Cytokine Induction of Fas Gene Expression in Insulin-Producing Cells Requires the Transcription Factors NF-κB and C/EBP
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Figures
- FIG. 1.
Cytokine regulation of Fas mRNA expression in rat β-cells. Rat β-cells were cultured for 6 or 24 h in control medium (C, no cytokine added) or with IL-1β (30 U/ml), IFN-γ (1,000 U/ml), or IL-1β + IFN-γ. Fas and GAPDH mRNA content was analyzed by RT-PCR. A: A representative experiment is shown. B: Shown are the relative Fas mRNA contents expressed as optical densities, corrected for GAPDH. The results are the means ± SE of three to five independent experiments. *P < 0.05, **P < 0.01 vs. control (ANOVA).
- FIG. 2.
Localization of the cytokine-responsive elements in the Fas promoter in rat β-cells. A: Schematic representation of the rat Fas promoter. Putative binding sites for transcription factors are indicated by boxes. Hatched boxes represent binding sites that were mutated in this study. The promoter fragments cloned upstream of the luciferase reporter gene are indicated in the lower part of the figure. B: β-Cells were transfected with the 5′ deleted promoter constructs shown in A and left untreated (control [C]) or treated with IL-1β for 16 h. Relative luciferase activity values are the means ± SE of five independent experiments. The relative luciferase activity value of the promoterless pGL3 vector is 0.0008. **P < 0.01 vs. respective control (ANOVA).
- FIG. 3.
Localization of the cytokine-responsive elements in the Fas promoter in RINm5F cells. A: The cells were transfected with the 5′ deleted promoter constructs shown in Fig. 2A and left untreated (control [C]) or treated with IL-1β for 16 h. Relative luciferase activity values are the means ± SE of four independent experiments. **P < 0.01 vs. respective control (ANOVA). B: The cells were transfected with the promoterless pGL3 vector, the wild-type (WT) pFas-811 luc, or the same construct with deletion of the NF-κB and C/EBP sites indicated in Fig. 2A (ΔNF–C/EBP), or with either the NF-κB site mutated (NFmut) or the C/EBP site mutated (C/EBPmut). The cells were treated as in A. Relative luciferase activity values are the means ± SE of three independent experiments. **P < 0.01 vs. respective control (ANOVA).
- FIG. 4.
NF-κB binding to the Fas promoter. A: EMSAs were performed in electrophoresis buffer Tris-borate with an oligonucleotide spanning from nucleotides −148 to −116 and containing the NF-κB and C/EBP sites (probe Fas NF-C/EBP) or with the C/EBP site mutated (probe Fas NF-C/EBPmut), and with nuclear extracts from RINm5F cells left untreated (control [C]) or treated with IL-1β for 30 min or 4 h. Arrows a and b indicate specific complexes; ns, nonspecific complexes; F, free probe. B: EMSAs were performed with the same probes as in A and with nuclear extracts from RINm5F cells treated with IL-1β for 30 min and with competing oligonucleotides (lanes 2–5 and 13) or specific antibodies (lanes 6–8, 10, 11, 14, 16, and 17) added to the reaction as indicated above the lanes. Arrowheads indicate supershifted complexes. The figures are representative of three similar experiments.
- FIG. 5.
C/EBP binding to the Fas promoter. EMSAs were performed in electrophoresis buffer Tris-glycine with an oligonucleotide spanning from nucleotides −134 to −116 and containing the C/EBP site (Fas C/EBP), using nuclear extracts from RINm5F cells treated with IL-1β for 2 h. Competing oligonucleotides (lanes 2 and 3) or specific antibodies (lanes 4–6) were added to the reaction as indicated above the lanes. Arrows c and d indicate specific complexes; ns, nonspecific complexes; , ◃, supershifted complexes. The figure is representative of two similar experiments.
- FIG. 6.
Effect of overexpression of NF-κB p65, NF-κB p50, and C/EBPβ on Fas promoter activity. A: RINm5F cells were cotransfected with the wild-type pFas-811 luc and 0.005–0.1 μg of the expression vector CMV-p65. The amount of DNA transfected was kept constant by adding the control vector CMV. Relative luciferase activity values are the means ± SE of four independent experiments. B: The wild-type (WT) pFas-811 luc or the same construct with deletion of the NF-κB and C/EBP sites indicated in Fig. 2A (ΔNF-C/EBP), or with either the NF-κB site mutated (NFmut) or the C/EBP site mutated (C/EBPmut), were cotransfected with 0.01 μg of CMV-p65 or the control plasmid CMV. Relative luciferase activity values are the means ± SE of three independent experiments. C and D: The wild-type pFas-811 luc was cotransfected with 0.01 μg of CMV-p65 and/or increasing amounts of CMV-p50 from 0.005–0.05 μg (in C) or increasing amounts of CMV-C/EBPβ from 0.0025 to 0.02 μg (in D). Relative luciferase activity values are the means ± SE of four independent experiments. *P < 0.05, **P < 0.01 vs. p65 alone (paired t test).
- FIG. 7.
Transient overexpression of NF-κB p65, NF-κB p50, and C/EBPβ in RINm5F cells. Cells were transfected with the expression vectors for p65, p50, or C/EBPβ alone, or for p65 in combination with p50 or C/EBPβ at a one-to-two ratio, or with the empty vector pCMV, as indicated above the lanes. Nuclear proteins were fractionated by SDS-PAGE and immunoblotted with antibodies specific for p65 or Oct-1 (used as control for protein loading) in A; p50 in B; and C/EBPβ in C. In A, untransfected cells exposed to IL-1β for 30 min (IL) were used as a positive control. Arrowheads indicate the expressed proteins. ns, A nonspecific band. The figure is representative of two similar experiments.
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