Miguel M. Murillo

Involvement of EGF receptor and c-Src in the survival signals induced by TGF-beta1 in hepatocytes.

Transforming growth factor beta1 (TGF-β1) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. Although solid data established the role of TGF-βs as suppressor factors in tumorigenic processes, in the context of an advanced stage of disease, TGF-βs could also play a pro-oncogenic role. We have previously shown that TGF-β1 induces both pro- and antiapoptotic signals in foetal rat hepatocytes. In this work, we have focused on its antiapoptotic mechanism. We show that TGF-β1 activates the epidermal growth factor receptor (EGFR) and phosphorylates c-Src. EGFR is required for Akt activation. Blocking EGFR signalling amplifies the apoptotic response to TGF-β1. TGF-β1 induced a rapid activation of the tumour necrosis factor-α-converting enzyme (TACE/ADAM (a disintegrin and metalloprotease) 17). Inhibitors of TACE considerably attenuated Akt activation, which suggests that TGF-β1 activates EGF signalling in hepatocytes by promoting shedding of EGF-like ligands. The activation of c-Src by TGF-β1 is EGFR dependent and is required for full Akt phosphorylation and cell survival. Inhibition of EGFR does not block the epithelial–mesenchymal transition (EMT) induced by TGF-β1 in hepatocytes, which indicates that activation of EGFR plays an essential role in impairing apoptosis, but it is dispensable for the EMT process.

Activation of NADPH oxidase by transforming growth factor-β in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-κB-dependent mechanism

The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.

The inhibition of the epidermal growth factor (EGF) pathway enhances TGF-β-induced apoptosis in rat hepatoma cells through inducing oxidative stress coincident with a change in the expression pattern of the NADPH oxidases (NOX) isoforms

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, through a mechanism mediated by reactive oxygen species (ROS) production. Numerous tumoral cells develop mechanisms to escape from the TGF-beta-induced tumor suppressor effects. In this work we show that in FaO rat hepatoma cells inhibition of the epidermal growth factor receptor (EGFR) with the tyrphostin AG1478 enhances TGF-beta-induced cell death, coincident with an elevated increase in ROS production and GSH depletion. These events correlate with down-regulation of genes involved in the maintenance of redox homeostasis, such as gamma-GCS and MnSOD, and elevated mitochondrial ROS. Nonetheless, not all the ROS proceed from the mitochondria. Emerging evidences indicate that ROS production by TGF-beta is also mediated by the NADPH oxidase (NOX) system. TGF-beta-treated FaO cells induce nox1 expression. However, the treatment with TGF-beta and AG1478 greatly enhanced the expression of another family member: nox4. NOX1 and NOX4 targeted knock-down by siRNA experiments suggest that they play opposite roles, because NOX1 knockdown increases caspase-3 activity and cell death, whilst NOX4 knock-down attenuates the apoptotic process. This attenuation correlates with maintenance of GSH and antioxidant enzymes levels. In summary, EGFR inhibition enhances apoptosis induced by TGF-beta in FaO rat hepatoma cells through an increased oxidative stress coincident with a change in the expression pattern of NOX enzymes.

Role of CXCR4/SDF-1α in the migratory phenotype of hepatoma cells that have undergone epithelial–mesenchymal transition in response to the transforming growth factor-β

Treatment of FaO rat hepatoma cells with TGF-beta selects cells that survive to its apoptotic effect and undergo epithelial-mesenchymal transitions (EMT). We have established a cell line (T beta T-FaO, from TGF-beta-treated FaO) that shows a mesenchymal, de-differentiated, phenotype in the presence of TGF-beta and is refractory to its suppressor effects. In the absence of this cytokine, cells revert to an epithelial phenotype in 3-4 weeks and recover the response to TGF-beta. T beta T-FaO show higher capacity to migrate than that observed in the parental FaO cells. We found that FaO cells express low levels of CXCR4 and do not respond to SDF-1 alpha. However, TGF-beta up-regulates CXCR4, through a NF kappaB-dependent mechanism, and T beta T-FaO cells show elevated levels of CXCR4, which is located in the presumptive migration front. A specific CXCR4 antagonist (AMD3100) attenuates the migratory capacity of T beta T-FaO cells on collagen gels. Extracellular SDF-1 alpha activates the ERKs pathway in T beta T-FaO, but not in FaO cells, increasing cell scattering and protecting cells from apoptosis induced by serum deprivation. Targeted knock-down of CXCR4 with specific siRNA blocks the T beta T-FaO response to SDF-1 alpha. Thus, the SDF-1/CXCR4 axis might play an important role in mediating cell migration and survival after a TGF-beta-induced EMT in hepatoma cells.

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

BackgroundThe 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)-β. We analyzed the oxidative stress induced by TGF-β and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs.ResultsWe found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-β 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-β/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.ConclusionThe data provide evidence that TGF-β 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.