Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive oxygen intermediates.
Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes.
Sánchez
A
A
Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Universidad Complutense de Madrid, Spain.
Alvarez
A M
AM
Benito
M
M
Fabregat
I
I
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D015342:DNA Probes; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005333:Fetus; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D018384:Oxidative Stress; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D015342:DNA Probes; D016166:Free Radical Scavengers; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D004247:DNA; D005978:Glutathione; D001205:Ascorbic Acid