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doi: 10.1038/cdd.2010.115pmid: 20865012
Autophagy is an evolutionarily conserved catabolic process that involves the invagination and degradation of cytoplasmic components through an autophagosomelysosome track. Autophagy functions as a quality control of cellular milieu and is implicated in a wide variety of pathological conditions. However, excessive or imbalanced autophagic flux may also be associated with cellular toxicity and may potentially contribute to the development of pathological conditions. Just as all membrane trafficking systems need to constantly strike a balance in their level of activation and inhibition to ensure proper spatial and temporal delivery of their cargo, autophagy must also be tightly regulated. Here, we provide an overview of the current knowledge regarding the negative regulation of mammalian autophagy in an effort to understand its physiological relevance and potential clinical importance.
Talos, F; Abraham, A; Vaseva, A V; Holembowski, L; Tsirka, S E; Scheel, A; Bode, D; Dobbelstein, M; Brück, W; Moll, U M
doi: 10.1038/cdd.2010.131pmid: 21076477
The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.
Sardina, J L; López-Ruano, G; Sánchez-Abarca, L I; Pérez-Simón, J A; Gaztelumendi, A; Trigueros, C; Llanillo, M; Sánchez-Yagüe, J; Hernández-Hernández, A
doi: 10.1038/cdd.2010.67pmid: 20523355
Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34+ cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22phox-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.
Mbaya, E; Oulès, B; Caspersen, C; Tacine, R; Massinet, H; Pennuto, M; Chrétien, D; Munnich, A; Rötig, A; Rizzuto, R; Rutter, G A; Paterlini-Bréchot, P; Chami, M
doi: 10.1038/cdd.2010.51
Smith, D M; Patel, S; Raffoul, F; Haller, E; Mills, G B; Nanjundan, M
doi: 10.1038/cdd.2010.53pmid: 20508647
Arsenic trioxide (As2O3), used to treat promyelocytic leukemia, triggers cell death through unknown mechanisms. To further our understanding of As2O3-induced death, we analyzed its effects on transforming growth factor-β (TGFβ) signaling mediators in ovarian cells. Dysregulated TGFβ signaling is a characteristic of ovarian cancers. As2O3 reduced the protein expression of EVI1, TAK1, SMAD2/3, and TGFβRII while increasing SnoN/SkiL. EVI1 protein was modulated by treatment with the proteasome inhibitors, MG132 and PS-341/Velcade, suggesting that degradation occurs through the ubiquitin-proteasome pathway. The sensitivity of ovarian cells to As2O3-induced apoptosis correlated with expression of multidrug resistance protein 1. Interestingly, expression of SnoN was similar to LC3-II (autophagy marker), which increased with induction of cytoplasmic vacuolation preceding apoptosis. These vesicles were identified as autophagosomes based on transmission electron microscopy and immunofluorescence staining with EGFP–LC3. The addition of N-acetyl-L-cysteine (ROS scavenger) to As2O3-treated cells reversed changes in SnoN protein and the autophagic/apoptotic response. In contrast to beclin-1 knockdown, siRNA targeting ATG5, ATG7, and hVps34 markedly reduced autophagy in As2O3-treated ovarian carcinoma cells. Further, treatment with SnoN siRNA markedly decreased LC3-II levels and increased PARP degradation (an apoptosis marker). Collectively, these findings suggest that As2O3 induces a beclin-1-independent autophagic pathway in ovarian carcinoma cells and implicates SnoN in promoting As2O3-mediated autophagic cell survival.
Namba, T; Tanaka, K-I; Ito, Y; Hoshino, T; Matoyama, M; Yamakawa, N; Isohama, Y; Azuma, A; Mizushima, T
doi: 10.1038/cdd.2010.64pmid: 20489727
Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a serious clinical concern and myofibroblasts have been suggested to have a major role, with it recently being revealed that some of these myofibroblasts are derived from lung epithelial cells through epithelial–mesenchymal transition (EMT). In this study, we examined the EMT-inducing abilities of drugs known to induce ILD clinically. EMT-like phenotypes were induced by A771726, an active metabolite of leflunomide having an inhibitory effect on dihydroorotate dehydrogenase (DHODH). Smad-interacting protein 1 (a transcription factor regulating EMT) and the Notch-signaling pathway but not transforming growth factor-β was shown to be involved in A771726-induced EMT-like phenotypes. When the cultures were supplemented with exogenous uridine, the A771726-induced EMT-like phenotypes and activation of the Notch-signaling pathway disappeared. Similarly, an A771726 analog without inhibitory activity on DHODH produced no induction, suggesting that this process is mediated through the inhibition of DHODH. In vivo, administration of leflunomide stimulated bleomycin-induced EMT-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fibrosis, both of which were suppressed by coadministration of uridine. Taken together, these findings suggest that leflunomide-dependent exacerbation of bleomycin-induced pulmonary fibrosis is mediated by stimulation of EMT of lung epithelial cells, providing the first evidence that drug-induced pulmonary fibrosis involves EMT of these cells.
Borrelli, S; Candi, E; Hu, B; Dolfini, D; Ravo, M; Grober, O M V; Weisz, A; Dotto, G P; Melino, G; Viganò, M A; Mantovani, R
doi: 10.1038/cdd.2010.59pmid: 20523354
Genetic experiments established that p63 is crucial for the development and maintenance of pluristratified epithelia. In the RNA interference (RNAi) screening for targets of p63 in keratinocytes, we identified the transcription factor, High Mobility Group (HMG) box protein 1 (HBP1). HBP1 is an HMG-containing repressor transiently induced during differentiation of several cell lineages. We investigated the relationship between the two factors: using RNAi, overexpression, chromatin immunoprecipitations and transient transfections with reporter constructs, we established that HBP1 is directly repressed by p63. This was further confirmed in vivo by evaluating expression in p63 knockout mice and in transgenics expressing p63 in basal keratinocytes. Consistent with these findings, expression of HBP1 increases upon differentiation of primary keratinocytes and HaCaT cells in culture, and it is higher in the upper layers of human skin. Inactivation of HBP1 by RNAi prevents differentiation of keratinocytes and stratification of organotypic skin cultures. Finally, we analyzed the keratinocyte transcriptomes after HBP1 RNAi; in addition to repression of growth-promoting genes, unexpected activation of differentiation genes was uncovered, coexisting with repression of other genes involved in epithelial cornification. Our data indicate that suppression of HBP1 is part of the growth-promoting strategy of p63 in the lower layers of epidermis and that HBP1 temporally coordinates expression of genes involved in stratification, leading to the formation of the skin barrier.
Quintavalle, C; Incoronato, M; Puca, L; Acunzo, M; Zanca, C; Romano, G; Garofalo, M; Iaboni, M; Croce, C M; Condorelli, G
doi: 10.1038/cdd.2010.65pmid: 20508645
Akt is a serine–threonine kinase that has an important role in transducing survival signals. Akt also regulates a number of proteins involved in the apoptotic process. To find new Akt interactors, we performed a two-hybrid screening in yeast using full-length Akt cDNA as bait and a human cDNA heart library as prey. Among 200 clones obtained, two of them were identified as coding for the c-FLIPL protein. c-FLIPL is an endogenous inhibitor of death receptor-induced apoptosis through the caspase-8 pathway. Using co-immunoprecipitation experiments of either transfected or endogenous proteins, we confirmed the interaction between Akt and c-FLIPL. Furthermore, we observed that c-FLIPL overexpression interferes with Gsk3-β phosphorylation levels. Moreover, through its effects on Gsk3β, c-FLIPL overexpression in cancer cells induced resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This effect was mediated by the regulation of p27Kip1 and caspase-3 expression. These results indicate the existence of a new mechanism of resistance to TRAIL in cancer cells, and unexpected functions of c-FLIPL.
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The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.
Despite advanced knowledge on the genetic basis of oxidative phosphorylation-related diseases, the molecular and/or cellular determinants for tissue-specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring before cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca2+ signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (Δψ mit) loss, [ATP] reduction and increased reactive oxygen species production. Cytosolic and mitochondrial Ca2+ overload are linked to increased endoplasmic reticulum (ER) Ca2+ leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca2+]mit is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca2+] activated on the one hand a compensatory Ca2+-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary function for Ca2+ signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency.