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Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins. [ABSTRACT FROM AUTHOR]

Activation of NF-κB (nuclear factor of kappa light chain gene enhancer in B cells) in response to DNA damage is considered to contribute to repair of genetic lesions, increased cell survival and cytokine release. The molecular mechanisms orchestrating this cytoplasmic event involve core components of the nuclear DNA damage response machinery, including ATM-kinase (ataxia telangiectasia mutated kinase) and PARP-1 (poly (ADP-ribose) polymerase 1). The physiological consequences of defective NF-κB activation in this context, however, remain poorly investigated. Here we report on the role of the 'p53-induced protein with a death domain', PIDD, which appears rate limiting in this process, as is PARP-1. Despite impaired NF-κB activation, DNA damage did not increase cell death or reduce clonal survival of various cell types lacking PIDD, such as mouse embryonic fibroblasts or stem and progenitor cells of the hematopoietic system. Furthermore, lymphomagenesis induced by γ-irradiation (IR) was unaffected by deficiency for PIDD or PARP-1, indicating that loss of DNA damage-triggered NF-κB signalling does not affect IR-driven tumorigenesis. However, loss of either gene compromised cytokine release after acute IR injury. Hence, we propose that NF-κB's most notable function after DNA damage in primary cells is related to the release of cytokines, thereby contributing to sterile inflammation. [ABSTRACT FROM AUTHOR]

Searching for selective tankyrases (TNKSs) inhibitors, a new small series of 6,8-disubstituted triazolo[4,3-b]piridazines has been synthesized and characterized biologically. Structure-based optimization of the starting hit compound NNL (3) prompted us to the discovery of 4-(2-(6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-ylamino)ethyl)phenol (12), a low nanomolar selective TNKSs inhibitor working as NAD isostere as ascertained by crystallographic analysis. Preliminary biological data candidate this new class of derivatives as a powerful pharmacological tools in the unraveling of TNKS implications in physiopathological conditions.

Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.

Background & Aims: Liver steatosis enhances ischemia/reperfusion (I/R) injury and is considered a primary factor in graft failure after liver transplantation. Although previous reports have shown a role for qualitative steatosis (macrovesicular vs. microvesicular) in hepatic I/R injury, no studies have compared side by side the specific contribution of individual lipids accumulating in fatty liver to I/R damage., Methods: We used nutritional and genetic models of micro and macrovesicular fatty livers exhibiting specific lipid profiles to assess their susceptibility to normothermic I/R injury., Results: Unlike choline-deficient (CD) diet-fed mice, characterized by predominant liver triglycerides/free fatty acids (TG/FFA) accumulation, mice fed a cholesterol-enriched (HC) diet, which exhibited enhanced hepatic cholesterol loading in mitochondria, were highly sensitive to I/R-induced liver injury. In vivo two-photon confocal imaging revealed enhanced mitochondrial depolarization and generation of reactive oxygen species following hepatic I/R in HC-fed but not in CD-fed mice, consistent with decreased mitochondrial GSH (mGSH) observed in HC-fed mice. Moreover, ob/ob mice, characterized by increased hepatic TG, FFA, and cholesterol levels, were as sensitive to I/R-mediated liver injury as mice fed the HC diet. Livers from ob/ob mice displayed increased StAR expression and mitochondrial cholesterol accumulation, resulting in mGSH depletion. Interestingly, atorvastatin therapy or squalene synthase inhibition in vivo attenuated StAR overexpression, mitochondrial cholesterol loading, and mGSH depletion, protecting ob/ob mice from I/R-mediated liver injury., Conclusions: Cholesterol accumulation, particularly in mitochondria, sensitizes to hepatic I/R injury, and thus represents a novel target to prevent the enhanced damage of steatotic livers to I/R-mediated damage., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect., Methodology/principal Findings: Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn's disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase., Conclusions: These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.

Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 belong to a family of enzymes that, using NAD(+) as a substrate, catalyze poly(ADP-ribosyl)ation of proteins. PARP-1 and PARP-2 catalytic activity is stimulated by DNA-strand breaks targeting mainly proteins involved in chromatin structure and DNA metabolism, providing strong support for a dual role of both PARP-1 and PARP-2 in the DNA damage response as DNA damage sensors and signal transducers to downstream effectors. The DNA damage response has important consequences for genomic stability and tumour development. In order to manipulate DNA damage responses to selectively induce tumour cell death, a considerable effort is centred on defining the molecular mechanisms that allow cells to detect, respond to, and repair DNA damage. PARP inhibitors that compete with NAD+ at the highly conserved enzyme active site are arisen as new potential therapeutic strategies as chemo- and radiopotentiation and for the treatment of cancers with specific DNA repair defects as single-agent therapies. In the present review, we highlight emerging information about the redundant and specific functions of PARP-1 and PARP-2 in genome surveillance and DNA repair pathways. Understanding these roles might provide invaluable clues to design new cancer therapeutic approaches. In addition, we provide an overview of ongoing clinical trials with PARP inhibitors and the value of PARP-1 and PARP-2 expression as prognostic biomarkers in cancer.

Unlabelled: Growth arrest-specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here we report an early increase in serum GAS6 levels after I/R exposure. Moreover, unlike wild-type (WT) mice, Gas6(-/-) mice were highly sensitive to partial hepatic I/R, with 90% of the mice dying within 12 hours of reperfusion because of massive hepatocellular injury. I/R induced early hepatic protein kinase B (AKT) phosphorylation in WT mice but not in Gas6(-/-) mice without significant changes in c-Jun N-terminal kinase phosphorylation or nuclear factor kappa B translocation, whereas hepatic interleukin-1β (IL-1β) and tumor necrosis factor (TNF) messenger RNA levels were higher in Gas6(-/-) mice versus WT mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes and thus protected them from hypoxia-induced cell death, whereas GAS6 diminished lipopolysaccharide-induced cytokine expression (IL-1β and TNF) in murine macrophages. Finally, recombinant GAS6 treatment in vivo not only rescued GAS6 knockout mice from severe I/R-induced liver damage but also attenuated hepatic damage in WT mice after I/R., Conclusion: Our data have revealed GAS6 to be a new player in liver I/R injury that is emerging as a potential therapeutic target for reducing postischemic hepatic damage.

Background: Hypoxia-mediated HIF-1alpha stabilization and NF-kappaB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth., Methodology/principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O(2)) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kappaB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts., Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kappaB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

The role of cholesterol in Alzheimer's disease (AD) has been linked to the generation of toxic amyloid beta peptides (Abeta). Using genetic mouse models of cholesterol loading, we examined whether mitochondrial cholesterol regulates Abeta neurotoxicity and AD pathology. Isolated mitochondria from brain or cortical neurons of transgenic mice overexpressing SREBP-2 (sterol regulatory element binding protein 2) or NPC1 (Niemann-Pick type C1) knock-out mice exhibited mitochondrial cholesterol accumulation, mitochondrial glutathione (mGSH) depletion and increased susceptibility to Abeta1-42-induced oxidative stress and release of apoptogenic proteins. Similar findings were observed in pharmacologically GSH-restricted rat brain mitochondria, while selective mGSH depletion sensitized human neuronal and glial cell lines to Abeta1-42-mediated cell death. Intracerebroventricular human Abeta delivery colocalized with mitochondria resulting in oxidative stress, neuroinflammation and neuronal damage that were enhanced in Tg-SREBP-2 mice and prevented upon mGSH recovery by GSH ethyl ester coinfusion, with a similar protection observed by intraperitoneal administration of GSH ethyl ester. Finally, APP/PS1 (amyloid precursor protein/presenilin 1) mice, a transgenic AD mouse model, exhibited mitochondrial cholesterol loading and mGSH depletion. Thus, mitochondrial cholesterol accumulation emerges as a novel pathogenic factor in AD by modulating Abeta toxicity via mGSH regulation; strategies boosting the particular pool of mGSH may be of relevance to slow down disease progression.

Nuclear factor (NF)-kappaB participates in ischemia/reperfusion (I/R) hepatic signaling, stimulating both protective mechanisms and the generation of inflammatory cytokines. After analyzing NF-kappaB activation during increasing times of ischemia in murine I/R, we observed that the nuclear translocation of p65 paralleled Src and IkappaB tyrosine phosphorylation, which peaked after 60 minutes of ischemia. After extended ischemic periods (90 to 120 minutes) however, nuclear p65 levels were inversely correlated with the progressive induction of oxidative stress. Despite this profile of NF-kappaB activation, inflammatory genes, such as tumor necrosis factor (TNF) and interleukin (IL)-1beta, predominantly induced by Kupffer cells, increased throughout time during ischemia (30 to 120 minutes), whereas protective NF-kappaB-dependent genes, such as manganese superoxide dismutase (Mn-SOD), expressed in parenchymal cells, decreased. Consistent with this behavior, gadolinium chloride pretreatment abolished TNF/IL-1beta up-regulation during ischemia without affecting Mn-SOD levels. Interestingly, specific glutathione (GSH) up-regulation in hepatocytes by S-adenosylmethionine increased Mn-SOD expression and protected against I/R-mediated liver injury despite TNF/IL-1beta induction. Similar protection was achieved by administration of the SOD mimetic MnTBAP. In contrast, indiscriminate hepatic GSH depletion by buthionine-sulfoximine before I/R potentiated oxidative stress and decreased both nuclear p65 and Mn-SOD expression levels, increasing TNF/IL-1beta up-regulation and I/R-induced liver damage. Thus, the divergent role of NF-kappaB activation in selective liver cell populations underlies the dichotomy of NF-kappaB in hepatic I/R injury, illustrating the relevance of specifically maintaining NF-kappaB activation in parenchymal cells.

Cholesterol metabolism is deregulated in carcinogenesis, and cancer cells exhibit enhanced mitochondrial cholesterol content whose role in cell death susceptibility and cancer therapy has not been investigated. Here, we describe that mitochondria from rat or human hepatocellular carcinoma (HC) cells (HCC) or primary tumors from patients with HC exhibit increased mitochondrial cholesterol levels. HCC sensitivity to chemotherapy acting via mitochondria is enhanced upon cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase or squalene synthase (SS), which catalyzes the first committed step in cholesterol biosynthesis. HCC transfection with siRNA targeting the steroidogenic acute regulatory protein StAR, a mitochondrial cholesterol-transporting polypeptide which is overexpressed in HCC compared with rat and human liver, sensitized HCC to chemotherapy. Isolated mitochondria from HCC with increased cholesterol levels were resistant to mitochondrial membrane permeabilization and release of cytochrome c or Smac/DIABLO in response to various stimuli including active Bax. Similar behavior was observed in cholesterol-enriched mitochondria or liposomes and reversed by restoring mitochondrial membrane order or cholesterol extraction. Moreover, atorvastatin or the SS inhibitor YM-53601 potentiated doxorubicin-mediated HCC growth arrest and cell death in vivo. Thus, mitochondrial cholesterol contributes to chemotherapy resistance by increasing membrane order, emerging as a novel therapeutic niche in cancer therapy.

The molecular mechanisms of hepatic ischemia/reperfusion (I/R) damage are incompletely understood. We investigated the role of ceramide in a murine model of warm hepatic I/R injury. This sphingolipid induces cell death and participates in tumor necrosis factor (TNF) signaling. Hepatic ceramide levels transiently increased after the reperfusion phase of the ischemic liver in mice, because of an early activation of acidic sphingomyelinase (ASMase) followed by acid ceramidase stimulation. In vivo administration of an ASMase inhibitor, imipramine, or ASMase knockdown by siRNA decreased ceramide generation during I/R, and attenuated serum ALT levels, hepatocellular necrosis, cytochrome c release, and caspase-3 activation. ASMase-induced ceramide generation activated JNK resulting in BimL phosphorylation and translocation to mitochondria, as the inhibition of ASMase by imipramine prevented these events. In contrast, blockade of ceramide catabolism by N-oleyolethanolamine (NOE), a ceramidase inhibitor, enhanced ceramide levels and potentiated I/R injury compared with vehicle-treated mice. Pentoxifylline treatment prevented TNF upregulation and ASMase activation. Furthermore, 9 of 11 mice treated with imipramine survived 7 days after total liver ischemia, compared with 4 of 12 vehicle-treated mice, whereas 8 of 8 NOE-treated mice died within 2 days of total liver ischemia. In conclusion, ceramide generated from ASMase plays a key role in I/R-induced liver damage, and its modulation may be of therapeutic relevance.