Changes in cIAP2, survivin and BimEL expression characterize the switch from autophagy to apoptosis in prolonged starvation.

BACKGROUND: Autophagy is a catabolic process involving the engulfment of cytoplasmic content within autophagosomes followed by their delivery to lysosomes. This process is a survival mechanism, enabling cells to cope with nutrient deprivation by degradation and recycling of macromolecules. Yet during continued stress such as prolonged starvation, a switch from autophagy to apoptosis is often detected. OBJECTIVE: In this work, we characterized the temporal dynamics of the transition from autophagy towards apoptosis with the aim of elucidating the molecular mechanism regulating the switch from survival autophagy to apoptotic cell death. RESULTS AND CONCLUSIONS: We defined an inverse relationship between apoptosis and autophagy spanning a period of 72 h, manifested by the sequential reduction in LC3 lipidation and the activation of caspase-3. The transition to apoptosis correlated with a selective decline in the mRNA and protein levels of two anti-apoptotic IAP family proteins, survivin and cIAP2 and a selective increase in the BH3-only protein, BimEL. This 'molecular signature' was common to several cell lines undergoing the switch from autophagy to apoptosis during prolonged starvation. Mechanistically, the increased BimEL protein levels resulted from its reduced binding to its specific E3 ligase, ßTrCP, leading to protein stabilization. Consistent with this, BimEL showed decreased phosphorylation at critical sites previously reported to be essential for binding to the E3 ligase. The decrease in the anti-apoptotic IAPs and the increase in the pro-apoptotic BimEL may thus constitute a molecular switch from autophagy to apoptosis during prolonged starvation.

DAPK2 is a novel regulator of mTORC1 activity and autophagy.

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca(2+)-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca(2+) levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.

Infestation of urban populations of the Northern white-breasted hedgehog, Erinaceus roumanicus, by Ixodes spp. ticks in Poland.

Infestation by the nest-dwelling Ixodes hexagonus Leach and the exophilic Ixodes ricinus (Linnaeus) (Ixodida: Ixodidae) on the Northern white-breasted hedgehog, Erinaceus roumanicus (Erinaceomorpha: Erinaceidae), was investigated during a 4-year study in residential areas of the city of Poznan, west-central Poland. Of 341 hedgehogs, 303 (88.9%) hosted 10 061 Ixodes spp. ticks encompassing all parasitic life stages (larvae, nymphs, females). Ixodes hexagonus accounted for 73% and I. ricinus for 27% of the collected ticks. Male hedgehogs carried significantly higher tick burdens than females. Analyses of seasonal prevalence and abundance of I. hexagonus revealed relatively stable levels of infestation of all parasitic stages, with a modest summer peak in tick abundance noted only on male hosts. By contrast, I. ricinus females and nymphs peaked in spring and declined steadily thereafter in summer and autumn, whereas the less abundant larvae peaked in summer. This is the first longterm study to evaluate the seasonal dynamics of both tick species on populations of wild hedgehogs inhabiting urban residential areas.

The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA.

Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Δ40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Δ40p53 protein levels and the subsequent transcriptional activation of the 14-3-3σ gene, a known target of Δ40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.

Life and death partners: apoptosis, autophagy and the cross-talk between them.

It is not surprising that the demise of a cell is a complex well-controlled process. Apoptosis, the first genetically programmed death process identified, has been extensively studied and its contribution to the pathogenesis of disease well documented. Yet, apoptosis does not function alone to determine a cell's fate. More recently, autophagy, a process in which de novo-formed membrane-enclosed vesicles engulf and consume cellular components, has been shown to engage in a complex interplay with apoptosis. In some cellular settings, it can serve as a cell survival pathway, suppressing apoptosis, and in others, it can lead to death itself, either in collaboration with apoptosis or as a back-up mechanism when the former is defective. The molecular regulators of both pathways are inter-connected; numerous death stimuli are capable of activating either pathway, and both pathways share several genes that are critical for their respective execution. The cross-talk between apoptosis and autophagy is therefore quite complex, and sometimes contradictory, but surely critical to the overall fate of the cell. Furthermore, the cross-talk is a key factor in the outcome of death-related pathologies such as cancer, its development and treatment.

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death.

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.

DAP-kinase (DAPk) is a Ser/Thr kinase that regulates cytoplasmic changes associated with programmed cell death. It is shown here that a GFP-DAPk fusion, which partially localized to actin stress fibers, induced extensive membrane protrusions. This phenotype correlated with changes in myosin-II distribution and with increased phosphorylation of the myosin-II regulatory light chain (RLC). A mutant lacking the cytoskeletal-interacting region (GFP-DAPkDeltaCyto) displayed diffuse cytoplasmic localization, and induced peripheral membrane blebbing, instead of the extensive protrusions. In contrast, deletion of the ankyrin repeats led to mislocalization of the kinase to focal contacts, where it failed to elicit any changes in cell morphology. While both wild-type DAPk and DAPkDeltaCyto induced RLC phosphorylation independently of the Rho-activated kinase ROCK, only the wild type led to increases in stress-fiber associated phospho-RLC. Thus, the precise intracellular localization of DAPk is critical for exposure to its substrates, including the RLC, which mediate varying morphologic changes.

Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors.

The presence of adenoviral cis-elements interfering with the activity of tissue-specific promoters has seriously impaired the use of transcriptional targeting adenoviruses for gene therapy purposes. As an approach to overcome this limitation, transcription terminators were previously employed in cultured cells to insulate a transgene promoter from viral activation. To extend these studies in vivo, we have injected into heart and skeletal muscle, adenoviruses containing the human growth hormone terminator and the cardiac-specific alpha-myosin heavy chain promoter (alphaMyHC) driving the chloramphenicol acetyltransferase (CAT) reporter gene. Promoterless CAT constructs were also tested to study interfering viral transcription and terminator activity. Here we demonstrate that the presence of a terminator can produce undesirable effects on the activity of heterologous promoters. Our analysis shows that in particular conditions, a terminator can reduce the tissue specificity of the transgene promoter. By RNAse protection assay performed on cardiac myocytes, we also show that adenoviral elements can direct high levels of autonomous transcription within the E1A enhancer region. This finding supports the model that passive readthrough of the transgene promoter is responsible for loss of selective expression.

The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism.

Death-associated protein kinase is a calcium/calmodulin serine/threonine kinase, which positively mediates programmed cell death in a variety of systems. Here we addressed its mode of regulation and identified a mechanism that restrains its apoptotic function in growing cells and enables its activation during cell death. It involves autophosphorylation of Ser(308) within the calmodulin (CaM)-regulatory domain, which occurs at basal state, in the absence of Ca(2+)/CaM, and is inversely correlated with substrate phosphorylation. This type of phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C(6)-ceramide. The substitution of Ser(308) to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca(2+)/CaM-independent substrate phosphorylation as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. Conversely, mutation to aspartic acid reduces the binding of the protein to CaM and abrogates almost completely the death-promoting function of the protein. These results are consistent with a molecular model in which phosphorylation on Ser(308) stabilizes a locked conformation of the CaM-regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device, which keeps death-associated protein kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes.

Many cell types undergo apoptosis under conditions of ischemia. Little is known, however, about the molecular pathways that mediate this response. A cellular and biochemical approach to elucidate such signaling pathways was undertaken in primary cultures of cardiac myocytes, a cell type that is especially sensitive to ischemia-induced apoptosis. Deprivation of serum and glucose, components of ischemia in vivo, resulted in myocyte apoptosis, as determined by nuclear fragmentation, internucleosomal cleavage of DNA, and processing of caspase substrates. These manifestations of apoptosis were blocked by zVAD-fmk, a peptide caspase inhibitor, indicating that caspase activity is necessary for the progression of apoptosis in this model. In contrast to control cells, apoptotic myocytes exhibited cytoplasmic accumulation of cytochrome c, indicating release from the mitochondria. Furthermore, both caspase-9 and caspase-3 were processed to their active forms in serum-/glucose-deprived myocytes. Caspase processing, but not cytochrome c release, was inhibited by zVAD-fmk, placing the latter event upstream of caspase activation. This evidence demonstrates that components of ischemia activate the mitochondrial death pathway in cardiac myocytes.

NF-kappaB inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis.

Toxins convert the hepatocellular response to tumor necrosis factor-alpha (TNF-alpha) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-alpha toxicity. Because nuclear factor-kappaB (NF-kappaB) activation confers resistance to TNF-alpha cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-alpha killing results from inhibition of NF-kappaB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-alpha cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-alpha-induced hepatocyte NF-kappaB activation but decreased NF-kappaB-dependent gene expression. Expression of an IkappaB superrepressor rendered RALA hepatocytes sensitive to TNF-alpha-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-alpha treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-kappaB activation was blocked. Although apoptosis was blocked by the NF-kappaB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-alpha-induced cytotoxicity. Thus NF-kappaB activation is the critical intracellular signal that determines whether TNF-alpha stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-alpha cytotoxicity, endogenous production of NO is not the mechanism by which NF-kappaB activation inhibits this death pathway.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction.