Expression of the iNOS gene is induced by proinflammatory cytokines in rat islets and β-cell lines. A, 832/13 cells were treated with 0.1 or 1 ng/mL of IL-1β alone or in combination with 0, 10, or 100 U/mL IFN-γ for 6 hours. B and C, INS-1E (B) or rat islets (C) were treated with 1 ng/mL (INS-1E) or 10 ng/mL (rat islets) IL-1β in the presence or absence of 100 U/mL IFN-γ for 6 hours as indicated on the x-axis. D, After a 6-hour stimulation with 1 ng/mL IL-1β, cells were treated with actinomycin D at time 0 (indicated by the dashed line), and total RNA was isolated after 0.5, 1, or 2 hours. A–D, Total RNA was isolated and iNOS mRNA levels were measured and normalized to those of the housekeeping gene, ribosomal S9 (RS9). **, P < .01 vs untreated (NT) (B), *, P < .05 vs NT (B and C), **, P < .01 vs no actinomycin D (D, white bar); n.s., not significant vs no actinomycin D (D, white bar). E and F, 832/13 cells were transfected with either −1 kb (E) or −3.6 kb (F) of the iNOS promoter upstream of the transcriptional start site fused to a luciferase reporter. At 24 hours posttransfection, cells were stimulated for 4 hours with 1 ng/mL IL-1β or 1 ng/mL IL-1β plus 100 U/mL IFN-γ, followed by measurement of promoter activity. **, P < .01 vs IL-1β (E); *, P < .05 vs IL-1β (F). Data are means ± SEM from 3 to 4 individual experiments.

IκBα attenuates cytokine-mediated activation of the iNOS gene. A, 832/13 cells were transfected with a wild-type iNOS promoter luciferase plasmid (−1 kb) or iNOS promoter with mutations in the proximal or distal NF-κB response elements or in the GAS element. At 24 hours posttransfection, cells were stimulated with 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 4 hours. B, 832/13 cells were transduced with increasing doses of an adenovirus expressing the IκBα S32A/S36A (IκBα^SR [superrepressor]), followed by collection of total protein and immunoblotting for IκBα with β-actin as the loading control. The highest concentration of virus shown in the immunoblot was used for the experiments shown in panels C and D. This blot is representative of 2 independent experiments. C, 832/13 cells were treated with recombinant adenoviruses expressing either β-galactosidase (βGAL) or IκBα^SR. At 24 hours posttransduction, cells were stimulated for 6 hours with either 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ. Relative iNOS mRNA levels were quantified and normalized to RS9. **, P < .01 vs corresponding white or black bar, respectively. D, 832/13 cells were transfected with the −1 kb promoter-luciferase construct, and 4 hours later were treated with the indicated adenoviruses, cultured overnight, and then exposed to either 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for an additional 4 hours. Promoter activity was measured by luciferase assay. **, P < .01 vs the corresponding white or black bar, respectively. E, 832/13 cells were treated with recombinant adenoviruses expressing either βGAL or 5 and 10 multiplicity of infection of IκBα^SR for 12 hours and then were stimulated with cytokines as indicated for an additional 12 hours. Doses used are the same as those shown in panel B above. F, Cells were treated with dimethylsulfoxide or 0.5, 1, or 2 μM TPCA and concurrently stimulated with IL-1β for 12 hours. E and F, Nitrite production was measured from the culture media using the Griess assay. #, P < .1 vs βGAL-IL-1β; **, P < .01 vs βGAL-IL-1β + IFN-γ (E); **, P < .01 vs DMSO-IL-1β (F). Data are means ± SEM from 3 to 4 individual experiments. NT, untreated.

Cytokine-stimulated expression of the iNOS gene requires the NF-κB subunits p65 and p50. A and C, 832/13 cells were transfected with siRNA duplexes targeting either p65 (A) or p50 (C) with a scrambled sequence as a control for siRNA transfection (siScramble). At 48 hours posttransfection, cells were exposed to either 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 6 hours. A and C, Total RNA was isolated, and iNOS transcript levels were normalized to that of RS9. *, P < .05 vs corresponding siScramble white or black bar, respectively (Α); **, P < .01 vs corresponding siScramble white or black bar, respectively (Α); n.s., not significant vs siScramble white or black bar, respectively (C). B and D, 832/13 cells were cotransfected with a −1 kb promoter-luciferase construct and siRNA duplexes targeting either p65 (B) or p50 (D). At 24 hours posttransfection, cells were stimulated with 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 4 hours. B and D, Promoter luciferase activity was measured. *, P < .05 vs corresponding siScramble white or black bar, respectively (D); **, P < .01 vs corresponding siScramble white or black bar, respectively (Β and D). Values are means ± SEM from 3 to 4 individual experiments.

IL-1β induces recruitment of p65 and p50 to regions in the iNOS gene promoter containing κB response elements. A, 832/13 cells were untreated (NT) or exposed to 1 ng/mL IL-1β for 30 minutes. Immunofluorescence assays were used to track nuclear localization of p65. The assay was performed on 3 independent occasions, and representative images are shown (scale bar corresponds to 50 μm). B, 832/13 cells were untreated (−) or stimulated with 1 ng/mL IL-1β for 30 minutes (+). Electrophoretic mobility shift assays were performed using nuclear extracts incubated in the presence or absence of increasing concentrations of an unlabeled (“cold”) competitor oligonucleotide. The arrow indicates the specific complex bound to the NF-κB DNA sequence from the iNOS gene promoter. C–F, 832/13 cells were untreated or stimulated with 1 ng/mL IL-1β for 15, 30, or 60 minutes. ChIP assays were conducted with antibodies that immunoprecipitated p65 (C and E) or p50 (D and F). Distal (C and D) and proximal (E and F) NF-κB elements (indicated by the arrows in the schematic diagrams) in the iNOS promoter were targeted for amplification in the recovered DNA by real-time PCR. *, P < .05 vs respective NT control (C and D); **, P < .01 vs respective NT control (E and F). All data in panels C to F represent means ± SEM from 3 to 4 individual experiments, whereas the image in panel B is representative of 3 independent experiments.

Pharmacological inhibition of JAK1 attenuates the IFN-γ-mediated potentiation of IL-1β-stimulated iNOS gene expression. A, 832/13 cells were stimulated with 100 U/mL IFN-γ for 15, 30, 60, 120, and 240 minutes. Whole-cell lysates were harvested and blotted for phospho-STAT1 (Y701) and total STAT1, IκBα, and β-actin as the loading control. B, 832/13 cells were pretreated for 1 hour with the indicated doses of the JAK inhibitor (JAKi) and then were incubated for an additional 6 hours with 1 ng/mL IL-1β alone or in combination with 100 U/mL IFN-γ. iNOS mRNA abundance was quantified and normalized to that of RS9. **, P < .01 vs dimethylsulfoxide (DMSO) and IL-1β and IFN-γ. C, 832/13 cells were transfected with a −1-kb iNOS-luciferase reporter plasmid. At 24 hours posttransfection, cells were pretreated for 1 hour with the indicated doses of a JAKi and then were stimulated for 4 hours with either 1 ng/mL IL-1β or IL-1β plus 10 U/mL IFN-γ. Promoter activity was measured at the end of the 4-hour period. **, P < .01 vs DMSO and IL-1β and IFN-γ. D, 832/13 cells were transfected concomitantly with a −1-kb promoter-luciferase construct and siRNA duplexes targeting STAT1. At 24 hours posttransfection cells, were stimulated with 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 4 hours. Promoter activity was assessed by luciferase assay. *, P < .05 vs siScramble-IL-1β; **, P < .01 vs siScramble-IL-1β + IFN-γ. Data are means ± SEM from 3 individual experiments.

GAS elements within the iNOS gene promoter regulate sensitivity to IL-1β. A, 832/13 cells were transfected with a −1-kb iNOS promoter-luciferase reporter construct containing either the WT GAS, a perfect cGAS, or a GASm. At 24 hours posttransfection cells were stimulated with either 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 4 hours. *, P < .05 vs WT for intragroup comparisons. B, 832/13 cells were transfected with either a −3.6-kb iNOS promoter-luciferase construct or a −3.6-kb construct wherein both the proximal and distal GAS elements are mutated (−3.6-kb GAS double mutant [GAS DM]). After overnight culture, cells were treated with either 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for 4 hours. **, P < .01. C, 832/13 cells were transfected with either a −1-kb WT iNOS promoter-luciferase reporter or a −1-kb cGAS promoter-luciferase construct. After overnight culture, cells were treated with either 0.1 or 1 ng/mL IL-1β in the presence or absence of 100 U/mL IFN-γ for 4 hours. *, P < .05; n.s., not significant. D, 832/13 cells were transfected with the indicated −1-kb plasmids. At 24 hours posttransfection, cells were stimulated with 1 ng/mL IL-1β alone or in the presence of 100 U/mL IFN-γ. **, P < .01 vs the respective pκBm control; n.s., not significant vs dκBm. A–D, Promoter activity was assessed by luciferase assay. Data are means ± SEM from 3 individual experiments.

Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble) or targeting STAT1 (siSTAT1). At 24 hours posttransfection, cells were treated with adenoviruses expressing either green fluorescent protein or p65 and cultured for 12 hours. *, P < .05 vs corresponding white bar. B, 832/13 cells were treated overnight with adenoviruses expressing either β-galactosidase (βGAL) or STAT1. Cells were untreated (NT) or stimulated for 15 minutes with either 1 ng/mL IL-1β or 100 U/mL IFN-γ. Whole-cell lysates were blotted for phospho-STAT1 (S727 and Y701), total STAT1, or tubulin as the loading control. The immunoblot was repeated on 2 separate occasions. C, 832/13 cells were transfected with a −1-kb cGAS construct; 4 hours posttransfection, cells were transduced with adenoviruses expressing either βGAL, STAT1, or STAT1^Y701F for overnight culture. These cells were then exposed to either 1 ng/mL IL-1β alone or IL-1β plus 100 U/mL γ-IFN for 4 hours. *, P < .05. D, 832/13 cells were transfected with cGAS −1-kb construct; 4 hours postplasmid transfection, cells were transduced with adenoviruses expressing βGAL, STAT1, or STAT1^S727A overnight. After overnight culture with plasmid and virus, these cells were exposed to1 ng/mL IL-1β alone or in combination with 100 U/mL IFN-γ for 4 hours. A, C, and D, Promoter activity was assessed by luciferase assay. E, Rat islets were transduced with the indicated adenoviruses. After overnight culture, islets were treated with 10 ng/mL IL-1β and 100 U/mL IFN-γ for 6 hours. iNOS mRNA abundance was normalized to RS9. F, 832/13 cells were untreated or treated for 15 minutes with 1 ng/mL IL-1β (β) or IL-1β plus 100 U/mL γ-IFN (βγ). Lysates were immunoprecipitated (IP) with antibodies against either normal rabbit serum (IgG) or p65 and then were separated by SDS-PAGE, followed by immunoblotting (IB) with antibodies targeting p65, STAT1, or IκBα (arrow indicates IκBα as the faster migrating band). G and H, 832/13 cells were stimulated for 15 minutes with IL-1β. ChIP assays were conducted using antibodies against IgG, STAT1 Y701, and STAT1 S727. The distal and proximal GAS elements were targeted for amplification by RT-PCR using the recovered DNA as template. **, P < .01 vs STAT1 S727 untreated (black bars). Data are means ± SEM from 3 individual experiments.

IL-1β promotes exchange of coactivators to regulate transcription of the iNOS gene promoter. A and B, 832/13 cells were stimulated with 1 ng/mL IL-1β for 15 minutes. ChIP assays were performed using antibodies against IgG (control), p65, CARM1, p300, and CBP. Proximal (A) and distal (B) NF-κB elements (indicated by the arrows in the schematic diagrams) in the iNOS promoter were targeted for amplification by real-time PCR. #, P < .1 vs NT for respective antibody; *, P < .05 vs NT for respective antibody; **, P < .01 vs NT for respective antibody. C, 832/13 cells were transfected with either a −1-kb iNOS promoter-luciferase construct or a −1-kb GASm construct. In addition, cells were cotransfected with the indicated combinations of p65, p300, and CARM1 and incubated for 24 hours. D, 832/13 cells were transfected with a −1-kb iNOS promoter-luciferase construct and concurrently with the combinations of p65, CBP, and CARM1 shown. Cells were cultured for a further 24 hours posttransfection. C and D, Promoter activity was measured by luciferase assay and normalized to protein content as quantified by BCA assay. *, P < .05. Data are means ± SEM from 3 individual experiments.

Coordinated chromatin remodeling by IL-1β correlates with recruitment of phosphorylated RNA Pol II. A–H, 832/13 cells were untreated (NT) or stimulated with 1 ng/mL IL-1β for 15, 30, or 60 minutes. ChIP assays were performed with antibodies that immunoprecipitated histone H3 phosphorylated on serine 10 (A and B), histone H3 methylated on lysine 4 (C), lysine 9 (D), and lysine 27 (E), histone H3 acetylated on lysine 14 (F), and RNA Pol II phosphorylated on the CTD tail at serine 5 (G) or serine 2 (H). Proximal (A, C, D, E, and F) and distal (B) NF-κB response elements in the iNOS gene promoter (indicated by arrows in the schematic diagrams) were targeted for amplification by real-time PCR. G and H, Primers targeting either the iNOS core promoter region or coding region were used for amplification in real-time RT-PCR experiments. n.s., not significant vs respective NT control; #, P < .1 vs respective NT control; *, P < .05 vs respective NT control; **, P < .01 vs respective NT control. Data are means ± SEM from 3 to 4 individual experiments.

Cytokine-mediated regulation of the iNOS gene occurs via a coactivator switching mechanism. A, The iNOS gene is silent in pancreatic β-cells in the absence of IL-1β and IFN-γ. RelA/p65 is held in the cytoplasm by regulatory proteins, such as IκBα, whereas the coregulator molecule CBP associates with the transcriptionally inactive iNOS gene promoter in the absence of cytokines. B, Upon β-cell exposure to IL-1β, the IκBα protein is subject to phosphorylation-induced degradation; p65 enters the nucleus and binds κB elements with its heterodimer partner p50. Signaling through the IL-1 receptor (IL-1R) also increases occupancy of Ser727-phosphorylated STAT1 at GAS elements within the iNOS gene promoter. IFN-γ, through JAK-STAT signaling mechanisms, promotes Tyr701 phosphorylation of STAT1, which amplifies the cellular response to IL-1β. These signaling events induce removal of CBP from the iNOS promoter concomitant with the arrival of the coactivators p300 and CARM1.The marked increase in iNOS transcript leads to increased iNOS protein abundance. iNOS catalyzes the production of nitric oxide (NO^•) and citrulline using arginine as a substrate. NO^• accumulation in pancreatic β-cells is linked to losses in insulin secretion and cellular viability.