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MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts themiR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose- and time-dependent manner. In turn,miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of themiR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated themiR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA andmiR-34a itself, providing a functionalmechanismformiR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging themiR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism- and apoptosis-related liver pathologies. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3-72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered alter PH by > 1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions. [ABSTRACT FROM AUTHOR]

The interplay between p53 and apoptosis in diseases such as cancer, neurodegeneration, ischemia and atherosclerosis underscores the need to understand the complexity of p53 networks. Here, we highlight recent studies of p53-induced apoptosis in human diseases, with a focus on the modulation of liver cell apoptosis. In addition, recent work has provided new insights into mechanisms underlying the antiapoptotic functions of the endogenous bile acid ursodeoxycholic acid (UDCA), suggesting that the finely tuned, complex control of p53 by Mdm2 is a key step in the UDCA modulation of deregulated, p53-triggered apoptosis. The effect of targeting cell death signaling proteins has been established in preclinical models of human diseases. Finally, we review recent therapeutic strategies and clinical applications of targeted agents, with a particular emphasis on the potential use of UDCA. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

Background/Aims: Ursodeoxycholic acid (UDCA) and its taurine-conjugated derivative, TUDCA, modulate cell death and cell cycle regulators, such as E2F-1 and p53. However, precise pathways underlying UDCA''s effects are not fully understood. The aim of this study was to identify specific cellular targets of UDCA. Methods: The expression profile of primary rat hepatocytes incubated with UDCA was determined using Affymetrix GeneChip Rat 230A arrays. Hybridization data were processed to identify genes with significant expression changes. RT-PCR and immunoblot analyses of a selected target confirmed microarray data. Results: The results showed that >440 genes were modulated with UDCA by >1.5-fold; ∼25% were significantly different from controls. Genes affected by UDCA included new regulatory molecules, such as Apaf-1. RT-PCR and immunoblotting confirmed a decrease in Apaf-1. Other altered genes were directly involved in cell cycle (cyclin D1, cadherin 1, HMG-box containing protein 1) and apoptosis (prothymosin-α) events. The E2F-1/p53/Apaf-1 pathway appears to be targeted by UDCA. Finally, transcripts for proteins with kinase activity and transcription factors were specifically modulated by TUDCA. Conclusions: This study expands our knowledge of the biological effects of UDCA in hepatocytes. Most of the identified genes represent novel potential targets of UDCA, which may ultimately explain its therapeutic properties. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Intracerebral hemorrhage (ICH) is a devastating acute neurological disorder, without effective treatment, in which a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we evaluated whether TUDCA can reduce brain injury and improve neurological function after ICH in rats. Administration of TUDCA before or up to 6 h after stereotaxic collagenase injection into the striatum reduced lesion volumes at 2 days by as much as 50%. Apoptosis was ≈50% decreased in the area immediately surrounding the hematoma and was associated with a similar inhibition of caspase activity. These changes were also associated with improved neurobehavioral deficits as assessed by rotational asymmetry, limb placement, and stepping ability. Furthermore, TUDCA treatment modulated expression of certain Bcl-2 family members, as well as NF-κB activity. In addition to its protective action at the mitochondrial membrane, TUDCA also activated the Akt-l/protein kinase Bα survival pathway and induced Bad phosphorylation at Ser-136. In conclusion, reduction of brain injury underlies the wide-range neuroprotective effects of TUDCA after ICH. Thus, given its clinical safety, TUDCA may provide a potentially useful treatment in patients with hemorrhagic stroke and perhaps other acute brain injuries associated with cell death by apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Submillimolar concentrations of cytotoxic bile acids (BAs) induce cell death via apoptosis. On the other hand, several cytoprotective BAs were shown to prevent apoptosis in the same concentration range. Still, the mechanisms by which BAs trigger these opposite signaling effects remain unclear. This study was aimed to determine if cytotoxic and cytoprotective BAs, at physiologically active concentrations, are able to modulate the biophysical properties of lipid membranes, potentially translating into changes in the apoptotic threshold of cells. Binding of BAs to membranes was assessed through the variation of fluorescence parameters of suitable derivatized BAs. These derivatives partitioned with higher affinity to liquid disordered than to the cholesterol-enriched liquid ordered domains. Unlabeled BAs were also shown to have a superficial location upon interaction with the lipid membrane. Additionally, the interaction of cytotoxic BAs with membranes resulted in membrane expansion, as concluded from FRET data. Moreover, it was shown that cytotoxic BAs were able to significantly disrupt the ordering of the membrane by cholesterol at physiologically active concentrations of the BA, an effect not associated with cholesterol removal. On the other hand, cytoprotective bile acids had no effect on membrane properties. It was concluded that, given the observed effects on membrane rigidity, the apoptotic activity of cytotoxic BAs could be potentially associated with changes in plasma membrane organization (e.g. modulation of lipid domains) or with an increase in mitochondrial membrane affinity for apoptotic proteins. [Copyright &y& Elsevier]