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. 2007 Feb 20:6:1.
doi: 10.1186/1476-5926-6-1.

TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells

Verena Proell  1 , Irene Carmona-Cuenca, Miguel M Murillo, Heidemarie Huber, Isabel Fabregat, Wolfgang Mikulits
Affiliations

TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells

Verena Proell et al. Comp Hepatol. .

Abstract

Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-beta. We analyzed the oxidative stress induced by TGF-beta and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs.

Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-beta/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense.

Conclusion: The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

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Figures

Figure 1
Figure 1
Cellular model of hepatic fibrosis. (A) Morphological changes of M1-4HSCs treated with TGF-β1 either for 72 hours or for long-term (myofibroblastoid M-HT) as analyzed by phase contrast microscopy. (B) Nuclear translocation of Smad2/3 as visualized by confocal immunofluorescence analysis. (C) Confocal immunofluorescence images after staining of cells with anti-desmin antibody.
Figure 2
Figure 2
TGF-β mediated accumulation of ROS associates with increased NADPH oxidase activity. (A) During the TGF-β dependent transdifferentiation of M1-4HSCs, ROS levels increase after 48 and 72 hours. (B) M-HTs show a reduction of ROS levels to about 50% as compared to untreated M1-4HSCs. (C) DPI inhibits TGF-β caused ROS accumulation in M1-4HSCs. (D) TGF-β treatment of M1-4HSCs induces NADPH oxidase activity after 48 hours. M-HTs display vast NADPH oxidase activity. For all situations, n = 3. * p < 0.05.
Figure 3
Figure 3
Expression profiling of oxidative stress components by semiquantitative RT-PCR. GCS, γ-glutamylcysteine synthetase; GSHPx, glutathione peroxidase; GSSG-R, glutathione reductase; SOD 1, Cu/Zn superoxide dismutase; SOD 2, mitochondrial superoxide dismutase. The constitutive expression of rhoA is shown as loading control.
Figure 4
Figure 4
Regulation of defense mechanisms against oxidative stress during the TGF-β driven transdifferentiation of M1-4HSCs to M-HTs. (A) SOD activity (n = 2). (B) Catalase activity (n = 4). (C) Glutathione levels (n = 3). * p < 0.05.
Figure 5
Figure 5
Steady state transcript levels of PDGF receptors and AP-1 components as analyzed by semiquantitative RT-PCR. The constitutive expression of rhoA is shown as loading control.
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