The Nuclear Export Signal of IκBα Drives RelB Oscillations in the Noncanonical NF-κB Pathway

The noncanonical NF-κB pathway regulates immune development and inflammation through RelB nuclear translocation, yet the dynamics of this process at the single-cell level remain poorly understood. Using live-cell imaging of RelB-Venus knock-in mouse embryonic fibroblasts, we show that LTβR or TWEAK stimulation induces four distinct RelB nuclear translocation patterns: oscillating, prolonged activation, transient activation, and non-responding. Approximately 40% of cells exhibited oscillatory behavior with a predominant period of 1.5-2.0 h, similar to RelA oscillations in the canonical pathway. Mechanistically, RelB oscillations required CRM1-mediated nuclear export, ongoing protein synthesis, NIK-dependent noncanonical signaling, and the IκBα nuclear export signal. Knockdown or knockout of Nfkb2 (encoding p100) induced spontaneous RelB oscillations without stimulation, identifying p100 as a threshold regulator that controls oscillation probability through cytoplasmic sequestration of RelB. Co-immunoprecipitation analysis revealed dynamic RelB-IκBα complex formation in both cytoplasmic and nuclear fractions following LTβR stimulation. Furthermore, disruption of RelB oscillations in Nfkbia^NES/NES cells was associated with impaired induction of NF-κB target genes. These findings provide the first experimental characterization of RelB oscillatory dynamics and reveal both conserved and pathway-specific mechanisms governing noncanonical NF-κB signaling.