The Nuclear Export Signal of IκBα Drives RelB Oscillations in the Noncanonical NF ‐

ABSTRACT

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.