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We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met(-/-) oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Met(flx/flx)), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Met(flx/flx) and Met(-/-) oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met(-/-) oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met(-/-) oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Met(flx/flx) oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met(-/-) oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met(flx/flx) oval cells, whereas no effect was observed in Met(-/-) oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.

Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway.

Epidermal growth factor (EGF) is a survival signal for transforming growth factor-beta (TGF-beta)-induced apoptosis in hepatocytes, phosphatidylinositol 3-kinase (PI 3-K) being involved in this effect. Here, we analyze the possible cross talks between EGF and TGF-beta signals to understand how EGF impairs the early pro-apoptotic events induced by TGF-beta. Data have indicated that neither SMAD nor c-Jun NH2 Terminal Kinase (JNK) activations are altered by EGF, which clearly interferes with events directly related to the radical oxygen species (ROS) production, impairing oxidative stress, p38 MAP kinase activation, and cell death. Activation of a NADPH-oxidase-like system, which is responsible for the early ROS production by TGF-beta, is completely inhibited by EGF, through a PI 3-K-dependent mechanism. Activity of RAC1 increases by TGF-beta, but also by EGF, and both act synergistically to get maximum effects. Fetal rat hepatocytes express nox4, in addition to nox1 and nox2, and TGF-beta clearly upregulates nox4. EGF blocks up-regulation of nox4 by TGF-beta. Interestingly, in the presence of PI 3-K inhibitors, EGF is not able to counteract the nox4 upregulation by TGF-beta. Taking together these results indicate that impairment of TGF-beta-induced NADPH oxidase activation by EGF is a RAC1-independent process and correlates with an inhibition of the mechanisms that address the increase of nox4 mRNA levels by TGF-beta.

Cannabinoids have been shown to inhibit the growth of a broad spectrum of tumour cells. However, the molecular mechanisms involved in that effect have not been completely elucidated. Here, we investigated the possible involvement of mitogen-activated protein kinases (MAPKs) in CB2 receptor-induced apoptosis of human leukaemia cells. Results show that stimulation of the CB2 receptor leads to p38 MAPK activation and that inhibition of this kinase attenuates CB2 receptor-induced caspase activation and apoptosis. These findings support a role for p38 MAPK in CB2 receptor-induced apoptosis of human leukaemia cells.

Treatment with transforming growth factor beta (TGF-beta) of hepatocytes from two different proliferative conditions, such as fetal development and adult liver regeneration, shows that regenerating cells respond to this cytokine in terms of growth inhibition, but are less sensitive than the fetal ones to the apoptosis induced by this factor. Regenerating TGF-beta treated cells show higher cell viability and lower percentage of apoptotic cells than the fetal treated ones. Furthermore, TGF-beta treated regenerating hepatocytes maintain a well-preserved parenchyma-like organization. Treatment with TGF-beta induces the loss of mitochondrial transmembrane potential in fetal but not in regenerating hepatocytes and activation of caspase-3 is lower in regenerating than in fetal cells. Regenerating hepatocytes show higher intracellular levels of some antiapoptotic proteins, such as Bcl-x(L) and c-IAP-1 and, interestingly, they present higher intracellular glutathione levels, which might provide of mechanisms to avoid potential dangerous effects of the oxidative stress-mediated apoptosis induced by TGF-beta. In fact, treatment with BSO (a glutathione synthesis inhibitor) restores the response of regenerating hepatocytes to TGF-beta in terms of cell death. In conclusion, increased levels of Bcl-x(L) and cIAP-1 and higher intracellular glutathione levels could confer resistance to the apoptosis induced by TGF-beta during liver regeneration.

Transforming growth factor-beta (TGF-beta) induces an oxidative stress process in hepatocytes that mediates its apoptotic activity. To determine the cellular source of the early reactive oxygen species (ROS) generated by fetal rat hepatocytes in response to TGF-beta, we used inhibitors that block different ROS-producing systems. Diphenyleneiodonium, which inhibits NADPH oxidase and other flavoproteins, completely blocked the increase in ROS induced by TGF-beta, coincidently with an impairment of caspase-3 activation and cell death. Rotenone, an inhibitor of the NADH dehydrogenase in mitochondrial complex I, attenuated, but did not completely inhibit, ROS-production, caspase activation, and cell death mediated by TGF-beta. No significant protection was observed with inhibitors of other ROS-producing systems, such as cytochrome P450 (metyrapone), cyclooxygenase (indomethacin), and xanthine oxidase (allopurinol). Additional experiments have indicated that two different mechanisms could be involved in the early ROS production by TGF-beta. First, an inducible (cycloheximide-inhibited) NADPH oxidase-like system could account for the extramitochondrial production of ROS. Second, TGF-beta could increase ROS by a rapid downregulation of antioxidant genes. In particular, intramitochondrial ROS would increase by depletion of MnSOD. Finally, glutathione depletion is a late event and it would be more the consequence than the cause of the increase in ROS induced by TGF-beta.

Transforming growth factor-beta (TGF-beta) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives concomitant with changes in morphology and phenotype, reminiscent of an epithelial mesenchymal transition (EMT) [Exp. Cell Res. 252 (1999) 281-291]. In this work, we have isolated the subpopulation that survives to TGF-beta-induced apoptosis, showing that these cells maintain the response to TGF-beta in terms of Smads activation and growth inhibition. Analyses of the intracellular signals that could impair the apoptotic effects of TGF-beta have indicated that the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway is activated in these resistant cells. Experiments in fetal rat hepatocytes have shown that TGF-beta is able to transiently activate PI 3-K/Akt by a mechanism independent of protein synthesis but dependent on a tyrosine kinase activity. Pro-apoptotic signals, such as oxidative stress and caspases, contribute to the loss of Akt at later times. Inhibiting PI 3-K sensitizes fetal hepatocytes (FH) to the apoptosis induced by TGF-beta and causes spontaneous death in the resistant cells. In conclusion, TGF-beta, as it is known for other cytokines, could be inducing pro-apoptotic and survival signals in hepatocytes, the balance among them will decide cell fate.

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.

Fetal rat hepatocytes treated with transforming growth factor beta (TGF-beta) die by apoptosis. However, a subpopulation of them survives and undergoes an epithelial mesenchymal transition (EMT). This transition also occurs upon incubation with fetal bovine serum. We have isolated the subpopulations that undergo EMT (TGF-beta-treated-fetal hepatocytes: TbetaT-FH; serum-treated-fetal hepatocytes: ST-FH) and show that they present high levels of vimentin and Snail expression and lack cytokeratin 18 and E-cadherin. Both TbetaT-FH and ST-FH cells require mitogens to grow and maintain the response to TGF-beta in terms of growth inhibition. However, they lack differentiation markers such as the liver-enriched transcription factors hepatocyte nuclear factor 4 (HNF-4) or HNF-1alpha and express the progenitor marker OV-6. Interestingly, the EMT process confers them resistance to the apoptotic effect of TGF-beta, with cells showing higher levels of active AKT and Bcl-x(L) than fetal hepatocytes. In summary, these cells are refractory to the apoptotic effects of TGF-beta, showing characteristics of liver progenitors and of some hepatocellular carcinoma cells.

We have studied the expression of XIAP, cIAP-1 and cIAP-2 in fetal rat hepatocytes and its possible regulation by pro-apoptotic stimuli (transforming growth factor-beta (TGF-beta)) and survival signals (epidermal growth factor (EGF)). The three forms of inhibitor of apoptosis proteins (IAPs) are expressed in fetal hepatocytes and only cIAP-1, but not XIAP or cIAP-2, is cleaved during TGF-beta-induced apoptosis. The pan-caspase inhibitor Z-VAD.fmk blocked this effect, which indicates that cIAP-1 is a caspase substrate. EGF plays a dual role in the regulation of IAPs expression. On one hand, it increases cIAP-1 and cIAP-2 basal expression and, on the other hand, it blocks the cleavage of cIAP-1 by caspases induced by TGF-beta.

The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells. [ABSTRACT FROM AUTHOR]

Abstract: Δ9-Tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat—that expresses CB2 as the unique CB receptor—to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Δ9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Δ9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation—an event typically related with the extrinsic apoptotic pathway—was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Δ9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway. [Copyright &y& Elsevier]

Epidermal growth factor (EGF) is a survival signal for transforming growth factor-beta (TGF-β)-induced apoptosis in hepatocytes, phosphatidylinositol 3-kinase (PI 3-K) being involved in this effect. Here, we analyze the possible cross talks between EGF and TGF-β signals to understand how EGF impairs the early pro-apoptotic events induced by TGF-β. Data have indicated that neither SMAD nor c-Jun NH2 Terminal Kinase (JNK) activations are altered by EGF, which clearly interferes with events directly related to the radical oxygen species (ROS) production, impairing oxidative stress, p38 MAP kinase activation, and cell death. Activation of a NADPH-oxidase-like system, which is responsible for the early ROS production by TGF-β, is completely inhibited by EGF, through a PI 3-K-dependent mechanism. Activity of RAC1 increases by TGF-β, but also by EGF, and both act synergistically to get maximum effects. Fetal rat hepatocytes express nox4, in addition to nox1 and nox2, and TGF-β clearly upregulates nox4. EGF blocks up-regulation of nox4 by TGF-β. Interestingly, in the presence of PI 3-K inhibitors, EGF is not able to counteract the nox4 upregulation by TGF-β. Taking together these results indicate that impairment of TGF-β-induced NADPH oxidase activation by EGF is a RAC1-independent process and correlates with an inhibition of the mechanisms that address the increase of nox4 mRNA levels by TGF-β. J. Cell. Physiol. 207: 322–330, 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Cannabinoids have been shown to inhibit the growth of a broad spectrum of tumour cells. However, the molecular mechanisms involved in that effect have not been completely elucidated. Here, we investigated the possible involvement of mitogen-activated protein kinases (MAPKs) in CB2 receptor-induced apoptosis of human leukaemia cells. Results show that stimulation of the CB2 receptor leads to p38 MAPK activation and that inhibition of this kinase attenuates CB2 receptor-induced caspase activation and apoptosis. These findings support a role for p38 MAPK in CB2 receptor-induced apoptosis of human leukaemia cells. [Copyright &y& Elsevier]

We have studied the expression of XIAP, cIAP-1 and cIAP-2 in fetal rat hepatocytes and its possible regulation by pro-apoptotic stimuli (transforming growth factor-β (TGF-β)) and survival signals (epidermal growth factor (EGF)). The three forms of inhibitor of apoptosis proteins (IAPs) are expressed in fetal hepatocytes and only cIAP-1, but not XIAP or cIAP-2, is cleaved during TGF-β-induced apoptosis. The pan-caspase inhibitor Z-VAD.fmk blocked this effect, which indicates that cIAP-1 is a caspase substrate. EGF plays a dual role in the regulation of IAPs expression. On one hand, it increases cIAP-1 and cIAP-2 basal expression and, on the other hand, it blocks the cleavage of cIAP-1 by caspases induced by TGF-β. [Copyright &y& Elsevier]

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-β type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response. [ABSTRACT FROM AUTHOR]