Abstract
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-α production and IKKβ 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-α. Ac2-26 also causes a decrease in IKKβ protein but not IKKβ mRNA, and this effect is inverted by lysosome inhibitor NH4CL. Furthermore, we demonstrate that Ac2-26 induces IKKβ 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 IKKβ degradation in lysosomes through chaperone-mediated autophagy (CMA). Interestingly, ANXA1 in the cytoplasm does not interact with IKKβ but with HSPB1, and Ac2-26 promotes HSPB1 binding to IKKβ. Furthermore, both ANXA1 and HSPB1 can interact with Hsc70 and LAMP-2A, but IKKβ only associates with LAMP-2A. Downregulation of HSPB1 or LAMP-2A reverses the degradation of IKKβ 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 IKKβ, which is degraded by CMA, thereby reducing TNF-α expression.
Highlights
Worldwide, stroke is a leading cause of death and disability with limited therapeutic options (Macrez et al, 2011), and ischemic stroke alone represents 80%–85% of acute strokes (Flynn et al, 2008)
Consistent with this finding, microglia cultured in neuronal conditioned medium (NCM) for 24 h exhibited a strong increase in TNF-α mRNA expression, which was reversed by Ac2-26 treatment and induced in the presence of NH4CL but not BOC-1 (Figure 1B)
We hypothesized that Ac2-26 affects TNF-α secretion and gene expression in microglia and that reduced TNF-α is associated with autophagy
Summary
Stroke is a leading cause of death and disability with limited therapeutic options (Macrez et al, 2011), and ischemic stroke alone represents 80%–85% of acute strokes (Flynn et al, 2008). Sudden occlusion of cerebral blood flow leads to energy depletion and necrotic neuronal death, which can trigger immune responses that lead to inflammatory cell activation. On one hand, lesioned neurons rapidly change their gene expression and generate multiple factors such as ATP and glutamate that stimulate nearby microglia activation and migration to protect the brain against ischemic and excitotoxic injury (Neumann, 2001; Lo et al, 2003; Luo et al, 2014). Excessively activated microglia release a large variety of proinflammatory mediators, including reactive oxygen species (ROS), IL-1 and TNF-α, which exacerbate tissue damage and contribute to the delayed exacerbation of ischemic brain deficits (Nathan and Ding, 2010; Graeber et al, 2011; Fernandes et al, 2014; Tuttolomondo et al, 2014). Blocking endogenous TNF-α, which is mainly generated by microglia (Zhang et al, 2013), significantly reduces focal ischemic brain injury and infarct size (Barone et al, 1997)
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