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.
Highlights
Transforming growth factor- (TGF-), a growth regulator of fetal hepatocytes in primary culture, regulates death of these cells
Cell death induced by TGF- 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- in these cells
Effect of Radical Scavengers on the Induction of Apoptosis by TGF- in Fetal Hepatocytes in Primary Culture—In order to know whether the increase in reactive oxygen species may be related to the fetal hepatocyte death, we examined the effects of antioxidants or radical scavengers, i.e. ascorbic acid, pyrrolidine carbodithioic acid (PDTC), N-acetyl-Lcysteine (NAC), superoxide dismutase, and nordihydroguaiaretic acid (NDGA) on the apoptosis induced by TGF- in the cells
Summary
(Received for publication, June 30, 1995, and in revised form, November 27, 1995). From the Departamento de Bioquımica y Biologıa Molecular, Instituto de Bioquımica (Consejo Superior de Investigaciones Cientıficas/Universidad Complutense de Madrid) and §Centro de Citometrıa de Flujo, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Peroxides and highly reactive free radicals can trigger cell death, and some investigators believe that oxidation is merely another metabolic disturbance that leads cells to respond to external stimuli, others propose a more central role for reactive oxygen species in cell death In this context, overexpression of bcl-2, a protooncogene that is unique among cellular genes in its ability to block apoptotic death in different cell types [16] decreases lipid peroxidation and can increase resistance to apoptotic killing by hydrogen peroxide, menadione, and depletion of glutathione [17]. High concentrations of this factor induce fetal hepatocyte death [26] All of these results suggest that primary cultured fetal hepatocytes are a good model in which to study the possible role of TGF- in the regulation of liver apoptosis during fetal life and the implication of oxygen radicals in its molecular mechanism
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