Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia.

Annexin A1 (ANXA1) is an endogenous protein with potent anti-inflammatory properties in the brain. Although ANXA1 has been predominantly studied for its binding to formyl peptide receptors (FPRs) on plasma membranes, little is known regarding whether this protein has an anti-inflammatory effect in the cytosol. Here, we investigated the mechanism by which the ANXA1 peptide Ac2-26 decreases high TNF-alpha production and IKK beta activity, which was caused by oxygen glucose deprivation/reperfusion (OGD/R)induced neuronal conditioned medium (NCM) in microglia. We found that exogenous Ac2-26 crosses into the cytoplasm of microglia and inhibits both gene expression and protein secretion of TNF-alpha. Ac2-26 also causes a decrease in IKK beta protein but not IKK beta mRNA, and this effect is inverted by lysosome inhibitor NH4CL. Furthermore, we demonstrate that Ac2-26 induces IKK beta accumulation in lysosomes and that lysosomal-associated membrane protein 2A (LAMP-2A), not LC-3, is enhanced in microglia exposed to Ac2-26. We hypothesize that Ac2-26 mediates IKK beta degradation in lysosomes through chaperone-mediated autophagy (CMA). Interestingly, ANXA1 in the cytoplasm does not interact with IKK beta but with HSPB1, and Ac2-26 promotes HSPB1 binding to IKK beta. Furthermore, both ANXA1 and HSPB1 can interact with Hsc70 and LAMP-2A, but IKK beta only associates with LAMP-2A. Downregulation of HSPB1 or LAMP-2A reverses the degradation of IKK beta induced by Ac2-26. Taken together, these findings define an essential role of exogenous Ac2-26 in microglia and demonstrate that Ac2-26 is associated with HSPB1 and promotes HSPB1 binding to IKK beta, which is degraded by CMA, thereby reducing TNF-alpha expression.