Expression of major
stress proteins is induced rapidly in ischemic tissues, a response that may protect cells from ischemic injury. We have shown previously that transcriptional induction of
heat-shock protein 70 by
hypoxia results from activation of
DNA binding of a preexisting, but inactive, pool of heat shock factor (HSF). To determine the intracellular signals generated in hypoxic or ischemic cells that trigger HSF activation, we examined the effects of
glucose deprivation and the metabolic inhibitor
rotenone on
DNA-binding activity of HSF in cultured C2 myogenic cells grown under normoxic conditions. Whole-
cell extracts were examined in gel mobility shift assays using a 39-bp synthetic
oligonucleotide containing a consensus heat-shock
element as probe.
ATP pools were determined by high-pressure liquid chromatography and intracellular pH (pHi) was measured using a fluorescent
indicator.
Glucose deprivation alone reduced the cellular
ATP pool to 50% of control levels but failed to activate HSF. However, 2 x 10(-4) M
rotenone induced
DNA binding of HSF within 30 min, in association with a fall in
ATP to 30% of control levels, and a fall in pHi from 7.3 to 6.9. Maneuvers (
sodium propionate and
amiloride) that lowered pHi to 6.7 without
ATP depletion failed to activate HSF. Conversely, in studies that lowered
ATP stores at normal pH (high K+/
nigericin) we found induction of HSF-
DNA binding activity. Our data indicate that the effects of
ATP depletion alone are sufficient to induce the
DNA binding of HSF when oxidative metabolism is impaired, and are consistent with a model proposed recently for transcriptional regulation of
stress protein genes during
ischemia.