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Chromatin organizer SATB1 and Wnt transducer β-catenin form a complex and regulate expression of GATA3 and TH2 cytokines in Wnt-dependent manner and orchestrate TH2 lineage commitment., In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of β-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/β -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with β-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon β-catenin signalling. GATA-3 is a T helper type 2 (TH2) specific transcription factor that regulates production of TH2 cytokines and functions as TH2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells, suggesting that SATB1 influences TH2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature TH2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for TH2 differentiation. Knockdown of β-catenin also produced similar results, confirming the role of Wnt/β-catenin signalling in TH2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits β-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating TH2 cells in a Wnt-dependent manner. SATB1 coordinates TH2 lineage commitment by reprogramming gene expression. The SATB1:β-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates TH2 lineage commitment by mediating Wnt/β-catenin signalling. This report identifies a new global transcription factor involved in β-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis., Author Summary In vertebrates the canonical Wnt signalling culminates in β-catenin moving into the nucleus where it activates transcription of target genes. Wnt/β-catenin signalling is essential for the thymic maturation and differentiation of naïve T cells. Here we show that SATB1, a T cell lineage-enriched chromatin organizer and global regulator, binds to β-catenin and recruits it to SATB1's genomic binding sites so that genes formerly repressed by SATB1 are upregulated by Wnt signalling. Some of the genes known to be regulated by SATB1 (such as genes encoding cytokines and the transcription factor GATA3) are required for differentiation of Th2 cells, an important subset of helper T cells. Specifically we show that siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells. Inhibiting Wnt signalling led to downregulation of GATA-3 and of signature TH2 cytokines such as IL-4, IL-10, and IL-13. Knockdown of β-catenin also produced similar results, thus together these data confirm the role of Wnt/β-catenin signalling in TH2 differentiation. Our data demonstrate that SATB1 orchestrates TH2 lineage commitment by modulating Wnt/β-catenin signalling.

Background Tamoxifen is still the most widely used drug in hormone therapy for the treatment of breast cancer. Its benefits in adjuvant treatment are well documented in controlled and randomized clinical studies, which have demonstrated an increase in disease-free intervals of patients with positive hormonal receptors. However, the mechanisms involved in endocrine resistance are not clear. Laboratory and clinical data now indicate that bi-directional molecular cross-talk between nuclear or membrane ER and growth factor receptor pathways may be involved in endocrine resistance. We recently found a functional interaction between α6β4 integrin and ErbB-3 receptor to maintain the PI3K/Akt survival pathway of mammary tumour cells. We sought to improve understanding of this process in order to provide the involvement of both receptors insight into mechanism of Tamoxifen resistance. Methods and Findings Using human breast cancer cell lines displaying different levels of α6β4 and ErbB-3 receptors and a series of 232 breast cancer biopsies from patients submitted to adjuvant Tamoxifen monotherapy for five years, we evaluated the functional interaction between both receptors in relationship to Tamoxifen responsiveness. In mammary carcinoma cells, we evidenced that the α6β4 integrin strongly influence Akt phosphorylation through ErbB-3 protein regulation. Moreover, the ErbB-3 inactivation inhibits Akt phosphorylation, induces apoptosis and inhibits in vitro invasion favouring Tamoxifen responsiveness. The analysis of human tumors revealed a significant relationship between α6β4 and ErbB-3 in P-Akt-positive and ERβ1-negative breast cancers derived from patients with lower disease free survival. Conclusions We provided evidence that a strong relationship occurs between α6β4 and ErbB-3 positivity in ERβ1-negative breast cancers. We also found that the association between ErbB-3 and P-Akt positivity mainly occurs in ERβ1-negative breast cancer derived from patients with lower DFS indicating that both receptors are clinically relevant in predicting the response to Tamoxifen.

Background We recently reported that colon tumor cells stimulate macrophages to release IL-1β, which in turn inactivates GSK3β and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells. Principal Findings Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1β by neutralizing IL-1β antibody, or silencing of IL-1β in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1β was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Δψ) and activation of caspases were prevented by macrophages or by recombinant IL-1β. Pharmacological inhibition of IL-1β release from macrophages by vitamin D3, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1β failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIκB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1β stabilized Snail in tumor cells in an NF-κB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1β, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL. Significance We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1β, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D3 halts this amplifying loop by interfering with the release of IL-1β from macrophages. Accordingly, vitamin D3 sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.

BACKGROUND: Low birth weight (LBW) is associated with increased future risk of insulin resistance and type 2 diabetes mellitus. The underlying molecular mechanisms remain poorly understood. We have previously shown that young LBW men have reduced skeletal muscle expression of PI3K p85alpha regulatory subunit and p110beta catalytic subunit, PKCzeta and GLUT4 in the fasting state. The aim of this study was to determine whether insulin activation of the PI3K/Akt and MAPK signalling pathways is altered in skeletal muscle of young adult men with LBW. METHODS: Vastus lateralis muscle biopsies were obtained from 20 healthy 19-yr old men with BW< or = 10th percentile for gestational age (LBW) and 20 normal birth weight controls (NBW), matched for physical fitness and whole-body glucose disposal, prior to (fasting state) and following a 4-hr hyperinsulinemic euglycemic clamp (insulin stimulated state). Expression and phosphorylation of selected proteins was determined by Western blotting. PRINCIPAL FINDINGS: Insulin stimulated expression of aPKCzeta (p

BACKGROUND: PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/-) T cells exhibit reduced activation and PKCtheta(-/-) mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIb)beta(3) in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIb)beta(3) activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/-) platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/-) platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIb)beta(3) activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced. CONCLUSIONS/SIGNIFICANCE: These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers.

Background: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. Methodology/Principal Findings: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the a2A-adrenoceptor (a2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14’304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of a2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. Conclusions/Significance: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.

Background Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg2+-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. Methodology and Principal Findings For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. Conclusion and Significance nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

A feedback loop maintains HER2 receptor signalling and cell survival in response to Herceptin treatment in HER2-positive breast cancers, but this Herceptin resistance may be bypassed by pan-HER inhibitors., Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)–positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2 phosphorylation during Herceptin treatment. The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors., Author Summary HER2 (ErbB2) is a surface protein and member of the epidermal growth factor receptor (EGFR) family that is overexpressed in approximately one-fifth of breast cancers. HER2-positive breast tumours tend to be very aggressive, and patients with this type of tumour have a poor prognosis. A therapeutic monoclonal antibody called trastuzumab (Herceptin) has been designed to block HER2 signalling and is used as a treatment for patients with HER2-positive breast cancer. However, recent studies have shown that Herceptin does not decrease HER2 activation. This may be why patients invariably develop resistance if treated with Herceptin monotherapy. To date, no study has explained why Herceptin cannot abolish HER2 signalling despite being an anti-HER2 monoclonal antibody. We have found that Herceptin switches on a feedback loop that increases the production of the ADAM17 protein, a protease that in turn releases the growth factors that activate HER (ErbB) receptors. These growth factors activate HER2 and also the other members of the HER receptor family—EGFR, HER3 and HER4—in such a way as to maintain HER2 activation and cell survival in HER2-positive breast cancer cells. We have found that when Herceptin is provided in combination with ADAM17 inhibitors, the feedback loop is abrogated in cells. Furthermore, a pan-HER inhibitor that decreases the activation of other HER receptors can also inhibit the feedback loop and decrease HER2 activation when used in combination with Herceptin. We further demonstrated that the combination therapy of Herceptin with a pan-HER inhibitor is more effective than Herceptin alone in an animal model of breast cancer. We believe our results offer treatment strategies that may help overcome acquired Herceptin resistance in patients with HER2-positive breast cancer.

BACKGROUND: The thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBalpha(-/-) neonates and an accumulation of early thymocyte subsets in PKBalpha(-/-) adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBalpha in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals. CONCLUSIONS/SIGNIFICANCE: This report highlights the specific requirements of PKBalpha for thymic development and opens up new prospects as to the mechanism downstream of PKBalpha in early thymocytes.

Background. Int-6 (integration site 6) was identified as an oncogene in a screen of tumorigenic mouse mammary tumor virus (MMTV) insertions. INT6 expression is altered in human cancers, but the precise role of disrupted INT6 in tumorigenesis remains unclear, and an animal model to study Int-6 physiological function has been lacking. Principal Findings. Here, we create an in vivo model of Int6 function in zebrafish, and through genetic and chemical-genetic approaches implicate Int6 as a tissue-specific modulator of MEK-ERK signaling. We find that Int6 is required for normal expression of MEK1 protein in human cells, and for Erk signaling in zebrafish embryos. Loss of either Int6 or Mek signaling causes defects in craniofacial development, and Int6 and Erk-signaling have overlapping domains of tissue expression. Significance. Our results provide new insight into the physiological role of vertebrate Int6, and have implications for the treatment of human tumors displaying altered INT6 expression.

Experimental work has shown that T cells of the immune system rapidly and specifically respond to antigenic molecules presented on the surface of antigen-presenting-cells and are able to discriminate between potential stimuli based on the kinetic parameters of the T cell receptor-antigen bond. These antigenic molecules are presented among thousands of chemically similar endogenous peptides, raising the question of how T cells can reliably make a decision to respond to certain antigens but not others within minutes of encountering an antigen presenting cell. In this theoretical study, we investigate the role of localized rebinding between a T cell receptor and an antigen. We show that by allowing the signaling state of individual receptors to persist during brief unbinding events, T cells are able to discriminate antigens based on both their unbinding and rebinding rates. We demonstrate that T cell receptor coreceptors, but not receptor clustering, are important in promoting localized rebinding, and show that requiring rebinding for productive signaling reduces signals from a high concentration of endogenous pMHC. In developing our main results, we use a relatively simple model based on kinetic proofreading. However, we additionally show that all our results are recapitulated when we use a detailed T cell receptor signaling model. We discuss our results in the context of existing models and recent experimental work and propose new experiments to test our findings., Author Summary T cells are essential players in the immune response to pathogens such as viruses and bacteria. They can be activated to respond when they recognize molecular signatures of infection (antigens) on the surface of antigen-presenting-cells of the immune system. The T cell response is highly specific (a particular T cell responds to only the right antigen), sensitive (a T cell will respond to as few as 1–10 antigens) and speedy (antigen binding may induce signaling within seconds). We wish to understand how the T cell, using its surface antigen receptors, is able perform this task. To do this, we developed mathematical models of antigens binding and unbinding from T cell surface receptors. Our primary finding is that T cells can discriminate antigens based on both their binding and unbinding rates from the T cell antigen receptor. We examine potential impacts of T cell antigen receptor clustering, T cell surface coreceptor molecules, and background self-antigens on this process.

Background: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression.Principal Findings: We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting.Conclusions: Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.

Analysis of the crystal structure of a phosphotransfer complex from the Rhodobacter sphaeroides chemotaxis pathway allowed reengineering of molecular recognition in a two-component signalling system., Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 Å crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA3, in complex with its cognate RR, CheY6. A methionine finger on CheY6 that nestles in a hydrophobic pocket in CheA3 was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA3, CheY6, and CheB2. Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA3-P to CheY6. Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA3-P. The structure presented here has allowed us to identify specificity determinants for the CheA–CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction., Author Summary The ability to respond to environmental stimuli is a universal feature of living cells. Evolution has created a vast array of signalling mechanisms that enable cells to react in many ways to extracellular changes. In bacteria, two-component signalling mechanisms, comprising a sensor protein kinase paired with its a cognate response regulator, are used widely to sense and respond to environmental changes. Some species of bacteria have over 150 different two-component pairs in a single cell, so the specificity between these pairs has to be tightly controlled to prevent “crossed wires” between signalling pathways. In this study, we have identified the determinants of this specificity in a two-component complex that controls the movement of Rhodobacter sphaeroides along a chemical gradient. By solving and analysing the crystal structure of this complex, we were able to pinpoint the amino acid residues that are crucially involved in formation of the complex. Knowledge of these crucial residues allowed us to convert noncognate response regulators into cognate response regulators simply by changing two amino acids. This reengineering of two-component signalling pathways paves the way for producing custom-designed circuits for applications in synthetic biology.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies., Author Summary Acquired immune deficiency syndrome (AIDS) remains a major health problem worldwide, affecting predominantly the adult population in the western world and in developing countries in particular. Despite a tremendous effort to develop a cure or a vaccine that confers protection against human immunodeficiency virus (HIV-1) infection, this has not been achieved in a satisfactory manner to date. Recent research efforts have suggested that the earliest immune responses activated after exposure to the virus have an influence on virus spread, containment and disease progression. In this study, a panel of donors who provided plasma samples collected over a time-frame spanning the period before and immediately after detection of HIV-1 infection permitted an insight into the activation of the earliest systemic immune responses. We describe increases in plasma levels of acute-phase reactants and proteolytically processed fragments that have anti-viral activity in vitro. These inductions occur prior to detection of HIV-1 virus in the blood and before the first increases in systemic cytokine levels, which may represent the earliest systemic host antiviral response activated following infection.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Wnt signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. Wnt can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans Wnt pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/12 branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and β-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the Wnt pathway to rearrangements of the cytoskeleton., This work was supported by an EU Training and Mobility of Researchers (TMR) Research Network grant (to RS), the Junta de Castilla y Leon (CSI03A08) (to JC), the Swiss National Science Foundation, and the Roche Research Fund. SJC was supported by a fellowship from the Canadian Institutes of Health Research and MOH was supported by the Ernst Hadorn Foundation.

In plants, auxin distribution and tissue patterning are coordinated via a feedback loop involving the auxin-regulated cell polarity factor ICR1 and the secretory machinery., Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning., Author Summary The coordination of different cells during pattern formation is a fundamental process in the development of multicellular organisms. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells demonstrates the importance of cell polarity for tissue patterning. The direction of auxin flow is determined by polar subcellular localization of auxin transport proteins called PINs, which facilitate auxin efflux. At the same time, an auxin-mediated positive feedback mechanism reinforces the polar distribution of PINs. However, the molecular mechanisms that underlie polar PIN localization are not well understood. In eukaryotic cells, the Rho family of small GTPases function as central regulators of cell polarity. We show that a Rho-interacting protein from plants, called ICR1, is required for recruitment via the secretory system of PIN proteins to polar domains in the cell membrane. As a result, ICR1 is required for directional auxin transport and distribution and thereby for proper pattern formation. In addition, both the expression and subcellular localization of ICR1 are regulated by auxin, suggesting that ICR1 could function in a positive feedback loop that reinforces auxin distribution. Thus, ICR1 forms an auxin-modulated link between cell polarity, protein secretion, and auxin-dependent tissue patterning.

Using genetic and molecular analyses, the authors identify Zyx as a positive regulator of Hippo signaling and characterize its role within the pathway., The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, we identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, we position the requirement for Zyx within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and we find that Zyx is required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. We also extend characterization of the Ajuba LIM protein Jub and determine that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. Our results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, we propose that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. Our identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling., Author Summary Processes that control cell numbers are essential during normal development, when they are required to generate organs of the correct size, and during cancinogenesis, when they influence tumor growth. The Hippo pathway is an intercellular signaling pathway that relays information about cell-cell contact and cell polarity to a signal transduction pathway that regulates the transcription of genes controlling cell numbers. The role of Hippo signaling in controlling growth is conserved from fruit flies to humans, but many aspects of the Hippo signal transduction pathway remain poorly understood. In this article, we identify Zyx as a previously unknown component of the Hippo pathway in Drosophila, and characterize its role within the pathway. We show that Zyx plays an essential role in a branch of Hippo signaling that involves the transmembrane receptor protein Fat and its target Dachs, which is a myosin family protein. Our results suggest a model in which Fat regulates the localization of Dachs, Dachs subsequently binds Zyx, stimulating its binding with the kinase Warts/Lats, and thereby regulates downstream signaling events. Zyx is conserved in vertebrates and we suggest that vertebrate Zyx proteins might also be involved in the regulation of Hippo signaling and, thereby, organ growth.

A simple feedback model of chemotaxis explains how new pseudopods are made and how eukaryotic cells steer toward chemical gradients., The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and—unexpectedly—lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants., Author Summary The efficiency, sensitivity, and huge dynamic range of eukaryotic cell chemotaxis have proven very hard to explain. Cells respond to shallow gradients of chemotactic molecules with directed movement, but the mechanisms remain elusive. Most current models predict that cells have an internal “compass” produced by processing the extracellular signal into an intracellular mechanism that points the cell towards the gradient and steers it in that direction. In this article, we present evidence that this internal compass does not exist; instead, the cell orients itself simply by making use of its pseudopods—the dynamic finger-like projections on the surface of the cell. We approached the question by making a computational model of the movement of a cell without a compass. In this model, the cell moves in a convincingly natural way simply by using its pseudopods, which respond to positive- and negative-feedback loops. The concentration of the chemoattractant molecule modulates the amount of positive feedback. Apart from this, no signal processing is necessary. This simple model reproduces many observations about normal chemotaxis. It also accurately predicts the angle at which new pseudopods split off from old ones, which had not been previously measured. The computational model thus demonstrates that pseudopod-based mechanisms are powerful enough to explain chemotaxis.

Lithium (Li(+)) is a common treatment for bipolar mood disorder, a major psychiatric illness with a lifetime prevalence of more than 1%. Risk of bipolar disorder is heavily influenced by genetic predisposition, but is a complex genetic trait and, to date, genetic studies have provided little insight into its molecular origins. An alternative approach is to investigate the genetics of Li(+) sensitivity. Using the social amoeba Dictyostelium, we previously identified prolyl oligopeptidase (PO) as a modulator of Li(+) sensitivity. In a link to the clinic, PO enzyme activity is altered in bipolar disorder patients. Further studies demonstrated that PO is a negative regulator of inositol(1,4,5)trisphosphate (IP(3)) synthesis, a Li(+) sensitive intracellular signal. However, it was unclear how PO could influence either Li(+) sensitivity or risk of bipolar disorder. Here we show that in both Dictyostelium and cultured human cells PO acts via Multiple Inositol Polyphosphate Phosphatase (Mipp1) to control gene expression. This reveals a novel, gene regulatory network that modulates inositol metabolism and Li(+) sensitivity. Among its targets is the inositol monophosphatase gene IMPA2, which has also been associated with risk of bipolar disorder in some family studies, and our observations offer a cellular signalling pathway in which PO activity and IMPA2 gene expression converge.

Kaposi's sarcoma (KS) lesions are complex mixtures of KS-associated herpesvirus (KSHV)-infected spindle and inflammatory cells. In order to survive the host immune responses, KSHV encodes a number of immunomodulatory proteins, including the E3 ubiquitin ligase K5. In exploring the role of this viral protein in monocytes, we made the surprising discovery that in addition to a potential role in down regulation of immune responses, K5 also contributes to increased proliferation and alters cellular metabolism. This ubiquitin ligase increases aerobic glycolysis and lactate production through modulation of cellular growth factor-binding receptor tyrosine kinase endocytosis, increasing the sensitivity of cells to autocrine and paracrine factors. This leads to an altered pattern of cellular phosphorylation, increases in Akt activation and a longer duration of Erk1/2 phosphorylation. Overall, we believe this to be the first report of a virally-encoded ubiquitin ligase potentially contributing to oncogenesis through alterations in growth factor signaling cascades and opens a new avenue of research in K5 biology., Author Summary Tumor viruses have proven to be valuable tools for dissecting the molecular mechanisms of transformation and cancer progression. Kaposi's sarcoma-associated herpesvirus (KSHV) infection is essential in driving at least three different neoplasias, including Kaposi's sarcoma (KS). Our understanding, however, of the molecular mechanism of KSHV-driven tumor progression is still limited and requires further examination. In this manuscript we demonstrate that the K5 E3 ubiquitin ligase of KSHV is able to alter monocyte metabolism, driving increased glucose consumption and lactate production, hallmarks of virtually every cancer. It is able to accomplish this through a modulation of selected receptor tyrosine kinases, whose normal role is to bind pro-growth factors. Indeed, this alteration in metabolism is coupled with increases in monocyte proliferation. Our study provides insights into the mechanisms of KSHV-driven oncogenesis, as well as a new tool for exploring the link between metabolism and cancer.

Retinoic acid (RA) is essential for the generation of GABAergic inhibitory neurons in the mouse forebrain, and RA treatment of embryonic stem cells induces the production of GABAergic neurons., Although retinoic acid (RA) has been implicated as an extrinsic signal regulating forebrain neurogenesis, the processes regulated by RA signaling remain unclear. Here, analysis of retinaldehyde dehydrogenase mutant mouse embryos lacking RA synthesis demonstrates that RA generated by Raldh3 in the subventricular zone of the basal ganglia is required for GABAergic differentiation, whereas RA generated by Raldh2 in the meninges is unnecessary for development of the adjacent cortex. Neurospheres generated from the lateral ganglionic eminence (LGE), where Raldh3 is highly expressed, produce endogenous RA, which is required for differentiation to GABAergic neurons. In Raldh3−/− embryos, LGE progenitors fail to differentiate into either GABAergic striatal projection neurons or GABAergic interneurons migrating to the olfactory bulb and cortex. We describe conditions for RA treatment of human embryonic stem cells that result in efficient differentiation to a heterogeneous population of GABAergic interneurons without the appearance of GABAergic striatal projection neurons, thus providing an in vitro method for generation of GABAergic interneurons for further study. Our observation that endogenous RA is required for generation of LGE-derived GABAergic neurons in the basal ganglia establishes a key role for RA signaling in development of the forebrain., Author Summary The vitamin A metabolite retinoic acid is an important signaling molecule needed for development of the central nervous system. Previous studies have shown a role for retinoic acid in regulating genes involved in the generation of motor neurons both in the hindbrain and spinal cord, but the role of retinoic acid in the forebrain has remained elusive. Here, we investigated mice that lack the ability to metabolize vitamin A into retinoic acid in the forebrain. Although no defects were observed in the generation of forebrain cortical neurons, we did observe a serious deficiency in GABAergic neurons, which provide inhibitory input to cortical neurons. Specifically, our results reveal that retinoic acid is required for forebrain neurons to activate an enzyme that converts glutamate to the inhibitory neurotransmitter GABA. We also find that retinoic acid treatment of human embryonic stem cells could stimulate production of GABAergic neurons. Deficiencies in GABAergic neurons have been associated with several neurological disorders, including Huntington's disease, autism, schizophrenia, and epilepsy. Knowledge of how GABAergic neurons are generated may aid efforts to treat these diseases.

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme., Author Summary Mosquitoes are vectors of human pathogens, such as Dengue virus and Malaria parasites, which profoundly affect health worldwide, killing millions of people annually. Recent studies have demonstrated that the presence of bacteria in the gut of mosquitoes is able to antagonize the establishment of pathogens. Therefore, mechanisms with the potential to regulate bacterial growth in the digestive tract of mosquitoes may hamper disease transmission. Here, we show that reactive oxygen species (ROS) are present in the gut epithelia of sugar-fed mosquitoes and are drastically reduced after blood feeding through a mechanism that involves activation of protein kinase C by heme. ROS levels are inversely correlated with the presence of bacteria in the midgut and therefore we investigated if ROS are involved in fighting bacterial infections in the gut. We discovered that mosquitoes producing low levels of ROS challenged with an oral bacterial infection exhibited increased mortality due to intense bacterial proliferation and epithelial cell damage. We propose that reduction in ROS levels after blood feeding is an adaptation to compensate for the ingestion of blood, a pro-oxidant meal. This finding has consequences for the understanding of disease transmission due to the ability of ROS to modulate the gut bacterial levels.

In frog and fish embryos, SNW1 is a protein required for the spatio-temporal activity of BMP signaling necessary for neural plate border formation and specification of neural crest tissue., Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification., Author Summary A subset of cells in the ectoderm of vertebrate embryos becomes the neural crest, which contributes to the bones and cartilage of the adult face. The neural crest arises in a location between the epidermis, which becomes the future skin, and the neural plate, which becomes the future central nervous system. Through our studies in both frog and fish embryos, we have discovered that the protein SNW-domain containing protein 1 (SNW1) is absolutely essential for defining the edge of the neural plate, where neural crest forms. SNW-domain containing proteins have been implicated in a variety of nuclear activities ranging from transcriptional regulation and elongation to RNA splicing. We show that SNW1 functions upstream of bone morphogenetic protein (BMP) receptors to regulate BMP activity, and is necessary for the activity of the BMP signaling pathway at the neural plate border where the neural crest is specified. In the absence of SNW1, BMP activity is reduced in this region and neural crest cells are lost. Given that SNW1 family proteins are highly conserved from nematodes to humans, SNW1's BMP regulatory function is likely conserved in other animals.

Background Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad. Methods and Results Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins - Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad. Conclusions In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST / nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST / nicotine associated head and neck cancer.

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses., Author Summary Dendritic cells (DCs) bear receptors specialized on recognition of patterns specific to pathogens (such as carbohydrates), which can either promote functional activation of these cells (such as TLRs), which renders them capable of efficiently presenting antigens to T cells, or, as DC-SIGN, endocytic uptake as essential for loading MHC molecules. Viruses such as HIV and measles virus (MV) exploit DC-SIGN for both their uptake into DCs and modulation of TLR signaling, yet how this is mechanistically exerted is poorly understood. We now show that DC-SIGN activates sphingomyelinases (SMases) which convert their sphingomyelin substrate into ceramides, thereby catalysing the formation of membrane platforms able to recruit and concentrate receptors and associated signaling components. We found DC-SIGN-dependent SMase activation as essential for DC-SIGN and thereby modulation of TLR signaling, but also for enhancement of MV uptake. This is mediated by a fast, transient recruitment of its entry receptor, CD150, from an intracellular storage compartment to the cell surface where it co-clusters in ceramide enriched platforms with DC-SIGN. The ability to segregate viral receptors into (or exclude them from) membrane microdomains, which, based on their biophysical properties, facilitate membrane fusion, proposes DC-SIGN-mediated SMAse activation as a central regulator of pathogen uptake into DCs.

Background Length and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface. Principal Findings In this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2. Conclusions/Significance As signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.

BACKGROUND: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. CONCLUSION/RELEVANCE: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Background The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. Methodology/Principal Findings Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3. Conclusions/Significance We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

Background Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis. Methodology/Principal Findings We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges. Conclusions/Significance Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.

Endomitosis is a form of mitosis in which both karyokinesis and cytokinesis are interrupted and is a hallmark of megakaryocyte differentiation. Very little is known about how such a dramatic alteration of the cell cycle in a physiological setting is achieved. Thrombopoietin-induced signaling is essential for induction of endomitosis. Here we show that calcium- and integrin-binding protein 1 (CIB1), a known regulator of platelet integrin α(IIb)β(3) outside-in signaling, regulates endomitosis. We observed that CIB1 expression is increased in primary mouse megakaryocytes compared to mononuclear bone marrow cells as determined by Western blot analysis. Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy. Overexpression of CIB1 in Dami cells resulted in multilobated nuclei and led to increased time for a cell to complete cytokinesis as well as increased incidence of furrow regression as observed by time-lapse microscopy. Additionally, we found that surface expression of integrin α(IIb)β(3,) an important megakaryocyte marker, was enhanced in CIB1 overexpressing cells as determined by flow cytometry. Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells. Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells. Furthermore, PMA treatment augmented the interaction between CIB1 and Plk3, which depended on the duration of treatment. These data suggest that CIB1 is involved in regulating endomitosis, perhaps through its interaction with Plk3.

Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes., Author Summary The rice blast fungus is a major pathogen of rice and a model for studying fungal-plant interactions. Like many other fungal pathogens, it can recognize physical and chemical signals present on the rice leaf surface and form a highly specialized infection structure known as appressorium. A well conserved signal transduction pathway involving the protein kinase gene PMK1 is known to regulate appressorium formation and plant penetration in this pathogen. However, it is not clear about the sensor genes that are involved in recognizing various plant surface signals. In this study we functionally characterize two putative sensor genes called MoMSB2 and MoSHO1. Genetic and biochemical analyses indicated that these two genes have overlapping functions in recognizing different physical and chemical signals present on the rice leaf surface for the activation of the Pmk1 pathway and appressorium formation. We found that primary alcohols, a major component of leaf waxes in grasses, can be recognized by the rice blast fungus as chemical cues. While MoMSB2 is critical for sensing hydrophobicity and precursors of cutin molecules of rice leaves, MoSHO1 appears to be more important than MoMSB2 for recognizing wax components.

BACKGROUND:Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. METHODOLOGY/PRINCIPAL FINDINGS:Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10% of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. CONCLUSIONS:This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials of endogenous redox modulation to enhance the cytotoxic effects of radiotherapy.

Background Akt/PKB is a promising anticancer therapeutic target, since abnormally elevated Akt activity is directly correlated to tumor development, progression, poor prognosis and resistance to cancer therapies. Currently, the unique role of each Akt isoform and their relevance to human breast cancer are poorly understood. Methodology/Principal Findings We previously found that Akt1, 2 and 3 are localized at specific subcellular compartments (the cytoplasm, mitochondria and nucleus, respectively), raising the possibility that each isoform may have unique functions and employ different regulation mechanisms. By systematically studying Akt-ablated MDA-MB231 breast cancer cells with isoform-specific siRNA, we here show that Akt2 is the most relevant isoform to cell proliferation and survival in our cancer model. Prolonged ablation of Akt2 with siRNA resulted in cell-cycle arrest in G0/G1 by downregulating Cdk2 and cyclin D, and upregulating p27. The analysis of the Akt downstream signaling pathways suggested that Akt2 specifically targets and activates the p70S6K signaling pathway. We also found that Akt2 ablation initially resulted in an increase in the mitochondrial volume concomitantly with the upregulation of PGC-1α, a regulator of mitochondrial biogenesis. Prolonged ablation of Akt2, but not Akt1 or Akt3, eventually led to cell death by autophagy of the mitochondria (i.e., mitophagy). Conclusions/Significance Collectively, our data demonstrates that Akt2 augments cell proliferation by facilitating cell cycle progression through the upregulation of the cell cycle engine, and protects a cell from pathological autophagy by modulating mitochondrial homeostasis. Our data, thus, raises the possibility that Akt2 can be an effective anticancer target for the control of (breast) cancer.

International audience; BACKGROUND: CDK11(p58) is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis. METHODOLOGY/PRINCIPAL FINDINGS: In addition to these previously described roles, our study shows that CDK11(p58) inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11(p58), which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression. CONCLUSION/SIGNIFICANCE: We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11(p58) could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.

The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway., Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors., Author Summary The highly conserved signaling pathway involving the transmembrane receptor Notch is essential for development, and misregulation of this pathway is linked to many diseases. We previously proposed that the Notch1 receptor is monoubiquitinated during its activation. With the aim of identifying a deubiquinating enzyme that could regulate Notch activation, we demonstrated that eIF3f, known previously as part of the multiprotein translation initiation factor eIF3 complex, harbors an enzymatic activity that acts on Notch. The activated form of Notch is able to interact with eIF3f only in the presence of the E3 ubiquitin ligase Deltex, and Notch needs to be deubiquitinated before it can be cleared and its intracellular domain can enter the nucleus and fulfill its transcriptional function. Our results further decipher the molecular mechanisms of Notch signaling activation, showing that ubiquitination and deubiquitination events are required. Additionally, we show that beyond acting as a translation initiation factor, eIF3f fulfills other functions and has an intrinsic enzymatic activity.

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells., National Institute of Environmental Health Sciences (ES002109), National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES015037), National Cancer Institute (U.S.) (CA026731), National Center for Research Resources (U.S.) (RR023783), Singapore-MIT Alliance for Research and Technology

Background Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes. Principal Findings To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates. Conclusions Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.

Background: Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters.\ud Methodology and Principal Findings: With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants.\ud Conclusion: These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in Drosophila.

Background S-nitrosation – the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins – is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. Methodology/Principal Findings The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. Conclusions We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.

Background Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis. Methodology and Principal Findings Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton. Conclusion and Significance Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules.

Background Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion. Methodology/Principal Findings To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation. Conclusions/Significance Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation.

Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.

Background: NFkB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFkB activation has not been previously described, although FGFs have been known to antagonize TNFa-induced apoptosis. Methodology/Principal Findings: Here, we demonstrate an interaction between FGFR4 and IKKb (Inhibitor of NFkB Kinase b subunit), an essential component in the NFkB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKb in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFa (Tumor Necrosis Factor a) to activate NFkB pathways, FGFR4 activation results in significant inhibition of NFkB signaling as measured by decreased nuclear NFkB localization, by reduced NFkB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKb kinase activity towards the substrate GST-IkBa in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFa-treated DU145 prostate cancer cells also leads to a decrease in IKKb activity, concomitant reduction in NFkB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFa treatment of DU145 cells, in comparison with TNFa alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFkB signaling. Conclusions/Significance: These results identify a compelling link between FGFR4 signaling and the NFkB pathway, and reveal that FGFR4 activation leads to a negative effect on NFkB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFkB signaling will not be limited to FGFR4 alone.

Background Hyaluronan (HA) is a glycosaminoglycan located in the interstitial space which is essential for both structural and cell regulatory functions in connective tissue. We have previously shown that HA synthesis is up-regulated in a rat model of experimental cardiac hypertrophy and that cardiac tissue utilizes two different HA synthases in the hypertrophic process. Cardiomyocytes and fibroblasts are two major cell types in heart tissue. The fibroblasts are known to produce HA, but it has been unclear if cardiomyocytes share the same feature, and whether or not the different HA synthases are activated in the different cell types. Methodology/Principal Findings This study shows, for the first time that cardiomyocytes can produce HA. Cardiomyocytes (HL-1) and fibroblasts (NIH 3T3) were cultivated in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2. HA concentration was quantified by ELISA, and the size of HA was estimated using dynamic light scattering. Cardiomyocytes synthesized HA but only when stimulated by PDGF-BB, whereas fibroblasts synthesized HA without addition of growth factors as well as when stimulated by any of the three growth factors. When fibroblasts were stimulated by the growth factors, reverse dose dependence was observed, where the highest dose induced the least amount of HA. With the exception of TGFB2, a trend of reverse dose dependence of HA size was also observed. Conclusions/Significance Co-cultivation of cardiomyocytes and fibroblasts (80%/20%) increased HA concentration far more that can be explained by HA synthesis by the two cell types separately, revealing a crosstalk between cardiomyocytes and fibroblasts that induces HA synthesis. We conclude that dynamic changes of the myocardium, such as in cardiac hypertrophy, do not depend on the cardiomyocyte alone, but are achieved when both cardiomyocytes and fibroblasts are present.

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk., National Science Foundation (U.S.) (CAREER Award), National Science Foundation (U.S.) (Graduate Fellowship)