THIOL REDUCING REAGENTS INHIBIT THE HEAT-SHOCK RESPONSE - INVOLVEMENTOF A REDOX MECHANISM IN THE HEAT-SHOCK SIGNAL-TRANSDUCTION PATHWAY

We evaluated the effects of thiol-reducing agents on the heat shock re sponse in human and rodent cells in culture. Using HeLa cells as an ex ample, we demonstrated that dithiothreitol (DTT, 2 mM) inhibited the h eat (42 degrees C) induced increase in the synthesis of heat shock pro teins (HSPs), abundance of mRNA of hsp 70, hsp 70 gene promoter activi ty, and the heat shock factor (HSF) DNA binding activity. This effect of DTT was specific and attributable to its reducing activity; oxidize d DTT was ineffective, and other thiol reducing compounds had the same effect as DTT. Time course and dose-response stud ies showed that DTT significantly inhibited the heat shock induction of heat shock elemen t binding activity with no preincubation and that 0.6 and 1-2 mM DTT g ave half-maximal and maximal inhibition, respectively. The effect of D TT was reversible; removal of the DTT-containing medium prior to heat shock rendered the cells fully responsive. Analysis of the effects of DTT on the regulation and function of HSF suggests that DTT blocked an early and important step in the activation process without having a d irect effect on the HSF protein. Thus, DTT inhibited the heat-induced trimerization, phosphorylation, and nuclear translocation of HSF and w as also effective against a number of other reagents that are known to activate HSF. On the other hand, DTT did not block the response induc ed by heat shock at 45 degrees C, and in vitro addition of DTT failed to modulate the DNA binding activity of activated HSF present in cell extracts, suggesting that the HSF protein itself is unlikely to be a d irect target of action of DTT. These results, together with the observ ation that activation of HSF DNA binding activity was attenuated under an anoxic condition and that hydrogen peroxide mimicked the effects o f heat shock, suggest the involvement of a redox mechanism as an early and important step in the heat shock signal transduction pathway.