Signal transduction pathways leading to apoptosis or necrosis in insulin-producing cells and prevention of cell death using gene transfer

Abstract: Signal transduction pathways potentially involved in destruction of insulin-producing β-cells in type-l diabetes and gene transfer to prevent β-cell death were studied in vitro. Adenovirus-polylysine/DNA complexes (AdpL) or polycationic liposomes mediated high reporter gene expression in dissociated primary human, fetal porcine, rat and mouse islet cells whereas gene transfer to intact islets was less efficient. Gene expression peaked after 5-7 days followed by a gradual decline.Both IL-1β and TNF-α activated transcription factor NF-κB in the rat insulin-producing cell line RINm5F without activating sphingomyelinase (SMase). However, ceramide and TNF-α potentiated IL-1β-induced activation of NF-κB and the inducible form of nitric oxide synthase (iNOS) as assessed by nitric oxide (NO) production. Moreover, the inhibitor of acid SMase, SR33557, suppressed these effects of TNF-α. Nα-p-tosyl-L-lysine chloromethyl ketone prevented IL-1β-induced activation of NF-κB and iNOS. Cytokines induced phosphorylation of p38 mitogen activated kinase (p38) and transcription factors ATF-1, CREB, CREM and ATF-2. The p38 inhibitor SB203580 counteracted CREB, CREM and ATF-1 phosphorylation and iNOS induction.In combination, but not alone, IL-1β, TNF-α and IFN-γ induced DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) after 24 h, both of which were counteracted by inhibition of iNOS. Cytokines induced morphological signs of both apoptosis and necrosis in rat islets and RINm5F cells whereas ceramide induced necrosis and early signs of apoptosis. The PARP-inhibitors nicotinamide and 3-aminobenzamide induced apoptosis in RINm5F cells and fetal but not adult rat islets. Hyperexpression of Bcl-2, achieved by stable transfection with a multicopy viral vector containing a bcl-2 cDNA insert, counteracted cleavage of PARP and both necrosis and apoptosis in RINm5F cells.In conclusion, AdpL or polycationic liposomes mediate transient expression of genes in islet cells. Full induction of NO production by cytokines may require basal acid SMase- and enhanced NF-κB- and p38 activities and promotes apoptosis or necrosis via the Bcl-2 preventable pathway in rat insulin-producing cells.