Susana Solá

Hepatocyte apoptosis, expression of death receptors, and activation of NF-κB in the liver of nonalcoholic and alcoholic steatohepatitis patients

OBJECTIVES:The increasing incidence of nonalcoholic (NASH) and alcoholic steatohepatitis (ASH), associated with lack of effective treatment, has prompted intensive studies on disease pathogenesis. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. In this study, we identified mechanisms of apoptosis induction in human steatohepatitis, and evaluated potential associations between apoptosis, liver pathology, and clinical presentation in NASH and ASH.METHODS:Hepatocyte apoptosis was evaluated by the TUNEL assay in 20 patients with NASH (all ambulatory), 40 with ASH (20 ambulatory, 20 hospitalized), and 20 controls. Liver biopsies were also graded for inflammation and fibrosis. Immunohistochemistry was performed for death receptors (Fas and TNF-R1), activated caspase-3, NF-κB p65, antiapoptotic Bcl-2 protein, and uncoupling protein 2 (UCP-2).RESULTS:TUNEL-positive hepatocytes were markedly increased in NASH (p < 0.05) and ASH (p < 0.01). Similar results were obtained for activated caspase-3, confirming the occurrence of apoptosis. The Fas receptor was upregulated in ASH, especially in hospitalized patients (p < 0.01), whereas TNF-R1 was expressed both in NASH and ASH (p < 0.01). In addition, patients with ASH showed a remarkable expression of active NF-κB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-κB p65 expression, which in turn coincided with apoptosis albeit Bcl-2 and UCP-2 expression.CONCLUSIONS:Liver injury in NASH and ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis correlates with active NF-κB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

miR-34a Regulates Mouse Neural Stem Cell Differentiation

Background MicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation. Methodology/Principal Findings miR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism. Conclusions Our results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.

Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis.

Objectives Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. Methods Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-κB inhibitor (IκB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. Results Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IκB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. Conclusion The results show that liver injury in NASH and ASH is associated with apoptosis and NF-κB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

BACKGROUND/AIMS Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. METHODS We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. RESULTS Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P<0.01), as well as disrupted protein mobility (P<0.001). Consistent with increased permeability, cytochrome c was released from the intermembrane space (P<0.01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P<0.01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. CONCLUSIONS UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation

BackgroundMicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated.ResultsThe expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells.ConclusionsIn conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.

p53 Is a Key Molecular Target of Ursodeoxycholic Acid in Regulating Apoptosis

p53 plays an important role in regulating expression of genes that mediate cell cycle progression and/or apoptosis. In addition, we have previously shown that the hydrophilic bile acid ursodeoxycholic acid (UDCA) prevents transforming growth factor β1-induced p53 stabilization and apoptosis in primary rat hepatocytes. Therefore, we hypothesized that p53 may represent an important target in bile acid-induced modulation of apoptosis and cell survival. In this study we demonstrated that UDCA reduces p53 transcriptional activity, thereby preventing its ability to induce Bax expression, mitochondrial translocation, cytochrome c release, and apoptosis in primary rat hepatocytes. More importantly, bile acid inhibition of p53-induced apoptosis was associated with decreased p53 DNA binding activity. Subcellular localization of p53 was also altered by UDCA. Both events appear to be related with increased association between p53 and its direct repressor, Mdm-2. In conclusion, these results further clarify the antiapoptotic mechanism of UDCA and suggest that modulation of Mdm-2/p53 interaction is a prime target for this bile acid.

Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid β-peptide-induced apoptosis of PC12 cells

Amyloid β‐peptide (Aβ)‐induced cell death may involve activation of the E2F‐1 transcription factor and other cell cycle‐related proteins. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates Aβ‐induced apoptosis by interfering with crucial events of the mitochondrial pathway. In this study, we examined the role of E2F and p53 activation in the induction of apoptosis by Aβ, and investigated novel molecular targets for TUDCA. The results showed that despite Bcl‐2 up‐regulation, PC12 neuronal cells underwent significant apoptosis after incubation with the active fragment Aβ (25–35), as assessed by DNA fragmentation, nuclear morphology and caspase‐3‐like activation. In addition, transcription through the E2F‐1 promoter was significantly induced and associated with loss of the retinoblastoma protein. In contrast, levels of E2F‐1, p53 and Bax proteins were markedly increased. Overexpression of E2F‐1 in PC12 cells was sufficient to induce p53 and Bax proteins, as well as nuclear fragmentation. Notably, TUDCA modulated Aβ‐induced apoptosis, E2F‐1 induction, p53 stabilization and Bax expression. Further, TUDCA protected PC12 cells against p53‐ and Bax‐dependent apoptosis induced by E2F‐1 and p53 overexpression, respectively. In conclusion, the results demonstrate that Aβ‐induced apoptosis of PC12 cells proceeds through an E2F‐1/p53/Bax pathway, which, in turn, can be specifically inhibited by TUDCA, thus underscoring its potential therapeutic use.

Tauroursodeoxycholic acid modulates p53-mediated apoptosis in Alzheimer's disease mutant neuroblastoma cells.

Early onset familial Alzheimers disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid β‐peptide (Aβ) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Aβ‐induced apoptosis by interfering with E2F‐1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild‐type APP, APP with the Swedish mutation (APPswe), or double‐mutated human APP and PS1 (APPswe/ΔE9), all exhibiting increased Aβ production and aggregation. Cell viability was decreased in APPswe and APPswe/ΔE9 but was partially reversed by z‐VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl‐2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild‐type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3′‐OH kinase pathway reduced TUDCA protection against p53‐induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53‐induced apoptosis and modulates expression of Bcl‐2 family.

p53 interaction with JMJD3 results in its nuclear distribution during mouse neural stem cell differentiation.

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.

Caspases and p53 Modulate FOXO3A/Id1 Signaling During Mouse Neural Stem Cell Differentiation

Neural stem cells (NSCs) differentiate into neurons and glia, and a large percentage undergoes apoptosis. The engagement and activity of apoptotic pathways may favor either cell death or differentiation. In addition, Akt represses differentiation by up‐regulating the inhibitor of differentiation 1 (Id1), through phosphorylation of its repressor FOXO3A. The aim of this study was to investigate the potential cross‐talk between apoptosis and proliferation during mouse NSC differentiation. We determined the time of neurogenesis and gliogenesis using neuronal β‐III tubulin and astroglial GFAP to confirm that both processes occurred at ∼3 and 8 days, respectively. p‐Akt, p‐FOXO3A, and Id1 were significantly reduced throughout differentiation. Caspase‐3 processing, p53 phosphorylation, and p53 transcriptional activation increased at 3 days of differentiation, with no evidence of apoptosis. Importantly, in cells exposed to the pancaspase inhibitor z‐VAD.fmk, p‐FOXO3A and Id1 were no longer down‐regulated, p53 phosphorylation and transcriptional activation were reduced, while neurogenesis and gliogenesis were significantly delayed. The effect of siRNA‐mediated silencing of p53 on FOXO3A/Id1 was similar to that of z‐VAD.fmk only at 3 days of differentiation. Interestingly, caspase inhibition further increased the effect of p53 knockdown during neurogenesis. In conclusion, apoptosis‐associated factors such as caspases and p53 temporally modulate FOXO3A/Id1 signaling and differentiation of mouse NSCs. J. Cell. Biochem. 107: 748–758, 2009.