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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 downregulation blocks the effect of FVIIa on cytokine-induced beta cell death. (a, b) MIN-6 cells were transfected with scrambled (Scr.) or Tf siRNA. At 48 or 72 h post transfection, cells were subjected to immunoblotting using TF and GAPDH antibodies. (c) At 48 h after siRNA transfection, MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). (d) MIN-6 cells were cultured in the absence or presence of TF antibody (Ab) 4515 (100 μg/ml, 1 h). Cells were left untreated or were treated with FVIIa (10 nmol/l) for 6 h and then cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SEM from three (a, b) or four (c, d) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
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Fig4: TF downregulation blocks the effect of FVIIa on cytokine-induced beta cell death. (a, b) MIN-6 cells were transfected with scrambled (Scr.) or Tf siRNA. At 48 or 72 h post transfection, cells were subjected to immunoblotting using TF and GAPDH antibodies. (c) At 48 h after siRNA transfection, MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). (d) MIN-6 cells were cultured in the absence or presence of TF antibody (Ab) 4515 (100 μg/ml, 1 h). Cells were left untreated or were treated with FVIIa (10 nmol/l) for 6 h and then cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SEM from three (a, b) or four (c, d) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test

Mentions: To determine whether the effects of FVIIa are dependent on TF/FVIIa, we downregulated TF protein levels (Fig. 4a, b). FVIIa potentiated cell death induced by the cytokine mixture in MIN-6 cells transfected with scrambled siRNA, when analysed using a cell-death ELISA (Fig. 4c). Tf siRNA prevented FVIIa-induced potentiation of cytokine-induced cell death but did not restore cell death to control levels (Fig. 4c). In addition, we used a TF antibody to block TF/FVIIa. We observed a decrease in cytokine-induced cell death after administration of TF antibody alone (Fig. 4d). Moreover, TF antibody counteracted the potentiation of cytokines induced by FVIIa but failed to restore cell death to control levels (Fig. 4d).Fig. 4


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 downregulation blocks the effect of FVIIa on cytokine-induced beta cell death. (a, b) MIN-6 cells were transfected with scrambled (Scr.) or Tf siRNA. At 48 or 72 h post transfection, cells were subjected to immunoblotting using TF and GAPDH antibodies. (c) At 48 h after siRNA transfection, MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). (d) MIN-6 cells were cultured in the absence or presence of TF antibody (Ab) 4515 (100 μg/ml, 1 h). Cells were left untreated or were treated with FVIIa (10 nmol/l) for 6 h and then cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SEM from three (a, b) or four (c, d) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
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Related In: Results  -  Collection

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Fig4: TF downregulation blocks the effect of FVIIa on cytokine-induced beta cell death. (a, b) MIN-6 cells were transfected with scrambled (Scr.) or Tf siRNA. At 48 or 72 h post transfection, cells were subjected to immunoblotting using TF and GAPDH antibodies. (c) At 48 h after siRNA transfection, MIN-6 cells were pre-treated with FVIIa (10 nmol/l) for 6 h followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). (d) MIN-6 cells were cultured in the absence or presence of TF antibody (Ab) 4515 (100 μg/ml, 1 h). Cells were left untreated or were treated with FVIIa (10 nmol/l) for 6 h and then cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA measuring absorbance (A405–A490). Results are means ± SEM from three (a, b) or four (c, d) individual experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test
Mentions: To determine whether the effects of FVIIa are dependent on TF/FVIIa, we downregulated TF protein levels (Fig. 4a, b). FVIIa potentiated cell death induced by the cytokine mixture in MIN-6 cells transfected with scrambled siRNA, when analysed using a cell-death ELISA (Fig. 4c). Tf siRNA prevented FVIIa-induced potentiation of cytokine-induced cell death but did not restore cell death to control levels (Fig. 4c). In addition, we used a TF antibody to block TF/FVIIa. We observed a decrease in cytokine-induced cell death after administration of TF antibody alone (Fig. 4d). Moreover, TF antibody counteracted the potentiation of cytokines induced by FVIIa but failed to restore cell death to control levels (Fig. 4d).Fig. 4

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