1501-P: Four Loci, Four Fates—Differential Regulation of IRF5 Promoter in Prodiabetogenic Stress

Introduction: Interferon regulatory factor (IRF)-5 is a transcription factor sitting at the crossroads of diverse cell processes. Emerging evidence supports its upregulation in response to different stimuli, however, the associated molecular mechanisms remain elusive.

Methods: Four distinct IRF5 regulatory loci 1A, 1B, 1C, & 1D were individually cloned into Luciferase-based reporter vectors. RAW 264.7 macrophage cells were transfected with each of four reporter constructs and cells were exposed for 24h to stress stimuli including 100 µM palmitate (saturated FA), 100 µM oleate (unsaturated FA), 100 µM H₂O₂ (ROS inducer), 100 ng/mL TNF-α (inflammatory cytokine), 25 mM glucose (hyperglycemia), or 30 ng/mL IFN-γ (inflammatory cytokine). Luciferase activity was measured and normalized to Renilla luciferase activity. In additional experiments, co-transfections were performed using each promoter construct and expression vectors for E2F, NRF2, and STAT3 to investigate their potential roles. Statistical analysis was performed using two-way ANOVA.

Results: Luciferase assays revealed a differential response of the four IRF5 promoter loci. IRF5-1C exhibited the least significant regulation in mouse macrophages. Alternatively, 1A, 1B, and 1D displayed the increased reporter activities (300-fold compared to the control construct). Palmitate and H₂O₂ treatments triggered activation at all four promoter sites, while oleate stimulated only the 1D promoter region. Interestingly, TNF-α, hyperglycemia, and IFN-γ failed to induce IRF5 promoter activity at any of these loci. Co-transfections with transcription factor E2F stimulated luciferase activity at all four promoter loci, NRF2 stimulated activity at loci 1A, 1B, and 1D, and STAT3 stimulated activity at loci 1A and 1B.

Conclusion: Our findings demonstrate that the four IRF5 promoter loci exhibit distinct responses to stimuli that mimic the pro-diabetogenic stress via the mechanism involving specific transcriptional regulators.