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Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive
A Sánchez1, A M Alvarez, M Benito
1Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Universidad Complutense de Madrid, Spain.
Abstract:
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.
Insights
Transforming growth factor-beta (TGF-beta) induces apoptosis in fetal hepatocytes via an oxidative stress pathway. This cell death mechanism, distinct from growth inhibition, involves reactive oxygen species and is partially mitigated by radical scavengers.
Area of Science:
- Hepatology
- Cell Biology
- Molecular Biology
Background:
- Transforming growth factor-beta (TGF-beta) regulates fetal hepatocyte growth and death.
- Distinct concentrations of TGF-beta are required for growth inhibition versus cell death.
Purpose of the Study:
- To investigate the mechanism of TGF-beta-induced apoptosis in fetal hepatocytes.
- To determine the role of oxidative stress in TGF-beta-mediated cell death.
Main Methods:
- Dose-response analysis of TGF-beta effects on fetal hepatocytes.
- Assessment of DNA fragmentation and DNA content.
- Measurement of reactive oxygen species and glutathione levels.
- Analysis of c-fos mRNA expression and AP-1 activity.
- Evaluation of radical scavengers' effects on cell death.
Main Results:
- TGF-beta induces apoptosis at 2.5 ng/ml, distinct from growth inhibition at 0.5 ng/ml.
- Apoptosis is characterized by DNA fragmentation and reduced DNA content.
- TGF-beta increases reactive oxygen species and decreases glutathione, indicating oxidative stress.
- High TGF-beta concentrations upregulate c-fos mRNA and AP-1 activity.
- Radical scavengers partially inhibit TGF-beta-induced apoptosis but not growth inhibition.
Conclusions:
- TGF-beta triggers apoptosis in fetal hepatocytes through an oxidative stress pathway.
- Oxidative stress is a key mediator of TGF-beta-induced programmed cell death in these cells.
- The mechanisms for TGF-beta-induced growth inhibition and apoptosis are distinct.