Induction of stress proteins in cultured myogenic cells. Molecular signals for the activation of heat shock transcription factor during ischemia.
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.
Benjamin
I J
IJ
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8573.
Horie
S
S
Greenberg
M L
ML
Alpern
R J
RJ
Williams
R S
RS
D000138:Acidosis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010085:Oxidative Phosphorylation / Q000187:drug effects; D011422:Propionates / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D012402:Rotenone / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D009838:Oligodeoxyribonucleotides; D011422:Propionates; D014157:Transcription Factors; D012402:Rotenone; D000584:Amiloride; D000255:Adenosine Triphosphate; C514135:sodium propionate; D005947:Glucose; C029658:propionic acid