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Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells.

Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Størling J, Rosenberg L, Billestrup N, Maysinger D, Mandrup-Poulsen T - Diabetes (2009)

Bottom Line: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation.Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Diabetology, Steno Diabetes Center, Gentofte, Denmark.

ABSTRACT

Objective: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.

Research design and methods: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.

Results: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.

Conclusions: Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

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Related in: MedlinePlus

IL-1β plus IFN-γ induces Bax-independent dephosphorylation of Bad Ser136 in INS-1 cells and rat islets. Bad Ser136 dephosphorylation after cytokine exposure was assessed by immunoblotting in INS-1 cells (A and C) or rat islets (B). C: Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. Data are presented as mean ± SE of four experiments (A), seven experiments (24 h) (B) or two experiments (6 h) (B), and seven experiments (C). Representative gels are shown, and actin and tubulin were used as loading controls. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.
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Figure 5: IL-1β plus IFN-γ induces Bax-independent dephosphorylation of Bad Ser136 in INS-1 cells and rat islets. Bad Ser136 dephosphorylation after cytokine exposure was assessed by immunoblotting in INS-1 cells (A and C) or rat islets (B). C: Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. Data are presented as mean ± SE of four experiments (A), seven experiments (24 h) (B) or two experiments (6 h) (B), and seven experiments (C). Representative gels are shown, and actin and tubulin were used as loading controls. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.

Mentions: Data are presented as means ± SE. Statistical significance was determined by Student's paired t test (on the planned comparisons reported) or by two-way ANOVA with Dunnett's post hoc test (time course experiments) (Figs. 3A and 5A), with P < 0.05 as the level of significance.


Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells.

Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Størling J, Rosenberg L, Billestrup N, Maysinger D, Mandrup-Poulsen T - Diabetes (2009)

IL-1β plus IFN-γ induces Bax-independent dephosphorylation of Bad Ser136 in INS-1 cells and rat islets. Bad Ser136 dephosphorylation after cytokine exposure was assessed by immunoblotting in INS-1 cells (A and C) or rat islets (B). C: Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. Data are presented as mean ± SE of four experiments (A), seven experiments (24 h) (B) or two experiments (6 h) (B), and seven experiments (C). Representative gels are shown, and actin and tubulin were used as loading controls. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712790&req=5

Figure 5: IL-1β plus IFN-γ induces Bax-independent dephosphorylation of Bad Ser136 in INS-1 cells and rat islets. Bad Ser136 dephosphorylation after cytokine exposure was assessed by immunoblotting in INS-1 cells (A and C) or rat islets (B). C: Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. Data are presented as mean ± SE of four experiments (A), seven experiments (24 h) (B) or two experiments (6 h) (B), and seven experiments (C). Representative gels are shown, and actin and tubulin were used as loading controls. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.
Mentions: Data are presented as means ± SE. Statistical significance was determined by Student's paired t test (on the planned comparisons reported) or by two-way ANOVA with Dunnett's post hoc test (time course experiments) (Figs. 3A and 5A), with P < 0.05 as the level of significance.

Bottom Line: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation.Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Diabetology, Steno Diabetes Center, Gentofte, Denmark.

ABSTRACT

Objective: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.

Research design and methods: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.

Results: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.

Conclusions: Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

Show MeSH
Related in: MedlinePlus