Involvement of reactive oxygen species in the induction of chemokine JE/MCP-1 gene by phorbol-12-myristate-13-acetate in Balb 3T3 cells

Cell Struct Funct. 1997 Apr;22(2):231-8. doi: 10.1247/csf.22.231.

Abstract

The induction of JE/MCP-1 gene by TPA was transcriptionally suppressed by antioxidants such as pyrrolidine dithiocarbamate (PDTC) or trimethylthiourea (TMTU) in Balb 3T3 cells, whereas that of other early response genes, c-fos or egr-1, was not affected by these agents. Induction of the JE gene by TNF alpha or serum was not completely inhibited by these antioxidants inhibited an increase in intracellular oxidized state of cells treated with TPA. Next we examined the transcriptional regulatory region of the rat JE gene to determine the genomic target of active oxygen species. The chloramphenicol acetyltransferase (CAT) reporter gene, containing the 5'-upstream region approximately 2.6 kb DNA from the cap site, was transfected into Balb 3T3 cells. The CAT activity induced by TPA increased in parallel with the endogenous JE and mRNA level, and the increase was inhibited by the antioxidants. The essential region for this response in the upstream region was within the -2.6 to -2.0 kb region, and further defined to -2,224 to -2,069 bp which contained and NF kappa B-binding element. Gel shift analysis indicated that the nuclear factors that bound to this essential element contained NF kappa B, and that NF kappa B activity was stimulated by TPA and inhibited by PDTC. These results suggest that active oxygen species are involved in induction of the JE gene caused by TPA in Balb 3T3 cells, through NF kappa B activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Antioxidants / pharmacology
  • Base Sequence
  • Binding Sites
  • Chemokine CCL2 / genetics*
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation* / drug effects
  • Mice
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Reactive Oxygen Species / metabolism*
  • Regulatory Sequences, Nucleic Acid
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Antioxidants
  • Chemokine CCL2
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • Tetradecanoylphorbol Acetate