Cytosolic heat shock protein 60, hypoxia, and apoptosis.
BACKGROUND
Heat shock protein (HSP)60 is an abundant protein found primarily in the mitochondria, though 15% to 20% is found in the cytosol. Previously we observed that HSP60 complexes with bax in the cytosol. Reduction in HSP60 precipitates translocation of bax to the mitochondria and apoptosis. We hypothesized that HSP60 would decrease with hypoxia/reoxygenation and that this would precipitate bax translocation to the mitochondria and release of cytochrome c.


RESULTS
Adult rat cardiac myocytes were studied at end-hypoxia and at 10 and 24 hours of reoxygenation. HSP60 levels were unchanged at end-hypoxia and decreased 33% and 40% at 10 and 24 hours of reoxygenation, whereas HSP72 increased 80% and 110%. Bax and bcl-2 decreased during reoxygenation. However, cytochrome c release occurred at end-hypoxia, before reoxygenation. Cell fractionation was done to analyze this further. In normal myocytes, bax and HSP60 were present in the cytosol, and bax coimmunoprecipitated with cytosolic HSP60. At end-hypoxia, mitochondrial HSP60 was unchanged, but cytosolic HSP60 had disappeared and was now in the plasma membrane fraction. Concurrently, bax was no longer in the cytosol but now in the mitochondria. Thus, although total HSP60 remained the same, it no longer complexed with bax, and bax was free to translocate to the mitochondria and precipitate apoptosis. Reduction in ATP had a similar effect.


CONCLUSIONS
These studies show that hypoxia results in disassociation of the HSP60-bax complex with translocation of cytosolic HSP60 to the plasma membrane and bax to the mitochondria. This is sufficient to trigger apoptosis.
Gupta
S
S
Baylor College of Medicine and the VA Medical Center, Houston, Tex, USA.
Knowlton
A A
AA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002458:Cell Fractionation; D015687:Cell Hypoxia / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D020552:Comet Assay; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009336:Necrosis; D010100:Oxygen / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D018834:Chaperonin 60; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D010100:Oxygen