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Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg 2+ ), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Annual influenza epidemics are associated with high incidence and mortality rates, and are an important cause of work absenteeism and productivity losses. For successful replication, influenza viruses have evolved as to counteract and/or take a part on host defense mechanisms. Manipulation of apoptosis is one of such mechanisms that have been subject of attention, particularly in relation to influenza type A viruses over the past years. However, this knowledge has not been extended to include influenza type B viruses. In this study, MDCK-SIAT1 cells were infected with influenza A and B strains and the kinetics and pathways of apoptosis post infection were studied, through LDH measurements, Hoechst dye staining, caspase activity assays and protein expression analysis. The resulting data points to a difference in induction of apoptosis profiles between influenza A and B strains. While influenza A strain induced apoptosis later in the course of infection and mainly by the intrinsic pathway, influenza B strain induced apoptosis early in infection by both intrinsic and extrinsic pathways. Also, data revealed the IκB/NF-κB pathway as the major contributor for the observed differences. The study of the virus-host interactions, particularly those that could have an impact on viral replication, are essential in both influenza A and B viruses, as they will allow the identification of viral/host targets common to both influenza types, which could affect viral replication. This information may prove useful for vaccine and antiviral research., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells., Scope of Review: This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome., Major Conclusions: Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision., General Significance: A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

p53/Mdm-2 interaction is a prime target of ursodeoxycholic acid (UDCA) for regulating apoptosis in primary rat hepatocytes. Here, we further explored the role of UDCA in downregulating p53 by Mdm-2. UDCA reduced the stability of p53 by decreasing protein half-life. Although proteasomal activity was slightly increased with UDCA, the effect was also observed for other bile acids. More importantly, immunoprecipitation assays revealed that UDCA promoted p53 ubiquitination, therefore leading to increased p53 degradation. In this regard, proteasome inhibition after UDCA pre-treatment resulted in accumulation of ubiquitinated p53, which in turn was prevented in cells overexpressing a mutated form of p53 that does not undergo Mdm-2 ubiquitination. The involvement of Mdm-2 in UDCA-mediated response was further confirmed by siRNA-mediated gene silencing experiments. Finally, the protective effect of UDCA against p53-induced apoptosis was abolished after inhibition of proteasome activity and prevention of p53 ubiquitination by Mdm-2. These findings suggest that UDCA protects cells from p53-mediated apoptosis by promoting its degradation via the Mdm-2-ubiquitin-proteasome pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: MicroRNAs (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., Results: The 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., Conclusions: In 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.

Nuclear steroid receptors (NSR) are ligand-activated transcription factors that play a key role in a variety of vital physiological phenomena including developmental or endocrine signaling, reproduction, and homeostasis. In addition, they are implicated in other important biological processes, such as apoptosis. Modulation of apoptosis by NSR is mostly associated with control of pro-apoptotic versus anti-apoptotic gene expression, and includes both induction and prevention of apoptosis depending on cell type. However, it is unclear how NSR can affect opposing expression of the same gene in different cells. Of note, recently described nongenomic mechanisms of NSR, in particular glucocorticoid receptor translocation to mitochondria, were suggested to be crucial steps for triggering apoptosis. NSR often act solely as nuclear transporters of other regulatory molecules, thus indirectly regulating several apoptosis-related genes. Curiously, NSR are thought to cooperate with the anti-apoptotic endogenous bile acid, ursodeoxycholic acid (UDCA), to prevent programmed cell death. In fact, as cholesterol-derived molecules and due to their chemical and structural similarities to steroid hormones, bile acids also modulate NSR activation. Although the precise link between NSR and UDCA remains unclear, we have demonstrated that the bile acid requires NSR for translocation to the cell nucleus as part of a ligand-receptor complex, using a mechanism similar to that of steroid hormones. Interestingly, other studies revealed that UDCA interacts with the glucocorticoid receptor as a novel and selective NSR modifier. The huge diversity of natural ligands and xenobiotics that bind to NSR and regulate their function represents one of the most exciting drug targets for potential therapeutic intervention. The next decade will almost certainly unveil the remarkable role of NSR in modulating cell fate in human health and disease.

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 beta1-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.

Apoptosis represents a universal and exquisitely efficient cellular suicide pathway essential for a variety of normal biological processes ranging from embryonic development to ageing. In fact, tissue homeostasis is dependent on the perfect balance between positive and negative signals that determines the decision between life and death. Therefore, any imbalance can result in a wide range of pathologic disorders associated with unwanted apoptosis or cell growth. During the apoptotic process, the molecular players interact closely with each other in ways relevant to accelerate or interrupt the cellular death process. In addition, two major pathways of apoptosis activation have been recognized as the "intrinsic" mitochondrial pathway and the "extrinsic" death receptor pathway. Although these pathways act independently to initiate apoptosis, a delicate balance and cross-talk between the extrinsic and intrinsic pathways is thought to occur in many cell types. Interestingly, we have shown that ursodeoxycholic acid (UDCA), an endogenous hydrophilic bile acid, is a potent inhibitor of apoptosis by either stabilizing the mitochondrial membrane or modulating the expression of specific upstream targets. Herein, we review the main effectors involved in the death machinery, describe how they interact to regulate apoptosis, and discuss the main pathways that control cell death and survival. Further, we address multiple interesting targets as well as the potential application of UDCA as a therapeutic modality for apoptosis-related disorders.

Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids modulate apoptosis and regulate cell-cycle effectors, including cyclin D1. In contrast, deoxycholic acid (DCA) induces cell death and cyclin D1. In this study, we explored the role of cyclin D1 in DCA-induced toxicity and further elucidated the antiapoptotic function of UDCA and TUDCA in primary rat hepatocytes. Cells were incubated with DCA and with or without UDCA or TUDCA for 8-30 h. In addition, hepatocytes were transfected with either an adenovirus expressing cyclin D1 or with a cyclin D1 reporter plasmid with or without bile acids. Finally, cells were cotransfected with short interfering RNA targeting p53. Unlike DCA, both UDCA and TUDCA reduced cyclin D1 expression and transcriptional activation, confirming our previous DNA microarray data. Furthermore, UDCA and TUDCA prevented DCA-induced cyclin D1 and cell death. Cyclin D1 overexpression increased DCA-induced Bax translocation, cytochrome c release, and apoptosis. However, UDCA and TUDCA were less efficient at decreasing cyclin D1 levels as well as DCA-induced changes with overexpression. Finally, after p53 silencing, the effects of cyclin D1 overexpression were almost completely abrogated, whereas UDCA and TUDCA cytoprotective potential was reestablished. In conclusion, cyclin D1 is a relevant player in modulating apoptosis by bile acids, in part through a p53-dependent mechanism.

Tauroursodeoxycholic acid (TUDCA) prevents amyloid beta-peptide (Abeta)-induced neuronal apoptosis, by modulating both classical mitochondrial pathways and specific upstream targets. In addition, activation of nuclear steroid receptors (NSRs), such as the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) differentially regulates apoptosis in the brain. In this study we investigated whether TUDCA, a cholesterol-derived endogenous molecule, requires NSRs for inhibiting Abeta-induced apoptosis in primary neurons. Our results confirmed that TUDCA significantly reduced Abeta-induced apoptosis; in addition, the fluorescently labeled bile acid molecule was detected diffusely in both cytoplasm and nucleus of rat cortical neurons. Interestingly, experiments using small interfering RNAs (siRNAs) revealed that, in contrast to GR siRNA, MR siRNA abolished the antiapoptotic effect of TUDCA. Abeta incubation reduced MR nuclear translocation while increasing nuclear GR levels. Notably, pretreatment with TUDCA markedly altered Abeta-induced changes in NSRs, including MR dissociation from its cytosolic chaperone, heat shock protein 90, and subsequent translocation to the nucleus. Furthermore, when a carboxy terminus-deleted form of MR was used, nuclear trafficking of both MR and the bile acid was abrogated, suggesting that they translocate to the nucleus as a steroid-receptor complex. Transfection experiments with wild-type or mutant MR confirmed that this interaction was required for TUDCA protection against Abeta-induced apoptosis. Finally, in cotransfection experiments with NSR response element reporter and overexpression constructs, pretreatment with TUDCA significantly modulated Abeta-induced changes in MR and GR transactivation. In conclusion, these results provide novel insights into the specific cellular mechanism of TUDCA antiapoptotic function against Abeta-induced apoptosis and suggest targets for potential therapeutic intervention.

Early onset familial Alzheimer's 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 beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-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/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 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.

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-kappaB inhibitor (IkappaB) 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 IkappaB 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-kappaB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.

The efficient removal of unwanted cells, such as senescent, damaged, mutated or infected cells is crucial for the maintenance of normal liver function. In fact, apoptosis has emerged as a potential contributor to the pathogenesis of a number of hepatic disorders, such as viral hepatitis, autoimmune diseases, ethanol-induced injury, cholestasis, and hepatocellular carcinoma. In contrast to the effect of cytotoxic bile acids in the liver, ursodeoxycholic acid (UDCA) has increasingly been used for the treatment of various liver disorders. The clinical efficacy of this hydrophilic bile acid was first recognized by its use in traditional Asian medicine. However, many studies have subsequently confirmed that UDCA improves liver function by three major mechanisms of action, including protection of cholangiocytes against the cytotoxicity of hydrophobic bile acids, stimulation of hepatobiliary secretion, and inhibition of liver cell apoptosis. UDCA acts as a potent inhibitor of the classical mitochondrial pathway of apoptosis, but also interferes with alternate and upstream molecular targets such as the E2F-1/p53 pathway. Together, there is growing evidence that this hydrophilic bile acid may modulate gene expression to prevent cell death. Curiously, as a cholesterol-derived molecule, UDCA interacts with nuclear steroid receptors, such as the glucocorticoid receptor. Nuclear steroid receptors play crucial roles in mediating steroid hormone signaling involved in many biological processes, including apoptosis. Here, we review the anti-apoptotic mechanisms of UDCA in hepatic cells, and discuss a potential involvement of nuclear steroid receptors in mediating the survival effects of UDCA.

Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by a nuclear steroid receptor (NSR)-dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the anti-apoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-beta1-induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-beta1-induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-beta1. Finally, using a fluorescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the anti-apoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis.

Neurotoxicity associated with increased glutamate release results in cell death through both necrotic and apoptotic processes. In addition, tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, is a strong modulator of apoptosis in several cell types. The aims of this study were to test the hypothesis that TUDCA reduces the apoptotic threshold induced by glutamate in rat cortical neurons and examine potential transduction pathways involved in both apoptotic signaling and neuroprotection by TUDCA. The results demonstrated that exposure of cortical neurons to glutamate induced cytochrome c release and caspase activation, as well as morphologic changes of apoptosis. These events were associated with down-regulation of antiapoptotic members of the Bcl-2 family, Bcl-2 and Bcl-x(L), and dephosphorylation of the serine/threonine protein kinase Akt. Pretreatment with TUDCA significantly reduced glutamate-induced apoptosis of rat cortical neurons. In addition, TUDCA induced marked phosphorylation and translocation of Bad from mitochondria to the cytosol. Moreover, inhibition of the phosphatidylinositol 3-kinase (PI3K) survival pathway abrogated the protective effects of TUDCA, including phosphorylation and translocation of Bad. In conclusion, TUDCA appears to modulate glutamate-induced neuronal apoptosis, in part, by activating a PI3K-dependent Bad signaling pathway. These data suggest that TUDCA may be beneficial in treating neurodegenerative disorders in which increased glutamate levels contribute to the pathogenesis of the disease.

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-kappaB 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-kappaB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-kappaB 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-kappaB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

Amyloid beta-peptide (Abeta)-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 Abeta-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 Abeta, 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 Abeta (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 Abeta-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 Abeta-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.

We have recently shown that both ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) prevent transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. In addition, activation of glucocorticoid (GR) and mineralocorticoid receptors (MR) inhibits apoptosis in various systems. UDCA induces a ligand-independent activation of the GR, thus potentially regulating a number of targets. In this study, we investigated the role of GR and MR during TGF-beta1-induced hepatocyte apoptosis, and identified additional antiapoptotic targets for UDCA. Our results showed that in primary hepatocytes, TGF-beta1 induced 40-50% decreases in gr and mr mRNA expression (p < 0.01), together with up to 10-fold reductions in their protein levels (p < 0.01). Notably, pretreatment with UDCA resulted in a significant upregulation of nuclear steroid receptors (p < 0.05), which coincided with 2- and 3-fold increases in the level of GR and MR nuclear translocation, respectively, when compared with that of TGF-beta1 alone (p < 0.05). Similarly, TUDCA induced GR and MR nuclear translocations (p < 0.05) and markedly prevented MR protein changes associated with TGF-beta1 (p < 0.05) without affecting GR protein levels. Moreover, when interference RNA was used to inhibit GR and MR, UDCA no longer protected hepatocytes against TGF-beta1-induced apoptosis. In fact, the protective effect of UDCA in TGF-beta1-associated caspase activation decreased from 65 to <10% when GR or MR function was blocked. Finally, the TGF-beta1-induced E2F-1/Mdm-2/p53 apoptotic pathway, normally inhibited by UDCA, was not regulated by the bile acid after GR or MR silencing. These results demonstrate that UDCA protects against apoptosis through an additional pathway that involves nuclear receptors GR and MR as key factors. Further, the E2F-1/Mdm-2/p53 apoptotic pathway appears to be a prime target for UDCA-induced steroid receptor activation.

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-beta (Abeta) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Abeta-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Abeta peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3 prime-OH kinase (PI3K) pathway with wortmannin did not markedly affect Abeta-induced cell death, suggesting that this signaling pathway is not crucial for Abeta-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Abeta-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Abeta-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Abeta-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

Increased levels of unconjugated bilirubin, the end-product of heme catabolism, are detrimental to the central nervous system. To examine the role of apoptosis in bilirubin-induced toxicity and to characterize the biochemical pathway of cell death, we exposed developing rat brain neurons to purified unconjugated bilirubin at concentrations below and above saturation of human serum albumin. Isolated neurons treated with bilirubin showed increased levels of apoptosis. Mitochondrial cytochrome c was extensively released and accumulated in cytosol. Consistent with this observation, caspase-3 was activated and the full-length substrate poly(ADP)ribose polymerase (PARP) degraded, even in the presence of very modestly elevated concentrations of bilirubin. In parallel, all events were prevented in cells preincubated with ursodeoxycholate. Further experiments showed that bilirubin diminished mitochondrial transmembrane potential (DeltaPsi(m)) and increased mitochondrial-associated Bax protein levels, while directly disrupting membrane lipid and protein structure. In conclusion, bilirubin induces mitochondrial depolarization and Bax translocation via physical interaction with membranes, mediating the mitochondrial pathway of apoptosis in neurons exposed to bilirubin. These results provide a novel insight into the mechanism of bilirubin-induced toxicity.

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2;-deoxyuridine 5;-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.