Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 3T3cells in response to heat shock

The early response genes, c-Fos and c-Jun, are induced by environmental str ess and are thought to modulate injury processes via the induction of AP-l- dependent target genes. AP-I activation is thought to be regulated by chang es in intracellular oxidation/reduction reactions involving the redox facto r-1 (Ref-l) protein. In this study, NIH 3T3 and HeLa cells were used to det ermine whether heat shock induces the AP-1 transcription factor via signali ng pathways involving Ref-l, Reverse transcriptase-polymerase chain reactio n analysis and immunoblotting demonstrated that c-Fos and c-Jun were induce d 2-10 h following heat shock, and this induction was accompanied by an inc rease in AP-1 DNA binding. Electrophoretic mobility shift assay extracts im munodepleted of Ref-l protein demonstrated that the increase in AP-1 DNA-bi nding activity following heating was dependent upon the presence of Ref-l a nd that Ref-l 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-l to immunodepleted extracts. The ability of Ref-l from hea ted 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 th at redox-sensitive signal transduction pathways involving Ref-l may mediate heat-induced alterations in AP-1 activation.