitric-oxide synthase (NOS) and the production of micromolar levels of nitric oxide by β-cells (
). Nitric oxide attenuates insulin secretion by inhibiting the oxidation of glucose to CO2 and the activity of mitochondrial iron-sulfur center containing enzymes such as aconitase and complexes of the electron transport system (
) that leads to the inhibition of glucose-induced insulin secretion due to the inability to generate sufficient levels of ATP to close the ATP-sensitive K+ channels, an event required for β-cell depolarization and Ca2+-dependent exocytosis (
). The ability of β-cells to recover from cytokine- and nitric oxide-induced damage is temporally limited. Following a 36-h exposure to IL-1, β-cells are no longer capable of recovering metabolic and secretory function, and the islets are committed to death (
). This dichotomy in the type of cell death that has been observed appears to reflect the temporal changes in the metabolic responses and the extent of DNA damage caused by nitric oxide. Following short exposures to cytokines, under conditions in which nitric oxide-mediated damage is reversible, biochemical assays indicate that cell death is necrotic in nature. At points in which β-cells no longer have the capacity to recover from this damage, cytokine-mediated β-cell death shifts to an apoptotic process that is associated with irreversible DNA damage and caspase activation (
). Our findings suggest that there is an alternative pathway because p53 knockdown does not modify GADD45α expression or GADD45α-dependent DNA repair in β-cells. Members of the Forkhead family of transcription factors are known to regulate genes involved in cell cycle, stress resistance, DNA repair, and apoptosis (
In this study we provide evidence that FoxO1 and its regulation by SIRT1 play primary roles in regulating the cellular responses to nitric oxide. Nitric oxide, produced in response to IL-1, or supplied exogenously, stimulates the nuclear localization of FoxO1 and the FoxO1-dependent expression of GADD45α and repair of nitric oxide-damaged DNA in β-cells. This protective response is controlled by the activity of SIRT1. Activators of SIRT1 accelerate, whereas inhibitors attenuate, the repair of nitric oxide-damaged DNA. Further, inhibition of SIRT1 is associated with the induction of apoptosis that is characterized by enhanced expression of the proapoptotic gene p53-up-regulated mediator of apoptosis (PUMA)
and caspase activation. These findings suggest that the fate of β-cells in response to nitric oxide is controlled by the cellular localization of FoxO1 and activity of SIRT1 to either promote a protective response or induce β-cell death by apoptosis.
The mechanisms regulating the response of cells to high output (micromolar levels) nitric oxide have yet to be fully identified. It has been reported that cytokines stimulate nitric oxide-dependent cell death by necrosis or apoptosis and in many cases in the same cell type (
). Others have shown that prolonged incubation with cytokines, such as IL-1, stimulates an apoptotic cascade that is associated with the induction of endoplasmic reticulum stress and can be prevented using inhibitors of NOS (
). Complicating these divergent responses are reports that β-cells have the capacity to restore metabolic function and repair damaged DNA if the source of NO is removed and the cells are allowed a reasonable amount of time to recover (8 h for maximal recovery/repair) (
) provide evidence in support of a molecular mechanism that may explain these divergent cellular responses to nitric oxide. The central contributors to these responses appear to be the Forkhead transcription factor FoxO1 and the regulation of FoxO1-dependent transcription by SIRT1. FoxO1 is a transcription factor that is activated in response to various stress stimuli to alert the cell to damage. The cellular response is to initiate repair/protective pathways or, when the damage is too extensive, induce cell death by apoptosis. Growth factors and hormones, such as insulin, stimulate Ser-256 phosphorylation by Akt, inactivating FoxO1 by sequestering this transcription factor in the cytoplasm (
). In response to nitric oxide (either endogenously produced following cytokine treatment or exogenously applied using a donor), FoxO1 is dephosphorylated on Ser-256 and translocates to the nucleus (FIGURE 1, FIGURE 2). Once in the nucleus, deacetylated FoxO1 directs the expression of genes involved in stress resistance and repair (
). The role of SIRT1 in the regulation of stress responses has been described previously; however, studies have yet to examine its role in the response of cells to nitric oxide. When active, SIRT1 functions to deacetylate FoxO1 resulting in the expression of protective stress-resistant genes (
). Consistent with a role for FoxO1 deacetylation in the repair pathway, inhibitors of SIRT1, which increase FoxO1 acetylation, attenuate the repair of nitric oxide-damaged DNA. Under these conditions, the extent of DNA damage is enhanced above the levels induced by nitric oxide alone. In contrast, the SIRT1 activator resveratrol enhances the rate of DNA repair by 3-fold (Fig. 5). The specificity of resveratrol has been questioned because it has been shown to activate targets in addition to SIRT1 (e.g. AMPK) (
), and the activation of these nonselective targets may contribute to the enhancement in DNA repair (Fig. 5C). Further, we have recently shown that nitric oxide is an activator of AMPK and that AMPK participates in the recovery of islet metabolic function (
). In an attempt to control for these potential nonselective actions of resveratrol, the effects of SIRT1 inhibition and activation on the repair of DNA damage have been evaluated. In addition, we have examined the effects of siRNA knockdown of SIRT1. Although we have taken both molecular and pharmacological approaches, it is possible that additional SIRT1-dependent or -independent factors, such as AMPK activation, also contribute to this DNA repair process.
In addition to activating this recovery response, SIRT1 protects β-cells from cytokine-mediated apoptosis. Consistent with previous studies (
), IL-1 fails to stimulate apoptosis following a 24-h incubation; however, when SIRT1 is inhibited, there is an accumulation of PUMA mRNA and the activation of caspases-3 and -9 by cleavage (Fig. 6). Under these conditions, the inhibition of SIRT1 does not modify IL-1 induced nitric oxide production, indicating that changes in nitric oxide levels are not responsible for the differential gene expression and caspase activation under conditions of SIRT1 inhibition. Rather, we favor changes in the activation state of SIRT1 as the mechanism governing whether FoxO1 activates a protective or apoptotic transcriptional response to nitric oxide. Recently, we have shown the induction of caspase-3 activity, caspase-3 cleavage, and PUMA mRNA accumulation when islets are cultured for 36 h with IL-1 (
). These effects, which are not observed following a 24-h incubation with the cytokine, are prevented by inhibitors of NOS. It is likely that as damage induced by nitric oxide becomes more extensive, SIRT1 is inhibited, resulting in a shift in the FoxO1 transcriptional program from protection to the induction of apoptosis, as evidenced by PUMA expression and caspase-3 and -9 activation. Currently, the mechanisms responsible for inhibition of SIRT1 by nitric oxide are unknown. One potential target is the activation of polyADP-ribose polymerase, which uses NAD+ as a substrate (
), and we show that nitric oxide attenuates the phosphorylation of FoxO1 on the Akt-mediated site, Ser-256 (Fig. 1). JNK is also a known activator of FoxO-dependent gene expression and JNK participates in the GADD45α-dependent repair of nitric oxide-mediated DNA damage (
), with FoxO1 in the regulation of expression of potential recovery factors such as GADD45α. Additional studies will be required to determine the mechanism of action of JNK in both the induction of a defense/repair response and the induction of apoptosis.
Overall, this study provides evidence that FoxO1 regulates both DNA repair and the induction of β-cell apoptosis in response to nitric oxide. The proposed mechanism that controls these pathways is outlined in Fig. 7. Central to this regulation of FoxO1 is the activity of the NAD+-dependent deacetylase SIRT1. We hypothesize that when SIRT1 is active, it deacetylates FoxO1, thereby directing a transcriptional program that attenuates apoptosis and stimulates the expression of factors that protect β-cells from nitric oxide. In contrast, when SIRT1 is less active, FoxO1 becomes more acetylated and directs a proapoptotic transcriptional program resulting in apoptosis. These findings provide a working model to explain how it is possible that nitric oxide induces a protective response as well as cell death by either necrosis or apoptosis in the same cell type and in response to the same stimuli.
We thank Colleen Kelly Bratcher for expert technical assistance and Dr. Michael Moxley for helpful discussion related to these studies.