Annexin <scp>A2</scp> Contributes to Release of Extracellular Vimentin in Response to Inflammation

Vimentin, an abundant intracellular cytoskeletal protein, is secreted into the extracellular space, where it can amplify tissue destruction in inflammatory diseases. The mechanisms by which inflammation promotes the release of extracellular vimentin (ECV) are not defined. In human subjects, we found > twofold higher levels of ECV in gingival crevicular fluid from periodontitis sites with inflammation compared with healthy sites. In cultures of human gingival fibroblasts (hGFs) treated with 1% serum or IL-1β (10 ng/mL) to model tissue injury or inflammation, respectively, we found that 1% serum increased ECV release > 11-fold while IL-1β further enhanced release 17-fold. Mass spectrometry of vimentin immunoprecipitates identified Annexin A2 (AnxA2), a Ca²⁺-dependent phospholipid-binding protein, as a potential binding protein of ECV, which was confirmed by immunoprecipitation of cultured hGFs and immunostaining of inflamed human gingiva. IL-1β treatment enhanced the abundance of AnxA2 and vimentin in membrane fractions prepared by sucrose gradients of hGF lysates. IL-1β increased colocalization of ECV and AnxA2 at the outer aspect of the plasma membrane of intact hGFs. Knockdown of AnxA2 with siRNA or inhibition of the unconventional secretory pathway reduced ECV release from hGFs. These findings indicate that the production of ECV by hGFs in response to inflammation is mediated by an AnxA2-dependent, unconventional secretory pathway that may play a role in amplification of the inflammatory response.