A NIK-SIX signaling axis controls inflammation by targeted silencing of noncanonical NF-κB

The non-canonical NF-κB signaling cascade is essential for lymphoid organogenesis, B-cell maturation, osteoclast differentiation, and inflammation in mammals, whereas dysfunction of this system is associated with human diseases, including immunological disorders and cancer. While controlled expression of NF-κB Inducing Kinase (NIK) is the rate-limiting step in non-canonical NF-κB activation, mechanisms of inhibition remain largely unknown. Here, we report the identification of the sine oculis homeobox homolog family transcription factors SIX1 and SIX2 as essential inhibitory components of the non-canonical NF-κB signaling pathway. The developmentally silenced SIX-proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit RelA and RelB trans-activation function in a negative feedback circuit. In support of a physiologically pivotal role for SIX-proteins in host immunity, human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/p53-driven lung adenocarcinoma cells from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents, small-molecule activators of the non-canonical NF-κB pathway. Collectively, our study reveals a NIK-SIX signaling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions. In this data set, we first analyzed NIK-stimulated genes by expressing NIK and Fluc (control experiment) in STAT1-deficient fibroblasts. Each group has 2 independent experiments. Second, we analyzed the SIX2-downregulated NIK-stimulated genes, FlucRFP/FlucBFP, NIKRFP/FlucBFP, NIKRFP/SIX2BFP, or FlucRFP/SIX2BFP lentivirus was transduced into WT fibroblasts. We then analyzed the whole genome transcription prfiles. Each group has 2 independent experiments. Third, WT and SIX1-/- SIX2-/- H1792 treated with mock or BV6/TNF for 24 hours. we analyzed the whole genome transcription profiles. Each group has 2 independent experiments.