Intracellular Stability of Precursor IL-33 is Maintained through Interaction with Importin-

Interleukin (IL)-33 is a member of the IL-1 family and a potent regulator of immunity. The precursor, or full-length (FLIL33), form behaves differently from the mature (MIL33) cytokine. MIL33 is a potent pro-Th2 cytokine acting through the T1/ST2 receptor, whereas FLIL33 is constitutively expressed and resides predominantly in the nucleus of epithelial and endothelial cells, as well as fibroblasts. Increasing evidence suggests that this intracellular FLIL33 can mediate inflammatory responses and wound healing in a non-Th2 and receptor-independent manner. Additionally, nuclear retention of FLIL33 is essential to maintain homeostasis and prevent unintentional sterile inflammation. FLIL33 has been shown to bind histones and chromatin, but the mechanism of nuclear localization remains elusive. Though FLIL33 contains a conserved bipartite nuclear localization sequence (NLS), this is not required for nuclear localization.;Importins are a family of proteins that act as chaperones for import of proteins through the nuclear pore complex (NPC). Here, importin-5 (IPO5) is identified as a unique intracellular binding partner of IL-33 based on co-immunoprecipitation of HA-tagged IL-33 from normal human lung fibroblasts (NHLF). It was hypothesized that IPO5 binds the N-terminal domain of FLIL33 and that nuclear localization of FLIL33 is dependent on IPO5. To test this hypothesis, HA-tagged FLIL33 and HA-tagged peptide fragments of FLIL33 were expressed in cell culture and immunoprecipitated. We show that FLIL33, but not MIL33, co-immunoprecipitates with IPO5 and that this interaction localizes to the N-terminal domain of FLIL33. Knockdown of IPO5 in NHLFs using RNA interference did not prevent nuclear localization of FLIL33. Instead, attenuation of IPO5 led to a significant decrease in the intracellular protein levels of overexpressed FLIL33, which was reversed by treatment of cells with bortezomib, a proteasome inhibitor. Furthermore, FLIL33 mutant proteins deficient in IPO5-binding remained intra-nuclear. These mutants also had reduced protein levels that were restored by proteasome inhibition with bortezomib. These data indicate that the interaction between FLIL33 and IPO5 localizes to specific segments of the FLIL33 protein, is not required for nuclear localization of FLIL33, and results in its protection from proteasome-dependent degradation.