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J Biol Chem, Vol. 274, Issue 24, 16959-16964, June 11, 1999

Redox Factor-1 (Ref-1) Mediates the Activation of AP-1 in HeLa and NIH 3T3 Cells in Response to Heat Shock

David A. DiamondDagger §, Azemat ParsianDagger , Clayton R. HuntDagger , Sam LofgrenDagger , Douglas R. SpitzDagger , Prabhat C. GoswamiDagger , and David GiusDagger §

From the Dagger  Section of Cancer Biology, Radiation Oncology Center, Mallinckrodt Institute of Radiology and the § Washington University School of Medicine, St. Louis, Missouri 63108

The early response genes, c-Fos and c-Jun, are induced by environmental stress and are thought to modulate injury processes via the induction of AP-1-dependent target genes. AP-1 activation is thought to be regulated by changes in intracellular oxidation/reduction reactions involving the redox factor-1 (Ref-1) protein. In this study, NIH 3T3 and HeLa cells were used to determine whether heat shock induces the AP-1 transcription factor via signaling pathways involving Ref-1. Reverse transcriptase-polymerase chain reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2-10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was confirmed by the restoration of heat-inducible DNA binding upon addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from heated cells to stimulate AP-1 DNA binding was abolished by chemical oxidation and restored by chemical reduction. These results indicate that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA binding and suggests that redox-sensitive signal transduction pathways involving Ref-1 may mediate heat-induced alterations in AP-1 activation.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.


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