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

J Cell Biochem. 2018 Jan;119(1):580-598. doi: 10.1002/jcb.26216. Epub 2017 Jul 11.

Abstract

Here, 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.

Keywords: ADJUVANT; BACILLUS THURINGIENSIS; Cry1Ac RECEPTORS; HSP70; MAPKs.

MeSH terms

  • Animals
  • Bacillus thuringiensis / chemistry*
  • Bacillus thuringiensis Toxins
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / pharmacology*
  • Endotoxins / chemistry
  • Endotoxins / pharmacology*
  • HSP70 Heat-Shock Proteins / metabolism*
  • Hemolysin Proteins / chemistry
  • Hemolysin Proteins / pharmacology*
  • MAP Kinase Signaling System / drug effects*
  • Macrophage Activation / drug effects*
  • Macrophages / cytology
  • Macrophages / metabolism*
  • Mice
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • RAW 264.7 Cells
  • p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

  • Bacillus thuringiensis Toxins
  • Bacterial Proteins
  • Endotoxins
  • HSP70 Heat-Shock Proteins
  • Hemolysin Proteins
  • insecticidal crystal protein, Bacillus Thuringiensis
  • Mitogen-Activated Protein Kinase 3
  • p38 Mitogen-Activated Protein Kinases