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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-mediated JNK activation promotes cytokine-induced beta cell death. (a) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with individual cytokines or a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 15 min. Cells were then lysed and subjected to immunoblotting using phospho- and total JNK antibodies. (b) MIN-6 cells were transfected with scrambled (Scr.) siRNA or Tf siRNA. At 72 h post transfection cells were stimulated with cytokine mixture for 15 min, lysed and subjected to immunoblotting using TF and JNK antibodies. (c) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with cytokine mixture for 72 h. Thirty minutes before the addition of FVIIa, p38 inhibitor SB203580 (10 μmol/l), MEK1/2 inhibitor PD98059 (10 μmol/l) and JNK inhibitor SP600125 (10 μmol/l) were added to the indicated groups. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SD from three (a, b) or four (c) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
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Fig6: TF/FVIIa-mediated JNK activation promotes cytokine-induced beta cell death. (a) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with individual cytokines or a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 15 min. Cells were then lysed and subjected to immunoblotting using phospho- and total JNK antibodies. (b) MIN-6 cells were transfected with scrambled (Scr.) siRNA or Tf siRNA. At 72 h post transfection cells were stimulated with cytokine mixture for 15 min, lysed and subjected to immunoblotting using TF and JNK antibodies. (c) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with cytokine mixture for 72 h. Thirty minutes before the addition of FVIIa, p38 inhibitor SB203580 (10 μmol/l), MEK1/2 inhibitor PD98059 (10 μmol/l) and JNK inhibitor SP600125 (10 μmol/l) were added to the indicated groups. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SD from three (a, b) or four (c) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test

Mentions: To identify the mechanisms behind the effects of TF/FVIIa on cytokine-induced beta cell death, we studied the consequences of cytokine and FVIIa treatment on mitogen-activated protein kinase (MAPK) activation. Treatment with cytokines resulted in increased phosphorylation of p38, ERK and JNK in islets and MIN-6 beta cells (Fig. 5). FVIIa did not alter ERK or p38 phosphorylation in response to cytokines, but augmented cytokine-induced JNK phosphorylation (Fig. 5), an event that was abolished by Tf siRNA (Fig. 6b). FVIIa did not affect phosphorylation of p38, ERK or JNK at basal conditions (Fig. 5).Fig. 5


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-mediated JNK activation promotes cytokine-induced beta cell death. (a) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with individual cytokines or a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 15 min. Cells were then lysed and subjected to immunoblotting using phospho- and total JNK antibodies. (b) MIN-6 cells were transfected with scrambled (Scr.) siRNA or Tf siRNA. At 72 h post transfection cells were stimulated with cytokine mixture for 15 min, lysed and subjected to immunoblotting using TF and JNK antibodies. (c) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with cytokine mixture for 72 h. Thirty minutes before the addition of FVIIa, p38 inhibitor SB203580 (10 μmol/l), MEK1/2 inhibitor PD98059 (10 μmol/l) and JNK inhibitor SP600125 (10 μmol/l) were added to the indicated groups. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SD from three (a, b) or four (c) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: TF/FVIIa-mediated JNK activation promotes cytokine-induced beta cell death. (a) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with individual cytokines or a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 15 min. Cells were then lysed and subjected to immunoblotting using phospho- and total JNK antibodies. (b) MIN-6 cells were transfected with scrambled (Scr.) siRNA or Tf siRNA. At 72 h post transfection cells were stimulated with cytokine mixture for 15 min, lysed and subjected to immunoblotting using TF and JNK antibodies. (c) MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with cytokine mixture for 72 h. Thirty minutes before the addition of FVIIa, p38 inhibitor SB203580 (10 μmol/l), MEK1/2 inhibitor PD98059 (10 μmol/l) and JNK inhibitor SP600125 (10 μmol/l) were added to the indicated groups. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SD from three (a, b) or four (c) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
Mentions: To identify the mechanisms behind the effects of TF/FVIIa on cytokine-induced beta cell death, we studied the consequences of cytokine and FVIIa treatment on mitogen-activated protein kinase (MAPK) activation. Treatment with cytokines resulted in increased phosphorylation of p38, ERK and JNK in islets and MIN-6 beta cells (Fig. 5). FVIIa did not alter ERK or p38 phosphorylation in response to cytokines, but augmented cytokine-induced JNK phosphorylation (Fig. 5), an event that was abolished by Tf siRNA (Fig. 6b). FVIIa did not affect phosphorylation of p38, ERK or JNK at basal conditions (Fig. 5).Fig. 5

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