Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 3T3 cells in response to heat shock.
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.
Diamond
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DA
Section of Cancer Biology, Radiation Oncology Center, Mallinckrodt Institute of Radiology, St. Louis, Missouri 63108, USA.
Parsian
A
A
Hunt
C R
CR
Lofgren
S
S
Spitz
D R
DR
Goswami
P C
PC
Gius
D
D
D016475:3T3 Cells; D000818:Animals; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D017781:Genes, Immediate-Early; D006367:HeLa Cells; D018869:Heat-Shock Response; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase