The Macrophage Activation Induced by Bacillus thuringiensis Cry1Ac Protoxin Involves ERK1/2 and p38 Pathways and the Interaction with Cell‐Surface‐HSP70

Abstract

ABSTRACTHere, we aimed to further characterize the mechanisms involved in protoxin (p) Cry1Ac‐induced macrophage activation. We demonstrated that pCry1Ac induces MAPK ERK1/2, p38, and JNK phosphorylation in RAW264.7 macrophages. Because MAPK activation is mainly triggered via ligand–receptor interactions, we focused on the identification of potential pCry1Ac‐receptor proteins. Flow cytometry and confocal analysis showed specific saturable pCry1Ac‐binding to the macrophage surface and evidenced its internalization via the clathrin‐pathway. We performed immunoprecipitation assays and identified by MALDI‐TOF‐TOF several possible pCry1Ac‐binding proteins, such as heat shock proteins (HSPs), vimentin, α‐enolase, and actin; whose interaction and presence was confirmed, respectively, by ligand blot and Western blot assays. We also detected cell‐surface (cs) pCry1Ac‐HSP70 colocalization, so HSP70 was chosen for further characterization. Co‐immunoprecipitation with HSP70 antibodies followed by Western blot confirmed the pCry1Ac‐HSP70 interaction. Furthermore, pretreatment of RAW264.7 cells with HSP70 antibodies reduced pCry1Ac‐induced ERK1 phosphorylation and MCP‐1 production; thus suggest the functional participation of csHSP70 in pCry1Ac‐induced macrophage activation. csHSP70 also was evaluated in peritoneal‐cavity (PerC) macrophages of untreated BALB/c mice, interestingly it was found that the predominant population namely large‐peritoneal‐macrophages (LPM) displayed csHSP70 + hi. Furthermore, the dynamics of PerC macrophage subsets, LPM, and small‐peritoneal macrophages (SPM) were evaluated in response to in vivo pCry1Ac stimuli in presence or not of phenylethynesulfonamide (PES) a functional HSP70 inhibitor. It was found that pCry1Ac increased the proportion of SPM CD11b + F4/80 + lowMHCII + csHSP70 + low and markedly reduced the amount of LPM CD11b + F4/80 + hiMHCII‐csHSP70 + hi; while PES, partially suppressed this pCry1Ac‐induced effect, further suggesting the participation of HSP70 in macrophage activation process. J. Cell. Biochem. 119: 580–598, 2018. © 2017 Wiley Periodicals, Inc.