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Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets.

Edén D, Siegbahn A, Mokhtari D - Diabetologia (2015)

Bottom Line: The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death.Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Clinical Chemistry, Science for Life Laboratory, University Hospital, Uppsala University, Entr. 61 3rd floor, S-751 85, Uppsala, Sweden.

ABSTRACT

Aims/hypothesis: Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro.

Methods: Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA.

Results: A combination of the cytokines IL-1β, TNF-α and IFN-γ induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.

Conclusions/interpretation: These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

No MeSH data available.


Related in: MedlinePlus

TF/FVIIa signalling augments cytokine-induced beta cell death independently of downstream coagulation. MIN-6 cells were pre-treated with FVIIa (10 nmol/l), FXa (100 nmol/l) or FVIIa+hirudin (10 U/ml) for 6 h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. (a) Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). (b) Cells were stained with PI and Hoechst 33 258 (HO), ~2,000 cells/group were counted and the number of PI-positive cells was determined by fluorescence microscopy. (c) MIN-6 cells were first pre-treated with FVIIa (10 nmol/l) for 6 h. Indicated groups were then treated with IL-1 Ra (500 ng/ml) for 30 min followed by treatment with individual cytokines or with cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). Results are means ± SEM from four experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
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Fig2: TF/FVIIa signalling augments cytokine-induced beta cell death independently of downstream coagulation. MIN-6 cells were pre-treated with FVIIa (10 nmol/l), FXa (100 nmol/l) or FVIIa+hirudin (10 U/ml) for 6 h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. (a) Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). (b) Cells were stained with PI and Hoechst 33 258 (HO), ~2,000 cells/group were counted and the number of PI-positive cells was determined by fluorescence microscopy. (c) MIN-6 cells were first pre-treated with FVIIa (10 nmol/l) for 6 h. Indicated groups were then treated with IL-1 Ra (500 ng/ml) for 30 min followed by treatment with individual cytokines or with cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). Results are means ± SEM from four experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test

Mentions: We next studied the effects of FVIIa on cytokine-induced beta cell death, evaluated by a cell-death ELISA and PI staining. Treatment of MIN-6 cells with cytokine mixture led to an increase in cell death (Fig. 2). FVIIa treatment alone had no significant effect on cell death (Fig. 2). Conversely, FVIIa pre-treatment significantly potentiated cytokine-induced cell death (Fig. 2). Addition of FXa had no effect on cytokine-induced beta cell death and the thrombin inhibitor hirudin failed to prevent the potentiation of cytokine-induced beta cell death induced by FVIIa (Fig. 2a).Fig. 2


Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets.

Edén D, Siegbahn A, Mokhtari D - Diabetologia (2015)

TF/FVIIa signalling augments cytokine-induced beta cell death independently of downstream coagulation. MIN-6 cells were pre-treated with FVIIa (10 nmol/l), FXa (100 nmol/l) or FVIIa+hirudin (10 U/ml) for 6 h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. (a) Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). (b) Cells were stained with PI and Hoechst 33 258 (HO), ~2,000 cells/group were counted and the number of PI-positive cells was determined by fluorescence microscopy. (c) MIN-6 cells were first pre-treated with FVIIa (10 nmol/l) for 6 h. Indicated groups were then treated with IL-1 Ra (500 ng/ml) for 30 min followed by treatment with individual cytokines or with cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). Results are means ± SEM from four experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: TF/FVIIa signalling augments cytokine-induced beta cell death independently of downstream coagulation. MIN-6 cells were pre-treated with FVIIa (10 nmol/l), FXa (100 nmol/l) or FVIIa+hirudin (10 U/ml) for 6 h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. (a) Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). (b) Cells were stained with PI and Hoechst 33 258 (HO), ~2,000 cells/group were counted and the number of PI-positive cells was determined by fluorescence microscopy. (c) MIN-6 cells were first pre-treated with FVIIa (10 nmol/l) for 6 h. Indicated groups were then treated with IL-1 Ra (500 ng/ml) for 30 min followed by treatment with individual cytokines or with cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA by measuring absorbance (A405–A490). Results are means ± SEM from four experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
Mentions: We next studied the effects of FVIIa on cytokine-induced beta cell death, evaluated by a cell-death ELISA and PI staining. Treatment of MIN-6 cells with cytokine mixture led to an increase in cell death (Fig. 2). FVIIa treatment alone had no significant effect on cell death (Fig. 2). Conversely, FVIIa pre-treatment significantly potentiated cytokine-induced cell death (Fig. 2). Addition of FXa had no effect on cytokine-induced beta cell death and the thrombin inhibitor hirudin failed to prevent the potentiation of cytokine-induced beta cell death induced by FVIIa (Fig. 2a).Fig. 2

Bottom Line: The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death.Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Clinical Chemistry, Science for Life Laboratory, University Hospital, Uppsala University, Entr. 61 3rd floor, S-751 85, Uppsala, Sweden.

ABSTRACT

Aims/hypothesis: Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro.

Methods: Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA.

Results: A combination of the cytokines IL-1β, TNF-α and IFN-γ induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.

Conclusions/interpretation: These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

No MeSH data available.


Related in: MedlinePlus