Protection against cytokine toxicity through endoplasmic reticulum and mitochondrial stress prevention by prostacyclin synthase overexpression in insulin-producing cells

Abstract

Proinflammatory cytokines play a crucial role in the pathogenesis of type 1 diabetes mellitus. One of the cytokine-regulated pathways mediating inflammation in this autoimmune disease is the arachidonic acid metabolism pathway, comprising both the induction of cyclooxygenases and the production of different prostaglandins. Cytokine toxicity is mediated in many cell types, including pancreatic beta cells through this pathway. Interestingly, some cell types have been shown to be insensitive to such toxicity, and this correlated with a high expression of prostacyclin synthase (PGIS). Using insulin-producing RINm5F cells as a model for pancreatic beta cells, PGIS was overexpressed and exhibited a large protective effect against cytokine toxicity. This protective effect of PGIS against cytokine toxicity correlated with a decreased activation of the transcription factor NFkappaB and the inducible NO synthase promoter as well as a reduced inducible NO synthase protein expression and nitrite production. A reduction in the cytokine-stimulated endoplasmic reticulum and mitochondrial stress was also found in the PGIS-overexpressing cells. Moreover, cytokine-induced caspase-3 activation and reduction of glucose oxidation and cell proliferation were suppressed. Thus, PGIS overexpression apparently protects insulin-producing cells against cytokine toxicity via suppression of endoplasmic reticulum and mitochondrial stress-mediated cell death pathways.

FIGURE 1.

Prostacyclin synthase expression in insulin-producing RINm5F cell clones. The cells were stably transfected with either the empty vector (control cells) or with the pcDNA3-hPGIS. A, gene expression of hPGIS was measured by quantitative real time reverse transcription-PCR. B, protein expression. *, p < 0.05 versus control cells, analysis of variance followed by Bonferroni. The basal expression of rat PGIS in the control as well as in all clones overexpressing PGIS was negligible (data not shown).

FIGURE 2.

Effects of PGIS overexpression in insulin-producing RINm5F cells on cytokine-stimulated caspase-3, -9, and -12 activity. RINm5F insulin-producing cells were incubated with IL-1β (600 units/ml) or a cytokine mixture (60 units/ml IL-1β, 185 units/ml TNFα, and 14 units/ml IFNγ), or camptothecin (0.5 μm) for 24 h. After cell lysis and extraction of the total intracellular protein, the activity of caspase-3 was measured using the DEVD cleavage method. Caspase-9 and -12 activation was measured using flow cytometry. The data are mean values from four independent experiments, each measured in at least two repetitions. *, p < 0.05 versus untreated; #, p < 0.05 versus control clone treated in the same way; analysis of variance followed by Bonferroni.

FIGURE 3.

Effects of PGIS overexpression in insulin-producing RINm5F cells on cytokine-induced CHOP gene expression. The cells were incubated with IL-1β (600 units/ml) or a cytokine mixture (60 units/ml IL-1β, 185 units/ml TNFα, and 14 units/ml IFNγ) for 24 h. Gene expression of CHOP was measured by quantitative real time reverse transcription-PCR. *, p < 0.05 versus untreated cells. Analysis of variance was followed by Bonferroni.

FIGURE 4.

Effects of PGIS overexpression in insulin-producing RINm5F cells on cytokine-stimulated iNOS protein expression. The cells were incubated with IL-1β (600 units/ml) or a cytokine mixture (60 units/ml IL-1β, 185 units/ml TNFα, and 14 units/ml IFNγ) for 24 h, and thereafter the samples were collected for Western-blotting. A representative blot from four independent experiments is shown. con, control. Mix, cytokine mixture.