Upregulation of the NADPH oxidase NOX4 by TGF-beta in hepatocytes is required for its pro-apoptotic activity☆
Introduction
Transforming growth factor-beta (TGF-β) plays a suppressor role in hepatocytes, where it regulates growth and differentiation and induces apoptosis [1], [2], [3]. Emerging evidence indicates that suppression of cell response to TGF-β might play important roles in the initiation of hepatocarcinogenesis [4], [5], [6], [7]. Thus, the antiproliferative and pro-apoptotic roles of TGF-β represent a “life-belt” that has to be evaded by tumor cells to gain malignancy. TGF-β-mediated cell death in hepatocytes requires the production of reactive oxygen species (ROS), which precedes the loss of mitochondrial-transmembrane potential, cytochrome c release and caspase activation [6], [8], [9], [10], [11]. Different mechanisms are involved in ROS production by TGF-β. Mitochondrial ROS might increase by suppression of antioxidant genes [12], [13] and an inducible NADPH oxidase-like system could account for extramitochondrial ROS production [13]. By treating cells with diphenyleneiodonium (DPI), a flavoprotein inhibitor widely used to block NADPH oxidase activity, all the apoptotic features induced by TGF-β are blocked [13]. These results indicate that NADPH oxidases might mediate ROS production by TGF-β, which is required for cell death.
For a long time, superoxide generation by a NADPH oxidase was considered to occur only in phagocytes. Over the last years, six homologues of the phagocyte catalytic NADPH oxidase (NOX2) were found: NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2 [14]. Activation mechanisms and tissue distribution of the different family members are markedly different. NOX1 and NOX2 are most likely activated through Rac1, in combination with other regulatory subunits [15]. NOX3, NOX4 and NOX5 appear to be Rac1-independent. Functional analysis of NOX4 has revealed unique characteristics when compared to other NADPH oxidases [16], [17]. NOX4 associates with the protein p22phox on internal membranes, where ROS generation occurs [16] and produces large amounts of superoxide anion constitutively. Modulation of its activity is only related to changes in gene transcription [14]. Among the different NOX isoforms, fetal rat hepatocytes express Nox1, Nox2 and Nox4 [18]. Incubating cells with TGF-β induces Nox4 and produces an increase in the protein levels of Rac1 [18]. Upregulation of Nox4, but not Rac1 activation, is impaired by anti-apoptotic signals, such as the epidermal growth factor (EGF) [18].
The aim of this work was to analyze the role of the different isoforms of NADPH oxidase in the intracellular signalling induced by TGF-β in hepatocytes leading to apoptosis, to later explore whether this mechanism might be altered in liver tumor cells.
Section snippets
Cell isolation and culture
Fetal rat hepatocytes were obtained by collagenase disruption of 20-day-old fetal Wistar rats and cultured in non-coated plastic dishes with arginine-free, ornithine-supplemented, M-199 medium as described [8]. After attachment and serum starvation for 12–24 h, factors were added. All animals received humane care according to the Institution’s guidelines. Fetal human hepatocytes were isolated and plated on collagen-coated plates using 10% FBS William’s E medium, supplemented with different
Regulation of Nox4 by TGF-β in fetal rat hepatocytes. Correlation with its pro-apoptotic activity
We first analyzed the relevance of the Rac-dependent NADPH oxidases on ROS production and cell death induced by TGF-β. By using NCS23766, a Rac GTPase-specific small molecule inhibitor [20], we observed impairment in the TGF-β-induced NADPH oxidase activation at very early times (1.5 h), but only a slight attenuation at later times (3 h) (Fig. 1A). Consequently, this inhibitor only blocked TGF-β-induced ROS production at short times (Fig. 1A). Interestingly, the Rac inhibitor was unable to impair
Discussion
TGF-β is an important growth inhibitor and apoptosis inducer in different cell types, including hepatocytes [22]. However, although there is no doubt about its role as a tumor suppressor [23], [24], it could also contribute to tumor progression and metastasis through the induction of other effects [24], such as epithelial mesenchymal transition (EMT) processes, which increase cell migration and invasion [25], [26]. Furthermore, TGF-β has long been believed to be a central mediator of the
Acknowledgements
We are very grateful to our laboratory members for helpful comments, in particular, to Drs. A. Sánchez and E. Bertran, for useful suggestions and critical reading of the manuscript. We are also grateful to A. Vázquez (Universidad Complutense de Madrid), M. Noriega (IDIBELL) and E. Castaño (Serveis Cientificotècnics, IDIBELL-UB) for their technical assistance. I. C.-C. is very grateful to the members of Dr. Fausto lab in Seattle, especially to R. Bauer, M. Johnson and J. Campbell, for their
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The authors who have taken part in the research of this paper declared that they do not have a relationship with the manufacturers of the materials involved either in the past or present and they did not receive funding from the manufacturers to carry out their research.