Inhibition of Cytokine-Induced NF-κB Activation by Adenovirus-Mediated Expression of a NF-κB Super-Repressor Prevents β-Cell Apoptosis
- Harry Heimberg1,
- Yves Heremans1,
- Christian Jobin2,
- Ruth Leemans1,
- Alessandra K. Cardozo1,
- Martine Darville1 and
- Décio L. Eizirik1
- 1Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
- 2Division of Digestive Diseases and Nutrition, University of North Carolina, Chapel Hill, North Carolina
Cytokine-induced β-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-κB (NF-κB) is activated by interleukin-1β (IL-1β), and its activity promotes the expression of several β-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1β + γ-interferon [IFN-γ])-induced expression of NF-κB in β-cell apoptosis, rat β-cells were infected with the recombinant adenovirus AdIκB(SA)2, which contained a nondegradable mutant form of inhibitory κB (IκB(SA)2, with S32A and S36A) that locks NF-κB in a cytosolic protein complex, preventing its nuclear action. Expression of IκB(SA)2 inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-κB. Cytokine-induced gene expression of several NF-κB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIκB(SA)2, as established by reverse transcriptase–polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, β-cell survival after IL-1β + IFN-γ treatment was significantly improved by IκB(SA)2 expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-κB activation, under in vitro conditions, has primarily a pro-apoptotic function in β-cells.
Address correspondence and reprint requests to H. Heimberg, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail:.
H.H. and Y.H. contributed equally to this work.
Received for publication 6 October 2000 and accepted in revised form 13 July 2001.
ANOVA, analysis of variance; FasL, ligand protein for Fas; GAPDH, glyceraldehyde-phosphate dehydrogenase; GFP, green fluorescent protein; IFN-γ, γ-interferon IκB, inhibitory κB; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; MnSOD, manganese superoxide dismutase; MOI, multiplicity of infection; NO, nitric oxide; PCR, polymerase chain reaction; PI, propidium iodide.