Le. Huang et al., THIOL REDUCING REAGENTS INHIBIT THE HEAT-SHOCK RESPONSE - INVOLVEMENTOF A REDOX MECHANISM IN THE HEAT-SHOCK SIGNAL-TRANSDUCTION PATHWAY, The Journal of biological chemistry, 269(48), 1994, pp. 30718-30725
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.