BCG Increased Membrane Expression of TRIM59 Through the TLR2/ TLR4/IRF5 Pathway in RAW264.7 Macrophages

Protein Pept Lett. 2017;24(8):765-770. doi: 10.2174/0929866524666170818155524.

Abstract

Objective: In our previous study, we showed that Bacillus Calmette-Guerin (BCG)- activated macrophages have the ability to directly kill tumor cells. One of the main properties of these macrophages is the high expression of tripartite motif family protein 59 (TRIM59). This study was conducted to investigate the mechanism of BCG-induced TRIM59 expression on macrophages and to identify the subcellular localization of TRIM59.

Methods: TRIM59 expression and TNF-α secretion were compared in RAW264.7 macrophage cells that were stimulated using BCG with or without Toll-like receptor 2/4 (TLR2/4)-neutralizing antibodies. Next, small interfering RNA (siRNA) was used to down-regulated interferon regulatory factor 5 (IRF5) gene expression in RAW264.7 cells. Transfected cells were stimulated with BCG, after which TRIM59 expression and TNF-α secretion were evaluated in cells pre-treated with siRNA or scramble control. After treatments, supernatants were co-cultured with MCA207, and cell viabilities were determined. Moreover, BCG-stimulated RAW264.7 cells were stained for TRIM59 and F4/80 expression.

Results: In this study, we showed that TRIM59 was expressed on the membrane of RAW264.7 cells. After blocking TLR2/4, treatment with BCG failed to induce the expression of TRIM59, IRF5, and TNF-α on RAW264.7 cells. In addition, down-regulation of IRF5 inhibited TRIM59 and TNF-α expression.

Conclusion: Our study showed that TRIM59 is a membrane protein, and that BCG treatment upregulated TRIM59 expression on macrophages via TLR2/4 and IRF5 pathways.

Keywords: BCG; IRF5; TLR2; TLR4; TRIM59; macrophages.

MeSH terms

  • Animals
  • Antibodies, Neutralizing / pharmacology
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Culture Media, Conditioned / isolation & purification
  • Culture Media, Conditioned / pharmacology
  • Gene Expression Regulation
  • Humans
  • Interferon Regulatory Factors / antagonists & inhibitors
  • Interferon Regulatory Factors / genetics*
  • Interferon Regulatory Factors / metabolism
  • Intracellular Signaling Peptides and Proteins
  • Macrophages / cytology
  • Macrophages / metabolism
  • Macrophages / microbiology
  • Mice
  • Mycobacterium bovis / chemistry*
  • Mycobacterium bovis / physiology
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction
  • Toll-Like Receptor 2 / antagonists & inhibitors
  • Toll-Like Receptor 2 / genetics*
  • Toll-Like Receptor 2 / metabolism
  • Toll-Like Receptor 4 / antagonists & inhibitors
  • Toll-Like Receptor 4 / genetics*
  • Toll-Like Receptor 4 / metabolism
  • Tripartite Motif Proteins
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Antibodies, Neutralizing
  • Carrier Proteins
  • Culture Media, Conditioned
  • Interferon Regulatory Factors
  • Intracellular Signaling Peptides and Proteins
  • Irf5 protein, mouse
  • RNA, Small Interfering
  • Tlr2 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 2
  • Toll-Like Receptor 4
  • Trim59 protein, mouse
  • Tripartite Motif Proteins
  • Tumor Necrosis Factor-alpha