Ac2-26 Induces IKKβ Degradation Through Chaperone-mediated Autophagy 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-αproduction and IKKβactivity,which was induced by oxygen glucose deprivation/reperfusion（OGD/R）induced neuronal conditioned medium（NCM）in microglia.Results:1.AC2-26 inhibits NCM-treated production of TNF-αfrom microglia:ELISA and RT-PCR were applied to detect protein secretion and gene expression of TNF-α.Results showed that high levels of TNF-αin the supernatant and in the mRNA after NCM,while administration of Ac2-26 decreased both levels and further inverted by lysosomal inhibitor NH₄CL.Western blot analyzed protein expression of TNF-αin microglia,and datas found that intracellular protein expression of TNF-αwas not significantly changed after different treatments.Confocal microscopy experiments on microglia revealed that most AC2-26 peptides were distributed diffusely through the cytoplasm.2.AC2-26 decreases NCM-induced IKKβactivity through autophagyWestern blot were applied to detect the expression of IKKβand p-IκB in microglia,and results showed that NCM-induced high levels of IKKβand p-IκB protein expression which inhibited by AC2-26.RT-PCR assessed mRNA levels of IKKβand no significant differences were found upon AC2-26 treatment despite the elevation for NCM treated alone.In addition,NH4CL clearly reversed the AC2-26-induced IKKβdeclination without affecting its mRNA.CO-IP analysis revealed that ANXA1 has no marked interaction with IKKβ.3.AC2-26 induces HSPB1 expression in microgliaWestern blot were applied to detect protein expression of HSPB1,and datas showed that HSPB1 increased in primary microglia treated with AC2-26 alone or before NCM.CO-IP analysis the interaction between ANXA1 and HSPB1,and results indicated that ANXA1physically associates with HSPB1.4.HSPB1 negatively regulates IKKβactivationCO-IP were applied to analysis the possible interaction between HSPB1 and IKKβ,and demonstrated that HSPB1 is associated with a few IKKβunder resting physiological conditions in microglia.In addition,NCM stimulation promoted HSPB1 to associate with IKKβin microglia,and AC2-26 further enhanced the interaction.Western blot were used to explore the interdependence between IKKβand HSPB1 in knockdown and overexpression experiments.Results showed that overexpression of GFP-HSPB1 reduced FLAG-IKKβboth in HeLa and BV-2 cells.Downregulated HSPB1 in microglia increased IKKβand TNF-αexpression.Meantime,knockdown of HSPB1 restored the loss of IKKβinduced by AC2-26.5.AC2-26 promotes CMA in microgliaWestern blot analyzed the autophagy markers and the results showed no variances in the conversion to LC3-II（a canonical autophagosome marker）and p62 accumulation,but increased LAMP-2A accompanied by CMA substrate protein GADPH accumulation.Both LC3-II and LAMP-2A were decreased in response to NCM exposure,and only LAMP-2A expression was restored by AC2-26.Confocal microscopy experiments on microglia further confirm that the increased LAMP-2A was mainly located in lysosomes.6.ANXA1 and HSPB1 assist the translocation of IKKβinto lysosomesProtein sequence analysis from NIH showed that IKKβ,ANXA1（AC2-26）and HSPB1 all bear putative CMA motifs in their amino acid sequence.CO-IP were applied to detect interaction between HSC70 and these proteins,and found an HSC70 associated wiht endogenous ANXA1,HSPB1 but not IKKβ,but all three proteins,including IKKβ,physically precipitated with HA-LAMP-2A.Knock down of HSC70 inhibited the interaction of LAMP-2A with three porteins.Knock down of ANXA1 completely blocked the interaction between LAMP-2A and IKKβ,while suppression of HSPB1 also presented weaker binding compared to control.7.Degradation of IKKβin lysosomes by CMALysosome isolated from rat brain tissue to assess the accumulation of IKKβ,ANXA1 and HSPB1 in lysosomes by immunoblotting,and all three proteins（IKKβ,HSPB1 and endogenous ANXA1）were detected to some extent in the isolated lysosomes.In addition,the amount of IKKβ,HSPB1 and endogenous ANXA1 in lysosomes was higher in AC2-26-treated tissue compared with control.ELISA and Western blot were applied to evaluate the contribution of CMA in IKKβdegradation,and depletion of LAMP-2A resulted in further accumulation of IKKβand subsequent TNF-αsecretion.In addition,by knocking down LAMP-2A before AC2-26 administration,the degradation of IKKβwas dramatically reduced compared with AC2-26 treatment alone.Consistently,knockdown of neither HSPB1 nor LAMP-2A affected IKKβmRNA.Conclusion:AC2-26 is associated with HSPB1 and promotes HSPB1 binding to IKKβ,which is degraded by CMA,thereby reducing TNF-αexpression.