FIGURE 6.

IRF-1 hampers STAT1 activation through the induction of SOCS-1. A, C, and D, INS-1E cells were left untransfected (NT) or transfected with 30 nm si-control, siIRF-1, or siIRF-1 primer 2. After 24 h of recovery, cells were left untreated or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 2, 4, 8, 16, or 24 h as indicated. A, phospho-STAT1, total STAT1, IRF-1, PTPN2, and α-tubulin protein expressions were evaluated by Western blot. Pictures are representative of five independent experiments. B, INS-1E cells were co-transfected with a STAT1 reporter and pRL-CMV alone (NT) or in combination with si-control, siIRF-1, or siIRF-1 primer 2. After 1 day of recovery, cells were left untreated or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 24 h as indicated. Results are mean relative luciferase unit (R.L.U.) ± S.E. of five independent experiments. C and D, IRF-1 and SOCS-1 mRNA expressions were assayed by real-time RT-PCR and normalized for the housekeeping gene GAPDH. Results are mean ± S.E. of four independent experiments. E and F, INS-1E cells were transfected with pCMV-Ctrl or pCMV-IRF-1. After overnight incubation, the cells were left untreated (time 0) or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 2, 4, 8, 16, or 24 h as indicated. E, IRF-1 and α-tubulin protein expressions were evaluated by Western blot in untreated cells 24 h after transfection. Pictures are representative of four independent experiments. F, SOCS-1 mRNA expression was assayed by real-time RT-PCR and normalized for the housekeeping gene GAPDH. Results are mean ± S.E. of four independent experiments. G–J, INS-1E cells were transfected with 30 nm si-control, siSOCS-1, or siSOCS-1 primer 2. After 24 h of recovery, cells were left untreated or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 24 h as indicated. SOCS-1, CXCL1, CXCL9, and CXCL10 mRNA expression was assayed by real-time RT-PCR and normalized for the housekeeping gene GAPDH. Results are mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus respective untreated control. §, p < 0.05; §§, p < 0.01; and §§§, p < 0.001 versus respective control treated with cytokines at the same time point, ANOVA followed by Student's t test with Bonferroni correction. K, schematic representation of the suggested regulatory loop controlled by IRF-1 in β-cells. IFN-γ binding to its receptor induces Jak-mediated STAT1 phosphorylation and dimerization (1) and its subsequent migration to the nucleus (2), where it stimulates the transcription of many genes, including chemokines and IRF-1. Once synthesized in the cytoplasm (3), IRF-1 also migrates to the nucleus (4) to stimulate the transcription of several genes including SOCS-1 (5). SOCS-1 may then interfere with Jak-mediated STAT1 phosphorylation (6), hence hampering STAT1 activation over time. In the absence of IRF-1 signaling, the defective SOCS-1 induction allows prolonged Jak-mediated STAT1 phosphorylation (7) and sustained STAT1 activity (8).