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1.  Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range 
Quantitative analysis of time-resolved data in primary erythroid progenitor cells reveals that a dual negative transcriptional feedback mechanism underlies the ability of STAT5 to respond to the broad spectrum of physiologically relevant Epo concentrations.
A mathematical dual feedback model of the Epo-induced JAK2/STAT5 signaling pathway was calibrated with extensive time-resolved quantitative data sets from immunoblotting, mass spectrometry and qRT–PCR experiments in primary erythroid progenitor cells.We show that the amount of nuclear phosphorylated STAT5 integrated for 60 min post Epo stimulation directly correlates with the fraction of surviving cells 24 h later.CIS and SOCS3 were identified as the most relevant transcriptional feedback regulators of JAK2/STAT5 signaling in primary erythroid progenitor cells. Applying the model, we revealed that CIS-mediated inhibitory effects are most important at low ligand concentrations, whereas SOCS3 inhibition is more effective at high ligand doses.The distinct modes of inhibition of CIS and SOCS3 at various Epo concentrations provide a strategy for achieving control of JAK2/STAT5 signaling over the entire range of physiological Epo concentrations.
Cells interpret information encoded by extracellular stimuli through the activation of intracellular signaling networks and translate this information into cellular decisions. A prime example for a system that is exposed to extremely variable ligand concentrations is the erythroid lineage. The key regulator Erythropoietin (Epo) facilitates continuous renewal of erythrocytes at low basal levels but also secures compensation in case of, e.g., blood loss through an up to 1000-fold increase in hormone concentration. The Epo receptor (EpoR) is expressed on erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage. Stimulation of these cells with Epo leads to rapid but transient activation of receptor and JAK2 phosphorylation followed by phosphorylation of the latent transcription factor STAT5. Although STAT5 is known to be an essential regulator of survival and differentiation of erythroid progenitor cells, a quantitative link between the dynamic properties of STAT5 signaling and survival decisions remained unknown. STAT5-mediated responses in CFU-E cells are modulated by multiple attenuation mechanisms that operate on different time scales. Fast-acting mechanisms such as depletion of Epo by rapid receptor turnover and recruitment of the phosphatase SHP-1 control the initial signal amplitude at the receptor level. Transcriptional feedback regulators such as suppressor of cytokine signaling (SOCS) family members CIS and SOCS3 operate at a slower time scale. Despite the ample knowledge of the individual components involved, only little is known about the specific contributions of these regulators in controlling dynamic properties of STAT5 in response to a broad range of input signals. Therefore, dynamic pathway modeling is required to understand the complex regulatory network of feedback regulators.
To address these questions, we established a dual negative feedback model of JAK2/STAT5 signaling in primary erythroid progenitor cells isolated from mouse fetal livers. We provide a large data set of JAK2/STAT5 signaling dynamics employing quantitative immunoblotting, mass spectrometry and quantitative RT–PCR measured under different perturbation conditions to calibrate our model (Figure 3). The structure of our model was constructed to comprise the minimal number of parameters necessary to explain the data. Thereby, we aimed at a model with fully identifiable parameters that are essential to obtain high predictive power. Parameter identifiability was analyzed by the profile likelihood approach. Applying this method, we could establish a dual negative feedback model of JAK2-STAT5 signaling with structurally and in most cases practically identifiable parameters.
A major bottle-neck in combining signal transduction events with cellular phenotypes is the discrepancy in the time scale and stimuli concentrations that are applied in the different experiments. The sensitivity of biochemical assays to determine phosphorylation events within minutes or hours after stimulation is usually lower than the threshold of sensitivity in assays to determine the physiological response after one or more days. Facilitated by the model, we were able to compute the integrated response of JAK2/STAT5 signaling components for experimentally unaddressable Epo concentrations. Our results demonstrate that the integrated response of pSTAT5 in the nucleus accurately correlates with the experimentally determined survival of CFU-E cells. This provides a quantitative link of the dependency of primary CFU-E cells on pSTAT5 activation dynamics. By correlation analysis, we could identify the early signaling phase (⩽1 h) of STAT5 to be the most predictive for the fraction of surviving cells, which was determined ∼24 h later. Thus, we hypothesize that as a general principle in apoptotic decisions, ligand concentrations translated into kinetic-encoded information of early signaling events downstream of receptors can be predictive for survival decisions 24 h later.
After the first hour of stimulation, it is important to constrain signaling to a residual steady-state level. Constitutive phosphorylation of the JAK2/STAT5 pathway has a crucial role in the onset of polycythemia vera (PV), a disease associated with Epo-independent erythroid differentiation. The two identified transcriptional feedback proteins, CIS and SOCS3, are responsible for adjusting the phosphorylation level of STAT5 after 1 h of stimulation. Since the Epo input signal can vary over a broad range of ligand concentrations, we asked how CIS and SOCS3 can facilitate control of STAT5 long-term phosphorylation levels over the entire physiological relevant hormone concentrations. By using model simulations, we revealed that the two feedbacks are most effective at different Epo concentration ranges. Predicted by our mathematical model, the major role of CIS in modulating STAT5 phosphorylation levels is at low, basal Epo concentrations, whereas SOCS3 is essential to control the STAT5 phosphorylation levels at high Epo doses (Figure 6). As a potential molecular mechanism of this dose-dependent inhibitory effect, we could identify the quantity of pJAK2 relative to pEpoR that increases with higher Epo concentrations. Since SOCS3 can inhibit JAK2 directly via its KIR domain to attenuate downstream STAT5 activation, SOCS3 becomes more effective with the relative increase of JAK2 activation. Hence, CIS and SOCS3 act in a concerted manner to ensure tight regulation of STAT5 responses over the broad physiological range of Epo concentrations.
In summary, our mathematical approach provided new insights into the specific function of feedback regulation in STAT5-mediated life or death decisions of primary erythroid cells. We dissected the roles of the transcriptionally induced proteins CIS and SOCS3 that operate as dual feedback with divided function thereby facilitating the control of STAT5 activation levels over the entire range of physiological Epo concentrations. The detailed understanding of the molecular processes and control distribution of Epo-induced JAK/STAT signaling can be further applied to gain insights into alterations promoting malignant hematopoietic diseases.
Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.
doi:10.1038/msb.2011.50
PMCID: PMC3159971  PMID: 21772264
apoptosis; erythropoietin; mathematical modeling; negative feedback; SOCS
2.  The Translational Regulators GCN-1 and ABCF-3 Act Together to Promote Apoptosis in C. elegans 
PLoS Genetics  2014;10(8):e1004512.
The proper regulation of apoptosis requires precise spatial and temporal control of gene expression. While the transcriptional and translational activation of pro-apoptotic genes is known to be crucial to triggering apoptosis, how different mechanisms cooperate to drive apoptosis is largely unexplored. Here we report that pro-apoptotic transcriptional and translational regulators act in distinct pathways to promote programmed cell death. We show that the evolutionarily conserved C. elegans translational regulators GCN-1 and ABCF-3 contribute to promoting the deaths of most somatic cells during development. GCN-1 and ABCF-3 are not obviously involved in the physiological germ-cell deaths that occur during oocyte maturation. By striking contrast, these proteins play an essential role in the deaths of germ cells in response to ionizing irradiation. GCN-1 and ABCF-3 are similarly co-expressed in many somatic and germ cells and physically interact in vivo, suggesting that GCN-1 and ABCF-3 function as members of a protein complex. GCN-1 and ABCF-3 are required for the basal level of phosphorylation of eukaryotic initiation factor 2α (eIF2α), an evolutionarily conserved regulator of mRNA translation. The S. cerevisiae homologs of GCN-1 and ABCF-3, which are known to control eIF2α phosphorylation, can substitute for the worm proteins in promoting somatic cell deaths in C. elegans. We conclude that GCN-1 and ABCF-3 likely control translational initiation in C. elegans. GCN-1 and ABCF-3 act independently of the anti-apoptotic BCL-2 homolog CED-9 and of transcriptional regulators that upregulate the pro-apoptotic BH3-only gene egl-1. Our results suggest that GCN-1 and ABCF-3 function in a pathway distinct from the canonical CED-9-regulated cell-death execution pathway. We propose that the translational regulators GCN-1 and ABCF-3 maternally contribute to general apoptosis in C. elegans via a novel pathway and that the function of GCN-1 and ABCF-3 in apoptosis might be evolutionarily conserved.
Author Summary
Apoptosis, also referred to as programmed cell death, is a crucial cellular process that eliminates unwanted cells during animal development and tissue homeostasis. Abnormal regulation of apoptosis can cause developmental defects and a variety of other human disorders, including cancer, neurodegenerative diseases and autoimmune diseases. Therefore, it is important to identify regulatory mechanisms that control apoptosis. Previous studies have demonstrated that the transcriptional induction of apoptotic genes can be crucial to initiating an apoptotic program. Less is known about translational controls of apoptosis. Here we report that the evolutionarily conserved C. elegans translational regulators GCN-1 and ABCF-3 promote apoptosis generally and act independently of the anti-apoptotic BCL-2 homolog CED-9. GCN-1 and ABCF-3 physically interact and maintain the phosphorylation level of eukaryotic initiation factor 2α, suggesting that GCN-1 and ABCF-3 act together to regulate the initiation of translation. We propose that the translational regulators GCN-1 and ABCF-3 maternally contribute to the proper execution of the apoptotic program.
doi:10.1371/journal.pgen.1004512
PMCID: PMC4125083  PMID: 25101958
3.  Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation. 
Molecular and Cellular Biology  1996;16(10):5450-5457.
Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.
PMCID: PMC231545  PMID: 8816458
4.  Apoptosis-Inducing Factor: Structure, Function, and Redox Regulation 
Antioxidants & Redox Signaling  2011;14(12):2545-2579.
Abstract
Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide-containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space whose specific enzymatic activity remains unknown. Upon an apoptotic insult, AIF undergoes proteolysis and translocates to the nucleus, where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner. Besides playing a key role in execution of caspase-independent cell death, AIF has emerged as a protein critical for cell survival. Analysis of in vivo phenotypes associated with AIF deficiency and defects, and identification of its mitochondrial, cytoplasmic, and nuclear partners revealed the complexity and multilevel regulation of AIF-mediated signal transduction and suggested an important role of AIF in the maintenance of mitochondrial morphology and energy metabolism. The redox activity of AIF is essential for optimal oxidative phosphorylation. Additionally, the protein is proposed to regulate the respiratory chain indirectly, through assembly and/or stabilization of complexes I and III. This review discusses accumulated data with respect to the AIF structure and outlines evidence that supports the prevalent mechanistic view on the apoptogenic actions of the flavoprotein, as well as the emerging concept of AIF as a redox sensor capable of linking NAD(H)-dependent metabolic pathways to apoptosis. Antioxid. Redox Signal. 14, 2545–2579.
Introduction
Multiple Forms of AIF
AIF precursor
Membrane-tethered mature AIFΔ1–54
Soluble apoptogenic AIFΔ1–102/118
AIF associated with the outer mitochondrial membrane
Splice variants AIF2, AIFsh, AIFsh2, and AIFsh3
Transcriptional Regulation
Phylogenetic Roots
Redox Properties of Recombinant AIF
Refolded murine AIFΔ1–120
Refolded human AIFsh2
Naturally folded murine AIFΔ1–53 and Δ1–101
AIF Structure
X-ray structures of murine and human AIFΔ1–120
X-ray structure of murine AIFΔ1–77
X-ray structure of reduced NAD-bound murine AIFΔ1–101
Redox-linked changes in the active site
Reorganization in the C-terminal domain
Conformational changes in the 509–559 peptide
Role of AIF in PCD
Apoptogenic effects of AIF in cell free systems and live cells
Release of mitochondrial AIF
Proteolysis of mature AIF
Release of truncated AIF into the cytoplasm
Release of AIF associated with the outer mitochondrial membrane
Cytoplasmic interactions of apoptogenic AIF
Pro-survival partners of AIF
Heat shock protein Hsp70
X-linked inhibitor of apoptosis protein
Pro-death partners of AIF
Eukaryotic translation initiation factor 3 subunit p44
T-cell ubiquitin ligand
Cyclophilin A
Phospholipid scramblase
Scythe
Nuclear effects of apoptogenic AIF
Transport of AIF to the nucleus
Interaction of AIF with DNA
Nuclear partners of AIF
Endonuclease G
Cyclophilin A
Histone H2AX
Relocation of AIF in late apoptosis
Apoptogenic properties of the AIF homologs
D. melanogaster
D. discoideum
Tetrahymena thermophila
S. cerevisiae
Vital Functions of Mitochondrial AIF
Role of AIF in mitochondrial respiration
Hq mouse phenotype
Tissue-specific AIF defects
Role of AIF in neurodegeneration, neurogenesis, and neuroprotection
AIF deficiency in lower eukaryotes
AIF and mitochondrial morphology
Mitochondrial abnormalities in telencephalon-specific AIFΔ mice
Association of AIF with the optic atrophy 1 protein
AIF isoform-specific cristae morphology
Human mitochondrial encephalomyopathy linked to the AIFΔ201 mutation
D. Involvement of AIF in regulation of cytoplasmic stress granules
Possible Redox Sensing Role of AIF
Concluding Remarks
doi:10.1089/ars.2010.3445
PMCID: PMC3096518  PMID: 20868295
5.  eIF4EBP3L Acts as a Gatekeeper of TORC1 In Activity-Dependent Muscle Growth by Specifically Regulating Mef2ca Translational Initiation 
PLoS Biology  2013;11(10):e1001679.
Muscle activity promotes muscle growth through the TOR-4EBP pathway by controlling the translation of specific mRNAs, including Mef2ca, a muscle transcription factor required for normal growth.
Muscle fiber size is activity-dependent and clinically important in ageing, bed-rest, and cachexia, where muscle weakening leads to disability, prolonged recovery times, and increased costs. Inactivity causes muscle wasting by triggering protein degradation and may simultaneously prevent protein synthesis. During development, muscle tissue grows by several mechanisms, including hypertrophy of existing fibers. As in other tissues, the TOR pathway plays a key role in promoting muscle protein synthesis by inhibition of eIF4EBPs (eukaryotic Initiation Factor 4E Binding Proteins), regulators of the translational initiation. Here, we tested the role of TOR-eIF4EBP in a novel zebrafish muscle inactivity model. Inactivity triggered up-regulation of eIF4EBP3L (a zebrafish homolog of eIF4EBP3) and diminished myosin and actin content, myofibrilogenesis, and fiber growth. The changes were accompanied by preferential reduction of the muscle transcription factor Mef2c, relative to Myod and Vinculin. Polysomal fractionation showed that Mef2c decrease was due to reduced translation of mef2ca mRNA. Loss of Mef2ca function reduced normal muscle growth and diminished the reduction in growth caused by inactivity. We identify eIF4EBP3L as a key regulator of Mef2c translation and protein level following inactivity; blocking eIF4EBP3L function increased Mef2ca translation. Such blockade also prevented the decline in mef2ca translation and level of Mef2c and slow myosin heavy chain proteins caused by inactivity. Conversely, overexpression of active eIF4EBP3L mimicked inactivity by decreasing the proportion of mef2ca mRNA in polysomes, the levels of Mef2c and slow myosin heavy chain, and myofibril content. Inhibiting the TOR pathway without the increase in eIF4EBP3L had a lesser effect on myofibrilogenesis and muscle size. These findings identify eIF4EBP3L as a key TOR-dependent regulator of muscle fiber size in response to activity. We suggest that by selectively inhibiting translational initiation of mef2ca and other mRNAs, eIF4EBP3L reprograms the translational profile of muscle, enabling it to adjust to new environmental conditions.
Author Summary
Most genes are transcribed into mRNA and then translated into proteins that function in various cellular processes. Initiation of mRNA translation is thus a fundamental control point in gene expression. Working in a zebrafish model, we have found that muscle activity (or inactivity) can differentially regulate the translation of specific mRNAs and thereby control the growth of skeletal muscle. Emerging evidence suggests that control of translational initiation of particular mRNAs by an intracellular signaling pathway acting through TORC1 is a major regulator of cell growth and function. We show here that muscle activity both activates the TORC1 pathway and suppresses the expression of a downstream TORC1 target—the translational inhibitor eIF4EBP3L. This removes a brake on translation of certain mRNAs. Conversely, we show that muscle inactivity can up-regulate this translational inhibitor, thereby causing reduced translation of these mRNAs. One of the mRNAs targeted in this manner by eIF4EBP3L is Mef2ca, which encodes a transcription factor that promotes assembly of muscle contractile apparatus. Our work thus reveals a mechanism by which muscle growth can be differentially influenced depending on the context of muscle activity (or lack thereof). If this pathway operates in people, it may help explain how exercise regulates muscle growth and performance.
doi:10.1371/journal.pbio.1001679
PMCID: PMC3797031  PMID: 24143132
6.  Translational Regulation of Specific mRNAs Controls Feedback Inhibition and Survival during Macrophage Activation 
PLoS Genetics  2014;10(6):e1004368.
For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the resolution of inflammation.
Author Summary
When macrophages encounter pathogens, they initiate inflammation by secreting pro-inflammatory factors such as the cytokine TNF. Because a prolonged or overshooting release of these factors is harmful for the organism, their production needs to be tightly controlled and shut off in due time. To ensure a rapid but transient inflammatory response, gene expression is regulated at multiple levels, including transcription, stability and translation of mRNAs. While control of transcription and mRNA stability has been studied extensively, little is known about translational regulation in macrophages. In this study, we measured the translation of all mRNAs expressed in mouse macrophages. Upon activation of macrophages with the bacterial cell wall component lipopolysaccharide, we found that many feedback inhibitors, which are important for dampening the inflammatory response, are translationally up-regulated. Translation of these mRNAs is repressed in resting cells and de-repressed after stimulation. In contrast to feedback inhibitors, most cytokines are primarily regulated by changes in mRNA abundance. Furthermore, we could show that one of the feedback inhibitors, IER3, protects macrophages from cell death during activation. Therefore, regulation at the level of translation is important for the induction of negative feedback loops and cellular survival.
doi:10.1371/journal.pgen.1004368
PMCID: PMC4063670  PMID: 24945926
7.  Regulation of protein translation initiation in response to ionizing radiation 
Background
Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR) blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR.
Methods
Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach.
Results
IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1.
Conclusion
Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells.
doi:10.1186/1748-717X-8-35
PMCID: PMC3577660  PMID: 23402580
lonizing radiation; Protein translation; Eukaryotic initiation factor; Akt; mTOR; Apoptosis; Mcl-1
8.  Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells 
PLoS Pathogens  2013;9(1):e1003100.
During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.
Author Summary
HIV-1 infected individuals become increasingly immunocompromised and susceptible to opportunistic infection during disease progression, which is associated with significant reduction of the dendritic cell number in the peripheral blood or secondary lymphoid tissues. Because dendritic cells are the most powerful antigen-presenting cells, their survival is critical for host defence and inadequate dendritic cell number will fail to induce effective host immune responses. Here we describe a mechanism that may at least partly explain why dendritic cells become significantly depleted in chronic HIV-1 infection. We found that after binding of the HIV-1 envelope protein gp120 to the dendritic cell surface protein DC-SIGN, the subsequent activation by CD40 ligation, or by exposure to bacterial product lipopolysaccharide or pro-inflammatory cytokines such as TNF-α and IL-1β, will lead to overexpression of pro-apoptotic molecule ASK-1, resulting in excessive dendritic cell death. We also confirmed that DC-SIGN(+) dendritic cells in the blood of HIV-1 infected individuals have actually been pre-sensitized by viral gp120, which exists in vast amount in the blood, for activation-induced exorbitant death. Our study thus reveals a previously unknown pathway for dendritic cell depletion and provides clues for potential therapeutic approaches to prevent DC depletion in chronic HIV infection.
doi:10.1371/journal.ppat.1003100
PMCID: PMC3561151  PMID: 23382671
9.  A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation 
Molecular and Cellular Biology  2000;20(2):496-506.
Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5′ untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.
PMCID: PMC85113  PMID: 10611228
10.  CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis 
Molecular Biology of the Cell  2013;24(15):2477-2490.
This study addresses the mechanisms by which CHOP directs gene regulatory networks and determines cell fate. Transcriptional expression of ATF5 is activated by both CHOP and ATF4 in the integrated stress response. CHOP and ATF5 control a switch to activate apoptotic genes and decrease cell survival in response to loss of proteostatic control.
Environmental stresses that disrupt protein homeostasis induce phosphorylation of eIF2, triggering repression of global protein synthesis coincident with preferential translation of ATF4, a transcriptional activator of the integrated stress response (ISR). Depending on the extent of protein disruption, ATF4 may not be able to restore proteostatic control and instead switches to a terminal outcome that features elevated expression of the transcription factor CHOP (GADD153/DDIT3). The focus of this study is to define the mechanisms by which CHOP directs gene regulatory networks that determine cell fate. We find that in response to proteasome inhibition, CHOP enhances the expression of a collection of genes encoding transcription regulators, including ATF5, which is preferentially translated during eIF2 phosphorylation. Transcriptional expression of ATF5 is directly induced by both CHOP and ATF4. Knockdown of ATF5 increases cell survival in response to proteasome inhibition, supporting the idea that both ATF5 and CHOP have proapoptotic functions. Transcriptome analysis of ATF5-dependent genes reveals targets involved in apoptosis, including NOXA, which is important for inducing cell death during proteasome inhibition. This study suggests that the ISR features a feedforward loop of stress-induced transcriptional regulators, each subject to transcriptional and translational control, which can switch cell fate toward apoptosis.
doi:10.1091/mbc.E13-01-0067
PMCID: PMC3727939  PMID: 23761072
11.  Regulation of Neuronal mRNA Translation by CaM-Kinase I Phosphorylation of eIF4GII 
The Journal of Neuroscience  2012;32(16):5620-5630.
Summary
Ca2+/CaM-kinases (CaMKs) are essential for neuronal development and plasticity, processes requiring de novo protein synthesis. Roles for CaMKs in modulating gene transcription are well established, but their involvement in mRNA translation is evolving. Here we report that activity-dependent translational initiation in cultured rat hippocampal neurons is enhanced by CaMKI-mediated phosphorylation of Ser1156 in eukaryotic initiation factor eIF4GII (4GII). Treatment with bicuculline or gabazine to enhance neuronal activity promotes recruitment of wild-type 4GII, but not the 4GII S1156A mutant or 4GI, to the heterotrimeric eIF4F (4F) complex that assembles at the 5' cap structure (m7GTP) of mRNA to initiate ribosomal scanning. Recruitment of 4GII to 4F is suppressed by pharmacological inhibition (STO-609) of CaM-kinase kinase, the upstream activator of CaMKI. Post-hoc in vitro CaMKI phosphorylation assays confirm that activity promotes phosphorylation of S1156 in transfected 4GII in neurons. Changes in cap-dependent and cap-independent translation were assessed using a bi-cistronic luciferase reporter transfected into neurons. Activity upregulates cap-dependent translation, and RNAi knockdown of CaMKIβ and γ isoforms, but not α or δ, led to its attenuation as did blockade of NMDA receptors. Furthermore, RNAi knockdown of 4GII attenuates cap-dependent translation and reduces density of dendritic filopodia and spine formation without effect on dendritic arborization. Taken together, our results provide a mechanistic link between Ca2+ influx due to neuronal activity and regulation of cap-dependent RNA translation via CaMKI activation and selective recruitment of phosphorylated 4GII to the 4F complex that may function to regulate activity-dependent changes in spine density.
doi:10.1523/JNEUROSCI.0030-12.2012
PMCID: PMC3346851  PMID: 22514323
12.  Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest 
PLoS Pathogens  2014;10(7):e1004217.
Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.
Author Summary
Like all viruses, Influenza A virus (IAV) is absolutely dependent on host-cell protein synthesis machinery. This dependence makes the virus vulnerable to the innate ability of cells to inhibit protein synthesis in response to various types of stress. This inhibition, termed translation arrest, helps cells survive adverse conditions by re-dedicating their energy to stress responses. When cells arrest translation, they form stress granules: depots of untranslated mRNAs and associated proteins. Translation arrest and formation of stress granules can be induced pharmacologically, and in this work we sought to determine whether stress granule induction would be effective in blocking IAV replication. Here we demonstrate that treatment of cells with inducers of stress granules at early times after infection resulted in blockade of viral protein synthesis and stopped viral replication. At later times post-infection, by contrast, IAV proteins prevented pharmacological induction of stress granules. We identified three viral proteins – more than in any virus to date – that work in concert to prevent stress granule formation. Taken together, our studies reveal a multipronged approach for viral suppression of translation arrest, and identify a window of opportunity early in infection when pharmacological induction of stress granules has a strong antiviral effect.
doi:10.1371/journal.ppat.1004217
PMCID: PMC4092144  PMID: 25010204
13.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
doi:10.1038/msb.2010.29
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
14.  Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q 
PLoS Biology  2013;11(5):e1001564.
The heterogeneous nuclear ribonucleoprotein Q2 competitively binds mRNA poly(A) tails to regulate translational and miRNA-related functions of PABP.
Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5′ m7G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs.
Author Summary
The regulation of mRNA translation and stability is of paramount importance for almost every cellular function. In eukaryotes, the poly(A) binding protein (PABP) is a central regulator of both global and mRNA-specific translation. PABP simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G). These interactions circularize the mRNA and stimulate translation. PABP also regulates specific mRNAs by promoting miRNA-dependent deadenylation and translational repression. A key step in understanding PABP's functions is to identify factors that affect its association with the poly(A) tail. Here we show that the cytoplasmic isoform of the mouse heterogeneous nuclear ribonucleoprotein Q (hnRNP-Q2/SYNCRIP), which exhibits binding preference to poly(A), interacts with the poly(A) tail by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to the poly(A) tail. Depleting hnRNP-Q2 stimulates translation in cell-free extracts and in cultured cells, in agreement with its function as translational repressor. In addition, hnRNP-Q2 impeded miRNA-mediated deadenylation and repression of target mRNAs, which requires PABP. Thus, competition from hnRNP-Q2 provides a novel mechanism by which multiple functions of PABP are regulated. This regulation could play important roles in various biological processes, such as development, viral infection, and human disease.
doi:10.1371/journal.pbio.1001564
PMCID: PMC3660254  PMID: 23700384
15.  The insulin receptor cellular IRES confers resistance to eIF4A inhibition 
eLife  2013;2:e00542.
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.
DOI: http://dx.doi.org/10.7554/eLife.00542.001
eLife digest
Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA (mRNA) in the nucleus, and then translation of that mRNA into a protein by ribosomes in the cytoplasm. These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions. This involves regulating the expression of the genes that code for these proteins.
When cells experience stressful events, such as a shortage of oxygen or nutrients, they reduce the synthesis of most proteins. This response is regulated, in part, by a signaling pathway known as the insulin and insulin-like receptor pathway. In particular, stressful events inhibit a protein complex called eIF4F, which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap. Despite this general inhibition, the production of certain other proteins—including the insulin receptor itself—is actually increased in response to stress.
Olson et al. have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor. The eIF4F complex is made up of three proteins, including one that binds to the 5′ cap and a helicase that unwinds the RNA. Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein. Olsen et al. now show that translation of this mRNA is also independent of the helicase. Instead, translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site, which allows ribosomes to bind to the mRNA without the influence of the 5′ cap.
Olson et al. reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells, suggesting this pathway represents a crucial regulatory process that has been conserved during evolution. A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors.
DOI: http://dx.doi.org/10.7554/eLife.00542.002
doi:10.7554/eLife.00542
PMCID: PMC3713452  PMID: 23878722
Foxo; IRES; Insulin receptor; PDCD4; eIF4A; IGFR; D. melanogaster; Mouse
16.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Background
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
Conclusions
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Background
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050094.
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050094
PMCID: PMC2386836  PMID: 18494554
17.  An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication 
PLoS Pathogens  2013;9(1):e1003147.
Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5′ and 3′ untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5′-UTRs lack internal ribosomal entry site function. However, the 5′-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5′-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5′-UTR. The L 5′-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5′-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a “weak” Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10–100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target.
Author Summary
Filoviruses (Ebola and Marburg viruses) are emerging zoonotic pathogens that cause lethal hemorrhagic fever in humans and have the potential to be employed as bioterrorism agents. Currently, approved therapeutics to treat filovirus infections are not available and new treatment strategies could be facilitated by improved mechanistic insight into the virus replication cycle. Compared to other related viruses, filovirus messenger RNAs have unusually long 5′ untranslated regions (UTRs) with undefined functions. In the Zaire ebolavirus (EBOV) genome, four of its seven messenger RNAs have 5′-UTRs with a small upstream open reading frame (uORF). We found that a uORF present in the EBOV polymerase (L) 5′-UTR suppresses L protein production and established a reporter assay to demonstrate that this uORF maintains L translation following the induction of an innate immune response; a phenomenon observed with several uORF-containing cellular messenger RNAs. The presence of the uORF is important for optimal virus replication, because a mutant virus lacking the upstream reading frame replicates less efficiently than a wildtype virus, an attenuation which is more pronounced following the induction of cellular stress. These studies define a novel mechanism by which filovirus upstream open reading frames modulate virus protein translation in the face of an innate immune response and highlight their importance in filovirus replication.
doi:10.1371/journal.ppat.1003147
PMCID: PMC3561295  PMID: 23382680
18.  MicroRNA Expression Profile in Human Macrophages in Response to Leishmania major Infection 
Background
Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts.
Methodology/Principal Findings
We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation.
Conclusions/Significance
Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.
Author Summary
Leishmania parasites belong to different species, each one characterized by specific vectors and reservoirs, and causes cutaneous or visceral disease(s) of variable clinical presentation and severity. In its mammalian host, the parasite is an obligate intracellular pathogen infecting the monocyte/macrophage lineage. Leishmania have developed ambiguous relationships with macrophages. Indeed, these cells are the shelter of invading parasites, where they will grow and eventually will reside in a silent state for life. But macrophages are also the cells that participate, through the induction of several pro-inflammatory mediators and antigen presentation, to shape the host immune response and ultimately kill the invader. To subvert these anti-parasite responses, Leishmania manipulate the host machinery for their own differentiation and survival. We aimed to evaluate the impact of L. major (the causative agent of zoonotic cutaneous leishmaniasis) infection on deregulation of non-coding miRNAs, a class of important regulators of gene expression. Our results revealed the implication of several miRNAs on macrophage fate upon parasite infection through regulation of different pathways, including cell death. Our findings provided a new insight for understanding mechanisms governing this miRNA deregulation by parasite infection and will help to provide clues for the development of control strategies for this disease.
doi:10.1371/journal.pntd.0002478
PMCID: PMC3789763  PMID: 24098824
19.  Post-transcriptional regulation in the myo1Δ mutant of Saccharomyces cerevisiae 
BMC Genomics  2010;11:690.
Background
Saccharomyces cerevisiae myosin type II-deficient (myo1Δ) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1Δ transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1Δ strain may provide a simplified paradigm for cell wall stress survival.
Results
To explore the post-transcriptional regulation of the myo1Δ stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1Δ strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1Δ strains. Deletion of GCN2 in myo1Δ abolished eIF2αp phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1Δ strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1Δ and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2α-P in myo1Δ strains.
Conclusions
Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1Δ strains. Inhibition of translation initiation factor eIF2α (eIF2α-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2α-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1Δ strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2α-P in myo1Δ strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1Δ stress response and possibly other stresses in yeast.
doi:10.1186/1471-2164-11-690
PMCID: PMC3017085  PMID: 21126371
20.  Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells 
PLoS Biology  2012;10(1):e1001245.
Cyclic oscillations in the level of phosphatidylinositol 3-phosphate in phagosomes, regulated by two phosphoinositide kinases and one phosphatase, are critical for phagosome maturation and degradation of apoptotic cells.
Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.
Author Summary
During animal development and in adulthood many cells are programmed to die by an active process called apoptosis. These dead or dying apoptotic cells are swiftly taken up by scavenger cells into membrane-bound compartments—phagosomes—where they are subsequently degraded when other intracellular organelles containing digestive enzymes fuse with phagosomes—a process called phagosome maturation. Phagocytosis of apoptotic cells is important for tissue remodeling in development and to prevent harmful inflammatory and autoimmune responses. In nematode worms—a model organism in which to study apoptosis—phagosome maturation is accompanied by two waves of the signaling molecule phosphatidylinositol 3-phosphate (PtdIns(3)P) in this compartment: one that forms soon after the formation of the phagosome and lasts for 10–15 minutes, and a second, weaker one 10 minutes later that lasts until the apoptotic cell is fully digested. In this study, we investigated the mechanism that regulates the timing and length of these two waves. We found that they are established by the sequential and combined action of three enzymes: two phosphoinositide 3-kinases, which add a phosphate group to the 3′ site of PtdIns, and one phosphoinositide 3-phosphatase, which removes it. We showed that inactivation of both kinases depleted phagosomes of PtdIns(3)P and resulted in the arrest of phagosome maturation and degradation of apoptotic cells. In addition, the timely turnover of PtdIns(3)P catalyzed by the phosphatase was critical for the step-wise progress of phagosome maturation. Our findings suggest that reversible phosphorylation of phophoinositides, catalyzed by distinct sets of kinases and phosphatases, might be a general mechanism to drive multi-step intracellular membrane trafficking events.
doi:10.1371/journal.pbio.1001245
PMCID: PMC3260314  PMID: 22272187
21.  mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8+ T Cells 
PLoS Pathogens  2014;10(10):e1004423.
Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.
Author Summary
Maintenance proteins of viruses establishing latent infections regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The Epstein-Barr virus maintenance protein, EBNA1, has recently been shown to contain unusual G-quadruplex structures within its repeat mRNA that reduces its translational efficiency. In this study we assess how modification of the EBNA1 mRNA repeat sequence to destabilize the native G-quadruplex structures and thereby increase translation, impacts on the activation of EBNA1-specific T cells in vivo. Mice primed with viral vectors encoding a more efficiently translated EBNA1 mRNA revealed increased trafficking of EBNA1-specific T cells, an enhanced functional profile and increased expression of transcription factors providing evidence for a potential link between mRNA translational efficiency and antigen presentation in vivo and the resultant impact on the functional programming of effector T cells. These findings suggest a novel approach to therapeutic development through the use of antisense strategies or small molecules targeting EBNA1 mRNA structure.
doi:10.1371/journal.ppat.1004423
PMCID: PMC4192603  PMID: 25299404
22.  Modifications of the 5′ Cap of mRNAs during Xenopus Oocyte Maturation: Independence from Changes in Poly(A) Length and Impact on Translation 
Molecular and Cellular Biology  1998;18(10):6152-6163.
The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3′ end of the mRNA can affect the state of the 5′ cap structure, m7G(5′)ppp(5′)G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5′ termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.
PMCID: PMC109201  PMID: 9742132
23.  Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study 
Respiratory Research  2010;11(1):45.
Background
Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD.
Methods
The extent of dendritic cell infiltration in COPD tissue specimens was determined using immunohistochemical localization of CD83+ cells (marker of matured myeloid dendritic cells), and CD1a+ cells (Langerhans cells). The extent of tissue infiltration with Langerhans cells was also determined by the relative expression of the CD207 gene in COPD versus control tissues. To determine mechanisms by which dendritic cells accumulate in COPD, complimentary studies were conducted using monocyte-derived human dendritic cells exposed to cigarette smoke extract (CSE), and dendritic cells extracted from mice chronically exposed to cigarette smoke.
Results
In human COPD lung tissue, we detected a significant increase in the total number of CD83+ cells, and significantly higher amounts of CD207 mRNA when compared with control tissue. Human monocyte-derived dendritic cells exposed to CSE (0.1-2%) exhibited enhanced survival in vitro when compared with control dendritic cells. Murine dendritic cells extracted from mice exposed to cigarette smoke for 4 weeks, also demonstrated enhanced survival compared to dendritic cells extracted from control mice. Acute exposure of human dendritic cells to CSE induced the cellular pro-survival proteins heme-oxygenase-1 (HO-1), and B cell lymphoma leukemia-x(L) (Bcl-xL), predominantly through oxidative stress. Although activated human dendritic cells conditioned with CSE expressed diminished migratory CCR7 expression, their migration towards the CCR7 ligand CCL21 was not impaired.
Conclusions
These data indicate that COPD is associated with increased numbers of cells bearing markers associated with Langerhans cells and mature dendritic cells, and that cigarette smoke promotes survival signals and augments survival of dendritic cells. Although CSE suppressed dendritic cell CCR7 expression, migration towards a CCR7 ligand was not diminished, suggesting that reduced CCR7-dependent migration is unlikely to be an important mechanism for dendritic cell retention in the lungs of smokers with COPD.
doi:10.1186/1465-9921-11-45
PMCID: PMC2867978  PMID: 20420706
24.  Reduced Secretion of YopJ by Yersinia Limits In Vivo Cell Death but Enhances Bacterial Virulence 
PLoS Pathogens  2008;4(5):e1000067.
Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow–derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis–infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.
Author Summary
The ability of bacterial pathogens to modulate death of infected host cells is an important virulence determinant. For pathogenic members of the genus Yersinia, the type III secreted effector protein YopJ/YopP is required for Yersinia-induced macrophage death. The YopJ protein is expressed by Y. pseudotuberculosis, while the ninety-four percent identical YopP protein is expressed by Y. enterocolitica. Y. enterocolitica infection also triggers YopP-dependent killing of dendritic cells, which are critical antigen presenting cells of the immune system. We demonstrate that in contrast to macrophages, dendritic cells are resistant to Y. pseudotuberculosis-mediated cytotoxicity. However, Y. pseudotuberculosis expressing YopP in place of YopJ was highly cytotoxic toward dendritic cells. This difference in cytotoxicity was attributable to a difference in the delivery of YopJ and YopP into mammalian cells. Furthermore, mutation of two amino acids at the N-terminus of YopJ enhanced its delivery and cytotoxicity. Remarkably, we found that enhancing the cytotoxicity of Y. pseudotuberculosis by expression of YopP led to its attenuation in a mouse model of Yersinia infection. This indicates that optimal virulence for a given pathogen requires careful regulation of virulence properties and highlights the potential evolutionary tradeoffs between cellular cytotoxicity and in vivo virulence.
doi:10.1371/journal.ppat.1000067
PMCID: PMC2361194  PMID: 18483548
25.  Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation. 
Molecular and Cellular Biology  1992;12(3):1239-1247.
Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.
Images
PMCID: PMC369556  PMID: 1545805

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