Resistance to TGF-beta-induced apoptosis in regenerating hepatocytes.

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.

P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli.

p38 alpha mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38 alpha knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38 alpha are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38 alpha-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38 alpha also have increased extracellular signal-regulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38 alpha. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the extracellular signal-regulated kinase MAP kinase pathway, may contribute to the decrease in both Bax and Fas expression in p38 alpha-/- cells. Thus, p38 alpha seems to sensitize cells to apoptosis via both up-regulation of proapoptotic proteins and down-regulation of survival pathways.

Sustained nitric oxide delivery delays nitric oxide-dependent apoptosis in macrophages: contribution to the physiological function of activated macrophages.

Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN-gamma accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress.

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.

The epithelial mesenchymal transition confers resistance to the apoptotic effects of transforming growth factor Beta in fetal rat hepatocytes.

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.

Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C delta.

Aplidin, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-delta) mediates the cytotoxic effect of Aplidin and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-delta show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-delta thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.

Differential role of PPAR gamma in the regulation of UCP-1 and adipogenesis by TNF-alpha in brown adipocytes.

Extracellular regulated kinases (ERKs) mediate the inhibitory effect of tumor necrosis factor alpha (TNF-alpha) on uncoupling protein-1 (UCP-1), but not on lipid accumulation. TNF-alpha-induced ERK-dependent peroxisome proliferator activator receptor gamma (PPAR gamma) phosphorylation could be responsible for UCP-1 downregulation. Thus, the negative effect of TNF-alpha on UCP-1 mRNA expression at 4-5 h, under basal conditions or in cells treated with the PPAR gamma agonist, rosiglitazone, was reversed by the MEK1 inhibitor PD98059. In contrast, fatty acid synthase and malic enzyme mRNA downregulation was not prevented. Moreover, rosiglitazone has no positive effect on adipogenic gene expression or lipid accumulation. Therefore, there is a differential regulation of thermogenic and adipogenic differentiation by PPAR gamma, which might account for the differences in the TNF-alpha regulation through ERKs.

Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase.

Both alleles of the XOG1 gene of Candida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity in C. albicans, although residual exoglucanase activity could still be detected. xog1 null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential for C. albicans growth. In addition, no differences were observed between wild-type and xog1 null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. The xog1 null strain is thus a suitable recipient for studying Candida gene expression using the exoglucanase as a reporter gene.