Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes.
Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF-beta) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Deltapsim), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF-beta mediates ROS production that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3. TGF-beta induces a decrease in the protein and mRNA levels of bcl-xL, an antiapoptotic member of the Bcl-2 family. In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family. EGF maintains Bcl-xL, preventing Deltapsim collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl-xL levels, Deltapsim collapse, cytochrome c release, and activation of caspase 3; in contrast, the presence of glutathione synthesis inhibitors such as BSO accentuated the effect. The incubation of fetal hepatocytes in the presence of ter-butyl-hydroperoxide alone produces a decrease in bcl-xL. These results indicate that during the apoptosis mediated by TGF-beta in fetal hepatocytes, ROS may be responsible for the decrease in bcl-xL mRNA levels that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3, culminating in cell death.
Herrera
B
B
Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Madrid, 28040 Madrid, Spain.
Alvarez
A M
AM
Sánchez
A
A
Fernández
M
M
Roncero
C
C
Benito
M
M
Fabregat
I
I
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D001205:Ascorbic Acid / Q000494:pharmacology; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D004815:Epidermal Growth Factor / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000975:Antioxidants; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494828:Bax protein, rat; C494813:Bcl2l1 protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011759:Pyrrolidines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013859:Thiocarbamates; D016212:Transforming Growth Factor beta; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C020972:pyrrolidine dithiocarbamic acid; D004815:Epidermal Growth Factor; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D001205:Ascorbic Acid