Cytokine Induction of Fas Gene Expression in Insulin-Producing Cells Requires the Transcription Factors NF-κB and C/EBP

Cytokine Induction of Fas Gene Expression in Insulin-Producing Cells Requires the Transcription Factors NF-κB and C/EBP Martine I. Darville and Décio L. Eizirik Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Abstract Fas-mediated cell death may play a role in the autoimmune destruction of pancreatic β-cells in type 1 diabetes. β-Cells do not express Fas under physiological conditions, but Fas mRNA and protein are induced in cytokine-exposed mouse and human islets, rendering the β-cells susceptible to Fas ligand–induced apoptosis. The aim of the present study was to investigate the molecular regulation of Fas by cytokines in rat β-cells and in insulin-producing RINm5F cells. Fas mRNA expression was increased 15-fold in fluorescence-activated cell sorting–purified rat β-cells exposed to interleukin (IL)-1β, whereas γ-interferon had no effect. Transfection experiments of rat Fas promoter-luciferase reporter constructs into purified rat β-cells and RINm5F insulinoma cells identified an IL-1β–responsive region between nucleotides −223 and −54. Inactivation of two adjacent NF-κB and C/EBP sites in this region abolished IL-1β–induced Fas promoter activity in RINm5F cells. Binding of NF-κB and C/EBP factors to their respective sites was confirmed by gel shift assays. In cotransfection experiments, NF-κB p65 transactivated the Fas promoter. NF-κB p50 and C/EBPβ overexpression had no effect by themselves on the Fas promoter activity, but when cotransfected with p65, each factor inhibited transactivation by p65. These results suggest a critical role for NF-κB and C/EBP factors in cytokine-regulation of Fas expression in insulin-producing cells. Footnotes Address correspondence and reprint requests to Dr. Martine I. Darville, Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090-Brussels, Belgium. E-mail: mdarv{at}mebo.vub.ac.be . Received for publication 17 October 2000 and accepted in revised form 2 May 2001. ANOVA, analysis of variance; CMV, control vector; EMSA, electrophoretic mobility shift assay; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; ICAM-1, intercellular adhesion molecule-1; IFN-γ, γ-interferon; IL, interleukin; iNOS, inducible form of nitric oxide synthesis; l -MA, N G -methyl- l -arginine; NO, nitric oxide; PCR, polymerase chain reaction; RT, reverse transcriptase; TNF, tumor ne