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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

FK506 inhibits cytokine-induced cleavage of capase-3 in rat islets, DEVDase activity, mitochondrial metabolic activity, and apoptosis in human islets. Cells were pre-exposed to FK506 (1 μmol/l) for 1 h before exposure to cytokines for 24 h. A: Cleavage of caspase-3 in rat islets was assessed by immunoblotting. Bars represent mean ± SE of four independent experiments, and representative gels are shown (actin is used as a loading control). B: DEVDase activity in human islets was measured as described in research design and methods. Bars represent mean ± SE of three independent experiments. C: Mitochondrial metabolic activity in human islets was measured using the MTT assay as described in research design and methods. Bars represent mean ± SE of four independent experiments. D: Apoptosis in human islets was measured with the cell death detection assay as described in research design and methods. Data are presented as means ± SE of three independent experiments. E: Phosphorylation of JNK was detected by immunoblotting in INS-1 cells exposed to IL-1β + IFN-γ with or without pre-exposure to FK506 for 1 h. A representative blot of two independent experiments is shown, and Ponceau staining was used to confirm equal protein loading. *P < 0.05; ** P < 0.01.
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Figure 7: FK506 inhibits cytokine-induced cleavage of capase-3 in rat islets, DEVDase activity, mitochondrial metabolic activity, and apoptosis in human islets. Cells were pre-exposed to FK506 (1 μmol/l) for 1 h before exposure to cytokines for 24 h. A: Cleavage of caspase-3 in rat islets was assessed by immunoblotting. Bars represent mean ± SE of four independent experiments, and representative gels are shown (actin is used as a loading control). B: DEVDase activity in human islets was measured as described in research design and methods. Bars represent mean ± SE of three independent experiments. C: Mitochondrial metabolic activity in human islets was measured using the MTT assay as described in research design and methods. Bars represent mean ± SE of four independent experiments. D: Apoptosis in human islets was measured with the cell death detection assay as described in research design and methods. Data are presented as means ± SE of three independent experiments. E: Phosphorylation of JNK was detected by immunoblotting in INS-1 cells exposed to IL-1β + IFN-γ with or without pre-exposure to FK506 for 1 h. A representative blot of two independent experiments is shown, and Ponceau staining was used to confirm equal protein loading. *P < 0.05; ** P < 0.01.

Mentions: Immunoblotting was performed as described previously (24,28). Briefly, lysate protein concentrations were measured by the Bradford method according to the manufacturer's instructions (Bio-Rad). Equivalent amounts of protein from each condition were immunoblotted as described by the manufacturer (Invitrogen), and the protein of interest was detected by chemiluminescence. Anti–cytochrome c (BD Biosciences) was used at a dilution of 1:1,000; anti–cleaved caspase-3 (Cell Signaling Technology) at 1:250–1,000; anti-Bax (Santa Cruz Biotechnology) at 1:800; anti–cytochrome c oxidase subunit IV (COX4) (BD Biosciences) at 1:500; pBads136 (Biosource) at 1:250; anti–phospho-JNK and total-JNK (Cell signaling) at 1:1,000; anti-actin (Abcam or Chemicon) at 1:10,000 or 1:1.000, respectively; anti-tubulin (Santa Cruz Biotechnology) at 1:1,000; and anti–cleaved caspase-9 (Cell Signaling Technology) at 1:500. Horseradish-peroxidase–conjugated anti-rabbit (1:2,000) and anti-mouse (1:1,000) were purchased from Cell Signaling Technology. Ponceau staining (Fluka biochemika) was used in Fig. 7E to show equal loading.


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)

FK506 inhibits cytokine-induced cleavage of capase-3 in rat islets, DEVDase activity, mitochondrial metabolic activity, and apoptosis in human islets. Cells were pre-exposed to FK506 (1 μmol/l) for 1 h before exposure to cytokines for 24 h. A: Cleavage of caspase-3 in rat islets was assessed by immunoblotting. Bars represent mean ± SE of four independent experiments, and representative gels are shown (actin is used as a loading control). B: DEVDase activity in human islets was measured as described in research design and methods. Bars represent mean ± SE of three independent experiments. C: Mitochondrial metabolic activity in human islets was measured using the MTT assay as described in research design and methods. Bars represent mean ± SE of four independent experiments. D: Apoptosis in human islets was measured with the cell death detection assay as described in research design and methods. Data are presented as means ± SE of three independent experiments. E: Phosphorylation of JNK was detected by immunoblotting in INS-1 cells exposed to IL-1β + IFN-γ with or without pre-exposure to FK506 for 1 h. A representative blot of two independent experiments is shown, and Ponceau staining was used to confirm equal protein loading. *P < 0.05; ** P < 0.01.
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Related In: Results  -  Collection

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Figure 7: FK506 inhibits cytokine-induced cleavage of capase-3 in rat islets, DEVDase activity, mitochondrial metabolic activity, and apoptosis in human islets. Cells were pre-exposed to FK506 (1 μmol/l) for 1 h before exposure to cytokines for 24 h. A: Cleavage of caspase-3 in rat islets was assessed by immunoblotting. Bars represent mean ± SE of four independent experiments, and representative gels are shown (actin is used as a loading control). B: DEVDase activity in human islets was measured as described in research design and methods. Bars represent mean ± SE of three independent experiments. C: Mitochondrial metabolic activity in human islets was measured using the MTT assay as described in research design and methods. Bars represent mean ± SE of four independent experiments. D: Apoptosis in human islets was measured with the cell death detection assay as described in research design and methods. Data are presented as means ± SE of three independent experiments. E: Phosphorylation of JNK was detected by immunoblotting in INS-1 cells exposed to IL-1β + IFN-γ with or without pre-exposure to FK506 for 1 h. A representative blot of two independent experiments is shown, and Ponceau staining was used to confirm equal protein loading. *P < 0.05; ** P < 0.01.
Mentions: Immunoblotting was performed as described previously (24,28). Briefly, lysate protein concentrations were measured by the Bradford method according to the manufacturer's instructions (Bio-Rad). Equivalent amounts of protein from each condition were immunoblotted as described by the manufacturer (Invitrogen), and the protein of interest was detected by chemiluminescence. Anti–cytochrome c (BD Biosciences) was used at a dilution of 1:1,000; anti–cleaved caspase-3 (Cell Signaling Technology) at 1:250–1,000; anti-Bax (Santa Cruz Biotechnology) at 1:800; anti–cytochrome c oxidase subunit IV (COX4) (BD Biosciences) at 1:500; pBads136 (Biosource) at 1:250; anti–phospho-JNK and total-JNK (Cell signaling) at 1:1,000; anti-actin (Abcam or Chemicon) at 1:10,000 or 1:1.000, respectively; anti-tubulin (Santa Cruz Biotechnology) at 1:1,000; and anti–cleaved caspase-9 (Cell Signaling Technology) at 1:500. Horseradish-peroxidase–conjugated anti-rabbit (1:2,000) and anti-mouse (1:1,000) were purchased from Cell Signaling Technology. Ponceau staining (Fluka biochemika) was used in Fig. 7E to show equal loading.

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