Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism

Abstract

The proinflammatory cytokines IL-1β and IFN-γ decrease functional islet β-cell mass in part through the increased expression of specific genes, such as inducible nitric oxide synthase (iNOS). Dysregulated iNOS protein accumulation leads to overproduction of nitric oxide, which induces DNA damage, impairs β-cell function, and ultimately diminishes cellular viability. However, the transcriptional mechanisms underlying cytokine-mediated expression of the iNOS gene are not completely understood. Herein, we demonstrated that individual mutations within the proximal and distal nuclear factor-κB sites impaired cytokine-mediated transcriptional activation. Surprisingly, mutating IFN-γ-activated site (GAS) elements in the iNOS gene promoter, which are classically responsive to IFN-γ, modulated transcriptional sensitivity to IL-1β. Transcriptional sensitivity to IL-1β was increased by generation of a consensus GAS element and decreased correspondingly with 1 or 2 nucleotide divergences from the consensus sequence. The nuclear factor-κB subunits p65 and p50 bound to the κB response elements in an IL-1β-dependent manner. IL-1β also promoted binding of serine-phosphorylated signal transducer and activator of transcription-1 (STAT1) (Ser727) but not tyrosine-phosphorylated STAT1 (Tyr701) to GAS elements. However, phosphorylation at Tyr701 was required for IFN-γ to potentiate the IL-1β response. Furthermore, coactivator p300 and coactivator arginine methyltransferase were recruited to the iNOS gene promoter with concomitant displacement of the coactivator CREB-binding protein in cells exposed to IL-1β. Moreover, these coordinated changes in factor recruitment were associated with alterations in acetylation, methylation, and phosphorylation of histone proteins. We conclude that p65 and STAT1 cooperate to control iNOS gene transcription in response to proinflammatory cytokines by a coactivator exchange mechanism. This increase in transcription is also associated with signal-specific chromatin remodeling that leads to RNA polymerase II recruitment and phosphorylation.

IFN-γ Priming of Macrophages Represses a Part of the Inflammatory Program and Attenuates Neutrophil Recruitment

“…Current literature mainly focuses on how IFN-g potentiates LPS-induced gene expression. For several typical M1 marker genes, it was very elegantly shown how STAT1, the transcription factor activated by IFN-g, helps NF-kB to increase inflammatory gene transcription (9,27,28). Although in our study we also found LPS-induced genes, including typical M1 markers like Nos2 and Il12b, to be upregulated, we identified an even larger proportion of LPS-induced genes that were repressed by prior IFN-g priming.…”

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

“…Accordingly, interfering with STAT1 abundance or activity diminishes production of the chemoattractant signals that promote immune cell invasion into islets. In addition, diminishing STAT1 activity also impacts the IL-1␤-mediated expression of the inducible nitric oxide synthase (iNOS) gene (8). The expression of iNOS and subsequent production of NO is strongly associated with losses in functional ␤-cell mass (15)(16)(17)70).…”

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB

“…Proinflammatory cytokines such as IL-1␤ and IFN␥ contribute significantly to losses in islet ␤-cell viability and insulin secretion. Islet ␤-cells exposed to IL-1␤ and IFN␥ undergo extensive genetic reprogramming, which includes transcriptional increases in the inducible nitric oxide synthase (iNOS) gene (13,26) and various genes encoding chemokines (8 -10, 12, 44).…”

“…IL-1␤ induces the expression of the iNOS gene, promoting marked accumulation of iNOS protein, a phenotype potentiated by the addition of IFN␥ (13,16,17,26). The active iNOS enzyme produces nitric oxide, a free radical signaling molecule that impacts numerous cellular functions (5,28,46).…”

“…Interestingly, CCL2, CCL20, and iNOS are all bona fide target genes controlled by the NF-B family of transcription factors (8,11,13). NF-B subunits include p65 (RelA), RelB, c-Rel, p50 (NF-B1), and p52 (NF-B2).…”