Showing papers in "Nature Cell Biology in 2008"
TL;DR: Tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test by incorporating an mRNA for a reporter protein into them, and it is demonstrated that messages delivered by microvesicle are translated by recipient cells.
Abstract: Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.
4,118 citations
TL;DR: It is found that all five members of the microRNA-200 family were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-β or to ectopic expression of the protein tyrosine phosphatase Pez, suggesting that downregulation of themicroRNAs may be an important step in tumour progression.
Abstract: Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumour metastasis. We found that all five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-β or to ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of the miR-200 family alone was sufficient to prevent TGF-β-induced EMT. Together, these microRNAs cooperatively regulate expression of the E-cadherin transcriptional repressors ZEB1 (also known as δEF1) and SIP1 (also known as ZEB2), factors previously implicated in EMT and tumour metastasis. Inhibition of the microRNAs was sufficient to induce EMT in a process requiring upregulation of ZEB1 and/or SIP1. Conversely, ectopic expression of these microRNAs in mesenchymal cells initiated mesenchymal to epithelial transition (MET). Consistent with their role in regulating EMT, expression of these microRNAs was found to be lost in invasive breast cancer cell lines with mesenchymal phenotype. Expression of the miR-200 family was also lost in regions of metaplastic breast cancer specimens lacking E-cadherin. These data suggest that downregulation of the microRNAs may be an important step in tumour progression. MicroRNAs are small, non-coding RNAs that modulate gene expression post-transcriptionally. In metazoa, they act predominantly to inhibit translation of their specific targets, but they also typically cause a modest reduction in the level of their target mRNAs 1,2 . Hundreds of microRNAs have been identified in vertebrates, with varying patterns of expression that range from ubiquitous to highly tissue- or developmental-stage-restricted. In some cases, an individual microRNA can act as a developmental switch by regulating a key target mRNA 3 . Speculating that switching between cell phenotypes that occurs during EMT may be specified to some extent by microRNAs, we searched for microRNAs whose expression changed during EMT. To this end, we used an in vitro model of EMT, which was generated by stable transfection of Madin Darby canine kidney (MDCK) epithelial cells with the protein tyrosine phosphatase Pez (PTP-Pez). Overexpression of PTP-Pez caused MDCK cells to undergo EMT, as indicated by loss of E-cadherin expression, gain in expression of the mesenchymal markers fibronectin, ZEB1 and SIP1, loss of cohesion, induction of cell motility and a change in cell morphology
3,640 citations
TL;DR: It is shown that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'), which can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.
Abstract: Aggressive human brain tumours (gliomas) often express a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII. Within each tumour only a small percentage of glioma cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. Here we show that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'). EGFRvIII expression in indolent glioma cells stimulates formation of lipid-raft related microvesicles containing EGFRvIII. Microvesicles containing this receptor are then released to cellular surroundings and blood of tumour-bearing mice, and can merge with the plasma membranes of cancer cells lacking EGFRvIII. This event leads to the transfer of oncogenic activity, including activation of transforming signalling pathways (MAPK and Akt), changes in expression of EGFRvIII-regulated genes (VEGF, Bcl-x(L), p27), morphological transformation and increase in anchorage-independent growth capacity. Thus, membrane microvesicles of cancer cells can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.
1,743 citations
TL;DR: Genetic studies in Drosophila show that Rag GTPases regulate cell growth, autophagy and animal viability during starvation, and establish a function of Rag G TPases in TORC1 activation in response to amino acid signals.
Abstract: TORC1 (target of rapamycin complex 1) has a crucial role in the regulation of cell growth and size. A wide range of signals, including amino acids, is known to activate TORC1. Here, we report the identification of Rag GTPases as activators of TORC1 in response to amino acid signals. Knockdown of Rag gene expression suppressed the stimulatory effect of amino acids on TORC1 in Drosophila melanogaster S2 cells. Expression of constitutively active (GTP-bound) Rag in mammalian cells activated TORC1 in the absence of amino acids, whereas expression of dominant-negative Rag blocked the stimulatory effects of amino acids on TORC1. Genetic studies in Drosophila also show that Rag GTPases regulate cell growth, autophagy and animal viability during starvation. Our studies establish a function of Rag GTPases in TORC1 activation in response to amino acid signals.
1,238 citations
TL;DR: Evidence is provided of a key signalling pathway involving HIF-1α and TWIST that promotes metastasis in response to intratumoural hypoxia that promotes epithelial–mesenchymal transition and metastastic phenotypes.
Abstract: Stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha) transcription complex, caused by intratumoural hypoxia, promotes tumour progression and metastasis, leading to treatment failure and mortality in different types of human cancers. The transcription factor TWIST is a master regulator of gastrulation and mesoderm-specification and was implicated recently as an essential mediator of cancer metastasis. Notably, HIF-1alpha- and TWIST-null mice show similarities in their phenotypes. Here, we have shown that hypoxia or overexpression of HIF-1alpha promotes epithelial-mesenchymal transition (EMT) and metastastic phenotypes. We also found that HIF-1 regulates the expression of TWIST by binding directly to the hypoxia-response element (HRE) in the TWIST proximal promoter. However, siRNA-mediated repression of TWIST in HIF-1alpha-overexpressing or hypoxic cells reversed EMT and metastastic phenotypes. Co-expression of HIF-1alpha, TWIST and Snail in primary tumours of patients with head and neck cancers correlated with metastasis and the worst prognosis. These results provide evidence of a key signalling pathway involving HIF-1alpha and TWIST that promotes metastasis in response to intratumoural hypoxia.
1,213 citations
French Institute of Health and Medical Research1, Institut Gustave Roussy2, University of Naples Federico II3, University of Paris-Sud4, University of Rome Tor Vergata5, Boston Children's Hospital6, Centre national de la recherche scientifique7, University of Gothenburg8, Foundation for Research & Technology – Hellas9, University of Ferrara10, Stony Brook University11, University of Graz12, University College London13
TL;DR: Evidence is provided of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53, which improved the survival of p 53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels.
Abstract: Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.
1,075 citations
TL;DR: It is found that humanmiR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miD-373 activity to migrate efficiently.
Abstract: MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes1,2 Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently3 and still remains largely unexplored4,5 To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44 We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs
971 citations
TL;DR: It is demonstrated that poly-SUMO chains can act as discrete signals from mono-SumOylation, in this case targeting a poly- SUMOylated substrate for ubiquitin-mediated proteolysis.
Abstract: In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.
806 citations
TL;DR: A model for adhesion assembly is developed that clarifies the relative contributions of myosin II and actin polymerization and organization and produces a model that shows that nascent adhesions assemble and are stable within the lamellipodium.
Abstract: Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an alpha-actinin-actin template that elongates centripetally from nascent adhesions. Alpha-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.
796 citations
TL;DR: It is demonstrated that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.
Abstract: miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.
776 citations
TL;DR: It is shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFβ-signalling (transforming growth factor β), constitutes a key factor that directs progenitor-cell sorting in germ-layer organization during gastrulation.
Abstract: 1. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/ TGFβ-signalling (transforming growth factor β), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation. 2 . Both cell adhesion and contraction have long been implicated in germ-layer formation; however, their relative contribution to these processes is still a matter of debate 3 . We therefore sought to quantify the specific adhesive and mechanical properties of the different progenitor types at the single-cell level and correlate these values to the actual sorting behaviour of progenitors in vitro and in vivo (for generation of different germ-layer progenitor
TL;DR: This study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression that is unique to ES cells.
Abstract: Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely and maintain pluripotency. These properties require transcription factors that specify the gene expression programme of ES cells. It has been possible to reverse the highly differentiated state of somatic cells back to a pluripotent state with a combination of four transcription factors: Klf4 is one of the reprogramming factors required, in conjunction with Oct4, Sox2 and c-Myc. Maintenance of self-renewal and pluripotency of ES cells requires Oct4, Sox2 and c-Myc, but Klf4 is dispensable. Here, we show that Kruppel-like factors are required for the self-renewal of ES cells. Simultaneous depletion of Klf2, Klf4 and Klf5 lead to ES cell differentiation. Chromatin immunoprecipitation coupled to microarray assay reveals that these Klf proteins share many common targets of Nanog, suggesting a close functional relationship between these factors. Expression analysis after triple RNA interference (RNAi) of the Klfs shows that they regulate key pluripotency genes, such as Nanog. Taken together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression that is unique to ES cells.
University of Nottingham1, Max Planck Society2, University of Tübingen3, University of Extremadura4, Institut national de la recherche agronomique5, Donald Danforth Plant Science Center6, Indiana University7, Ghent University8, Duke University9, Umeå University10, Bard College at Simon's Rock11, John Innes Centre12
TL;DR: It is described how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells in cortical and epidermal cells directly overlaying new primordia.
Abstract: Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia
TL;DR: It is reported that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery, and this interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of Autophagic cargo.
Abstract: Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome–endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG–Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy — autophagosome formation and maturation — but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.
TL;DR: It is reported that telomeric repeats are transcribed by DNA-dependent RNA polymerase II, which, in turn, interacts with the TRF1 shelterin protein, suggesting that TelRNAs may regulate telomerase activity at chromosome ends.
Abstract: Mammalian telomeres consist of non-coding TTAGGG repeats that are bound by the multi-protein complex 'shelterin', thus protecting chromosome ends from DNA repair mechanisms and degradation. Mammalian telomeric chromatin is enriched for the constitutive heterochromatin marks H3K9me3, H4K20me3 and HP1 (refs 2, 3, 4, 5, 6, 7). Similar to pericentric heterochromatin, telomeric heterochromatin is thought to be fundamental for the maintenance of chromosomal integrity. Here, we report that telomeric repeats are transcribed by DNA-dependent RNA polymerase II, which, in turn, interacts with the TRF1 shelterin protein. Telomeric RNAs (TelRNAs) contain UUAGGG repeats, are polyadenylated and are transcribed from the telomeric C-rich strand. Transcription of mammalian telomeres is regulated by several mechanisms, including developmental status, telomere length, cellular stress, tumour stage and chromatin structure. Using RNA-flourescent in situ hybridization (FISH), we show that TelRNAs are novel structural components of telomeric chromatin. Importantly, we provide evidence that TelRNAs block the activity of telomerase in vitro, suggesting that TelRNAs may regulate telomerase activity at chromosome ends. Our results indicate that TelRNAs are novel components of mammalian telomeres, which are anticipated to be fundamental for understanding telomere biology and telomere-related diseases, such as cancer and ageing.
TL;DR: It is proposed that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize, and an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours.
Abstract: Transmission of HIV-1 via intercellular connections has been estimated as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.
TL;DR: This model provides a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells, and suggests that once the H3K 27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, thereby preserving chromatin structure and transcriptional programs.
Abstract: Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.
TL;DR: It is shown that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae, and a link between ubiquitination and the regulated degradation of mature Ribosome-associated proteins by Autophagy is suggested.
Abstract: Eukaryotic cells use autophagy and the ubiquitin–proteasome system (UPS) as their major protein degradation pathways Whereas the UPS is required for the rapid degradation of proteins when fast adaptation is needed, autophagy pathways selectively remove protein aggregates and damaged or excess organelles1 However, little is known about the targets and mechanisms that provide specificity to this process Here we show that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae Surprisingly, this degradation not only occurs by a non-selective mechanism, but also involves a novel type of selective autophagy, which we term 'ribophagy' A genetic screen revealed that selective degradation of ribosomes requires catalytic activity of the Ubp3p/Bre5p ubiquitin protease Although ubp3Δ and bre5Δ cells strongly accumulate 60S ribosomal particles upon starvation, they are proficient in starvation sensing and in general trafficking and autophagy pathways Moreover, ubiquitination of several ribosomal subunits and/or ribosome-associated proteins was specifically enriched in ubp3Δ cells, suggesting that the regulation of ribophagy by ubiquitination may be direct Interestingly, ubp3Δ cells are sensitive to rapamycin and nutrient starvation, implying that selective degradation of ribosomes is functionally important in vivo Taken together, our results suggest a link between ubiquitination and the regulated degradation of mature ribosomes by autophagy
TL;DR: PML is identified as the first protein degraded by SUMO-dependent polyubiquitination, as it recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, which could physically integrate thesumOylation, ubiquItination and degradation pathways.
Abstract: In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.
TL;DR: This study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS–ERK and MDM2 and downregulates Forkhead box O 3a by directly interacting with and phosphorylating FOXO 3a at Ser 294, Ser 344 and Ser 425.
Abstract: The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that Erk downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the non-phosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.
TL;DR: It is demonstrated that in response to TGFβ stimulation the transcriptional regulator TAZ binds heteromeric Smad2/3–4 complexes and is recruited to T GFβ response elements and defines a hierarchical system regulating Smad nuclear accumulation and coupling to the transcriptionAL machinery.
Abstract: TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal
TL;DR: It is shown that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S 100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion.
Abstract: A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.
TL;DR: It is shown that anisotropy of cortical tension at apical cell junctions is sufficient to drive tissue elongation in Drosophila melanogaster embryos, and the contribution of subcellular tensile activity polarizing junction remodelling and the permissive role of vertex fluctuations during tissues elongation is delineated.
Abstract: The morphogenesis of developing embryos and organs relies on the ability of cells to remodel their contacts with neighbouring cells. Using quantitative modelling and laser nano-dissection, we probed the mechanics of a morphogenetic process, the elongation of Drosophila melanogaster embryos, which results from polarized cell neighbour exchanges. We show that anisotropy of cortical tension at apical cell junctions is sufficient to drive tissue elongation. We estimated its value through comparisons between in silico and in vivo data using various tissue descriptors. Nano-dissection of the actomyosin network indicates that tension is anisotropically distributed and depends on myosin II accumulation. Junction relaxation after nano-dissection also suggests that cortical elastic forces are dominant in this process. Interestingly, fluctuations in vertex position (points where three or more cells meet) facilitate neighbour exchanges. We delineate the contribution of subcellular tensile activity polarizing junction remodelling, and the permissive role of vertex fluctuations during tissue elongation.
TL;DR: It is shown that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5, and this effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4–β-catenin transcriptional repressor complex.
Abstract: Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4–β-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of β-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.
TL;DR: The results suggest that a positive-feedback loop exists, whereby glycolysis drives IKK–NF-κB activation, and that hyperactivation of this loop by loss of p53 is important in oncogene-induced cell transformation.
Abstract: Cancer cells use aerobic glycolysis preferentially for energy provision and this metabolic change is important for tumour growth. Here, we have found a link between the tumour suppressor p53, the transcription factor NF-kappaB and glycolysis. In p53-deficient primary cultured cells, kinase activities of IKKalpha and IKKbeta and subsequent NF-kappaB activity were enhanced. Activation of NF-kappaB, by loss of p53, caused an increase in the rate of aerobic glycolysis and upregulation of Glut3. Oncogenic Ras-induced cell transformation and acceleration of aerobic glycolysis in p53-deficient cells were suppressed in the absence of p65/NF-kappaB expression, and were restored by GLUT3 expression. It was also shown that a glycolytic inhibitor diminished the enhanced IKK activity in p53-deficient cells. Moreover, in Ras-expressing p53-deficient cells, IKK activity was suppressed by p65 deficiency and restored by GLUT3 expression. Taken together, these data indicate that p53 restricts activation of the IKK-NF-kappaB pathway through suppression of glycolysis. These results suggest that a positive-feedback loop exists, whereby glycolysis drives IKK-NF-kappaB activation, and that hyperactivation of this loop by loss of p53 is important in oncogene-induced cell transformation.
TL;DR: It is reported that the ubiquitin ligase (E3) TRAF6 interacts with a consensus motif present in TβRI, which is required for TGF-β-induced autoubiquitylation of TRAF 6 and subsequent activation of the TAK1–p38/JNK pathway, which leads to apoptosis.
Abstract: Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates embryonic development and tissue homeostasis; however, aberrations of its activity occur in cancer. TGF-beta signals through its Type II and Type I receptors (TbetaRII and TbetaRI) causing phosphorylation of Smad proteins. TGF-beta-associated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, was originally identified as an effector of TGF-beta-induced p38 activation. However, the molecular mechanisms for its activation are unknown. Here we report that the ubiquitin ligase (E3) TRAF6 interacts with a consensus motif present in TbetaRI. The TbetaRI-TRAF6 interaction is required for TGF-beta-induced autoubiquitylation of TRAF6 and subsequent activation of the TAK1-p38/JNK pathway, which leads to apoptosis. TbetaRI kinase activity is required for activation of the canonical Smad pathway, whereas E3 activity of TRAF6 regulates the activation of TAK1 in a receptor kinase-independent manner. Intriguingly, TGF-beta-induced TRAF6-mediated Lys 63-linked polyubiquitylation of TAK1 Lys 34 correlates with TAK1 activation. Our data show that TGF-beta specifically activates TAK1 through interaction of TbetaRI with TRAF6, whereas activation of Smad2 is not dependent on TRAF6.
TL;DR: It is shown that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells, and that unciliated cells activate transcription only in response to Wnt stimulation, but do so much more robustly than ciliated cells.
Abstract: Primary cilia are microtubule-based organelles involved in signal transduction and project from the surface of most vertebrate cells. Proteins that can localize to the cilium, for example, Inversin and Bardet-Biedl syndrome (BBS) proteins, are implicated in both beta-catenin-dependent and -independent Wnt signalling. Given that Inversin and BBS proteins are found both at the cilium and elsewhere in the cell, the role of the cilium itself in Wnt signalling is not clear. Using three separate mutations that disrupt ciliogenesis (affecting Kif3a, Ift88 and Ofd1), we show in this study that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells. Interestingly, unciliated cells activate transcription only in response to Wnt stimulation, but do so much more robustly than ciliated cells. Loss of Kif3a, but not other ciliogenic genes, causes constitutive phosphorylation of Dishevelled (Dvl). Blocking the activity of casein kinase I (CKI) reverses this constitutive Dvl phosphorylation and abrogates pathway hyper-responsiveness. These results suggest that Kif3a restrains canonical Wnt signalling both by restricting the CKI-dependent phosphorylation of Dvl and through a separate ciliary mechanism. More generally, these findings reveal that, in contrast to its role in promoting Hedgehog (Hh) signalling, the cilium restrains canonical Wnt signalling.
TL;DR: It is reported that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses.
Abstract: Stress-induced MAPK-dependent apoptosis is inhibited by the formation of stress granules, which sequester and inactivate the MTK1 activator RACK1. When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins1. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1–SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.
TL;DR: Evidence that progerin interferes with the function of human mesenchymal stem cells (hMSCs) is provided and it is found that expression of proger in hMSCs activates major downstream effectors of the Notch signalling pathway.
Abstract: The premature-ageing disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A1,2. Progerin is also expressed sporadically in wild-type cells and has been linked to physiological ageing3. Cells from HGPS patients exhibit extensive nuclear defects, including abnormal chromatin structure4,5 and increased DNA damage6. At the organismal level, HGPS affects several tissues, particularly those of mesenchymal origin7. How the cellular defects of HGPS cells lead to the organismal defects has been unclear. Here, we provide evidence that progerin interferes with the function of human mesenchymal stem cells (hMSCs). We find that expression of progerin activates major downstream effectors of the Notch signalling pathway. Induction of progerin in hMSCs changes their molecular identity and differentiation potential. Our results support a model in which accelerated ageing in HGPS patients, and possibly also physiological ageing, is the result of adult stem cell dysfunction and progressive deterioration of tissue functions.
TL;DR: Characterization of endogenous Nanog and Oct4 protein complexes in mouse ES cells found that these transcription factors interact with each other and associate with proteins from multiple repression complexes, including the NuRD, Sin3A and Pml complexes.
Abstract: Nanog and Oct4 are essential transcription factors that regulate self-renewal and pluripotency of ES cells. However, the mechanisms by which Nanog and Oct4 modulate ES cell fate remain unknown. Through characterization of endogenous Nanog and Oct4 protein complexes in mouse ES cells, we found that these transcription factors interact with each other and associate with proteins from multiple repression complexes, including the NuRD, Sin3A and Pml complexes. In addition, Nanog, Oct4 and repressor proteins co-occupy Nanog-target genes in mouse ES cells, suggesting that Nanog and Oct4 together may communicate with distinct repression complexes to control gene transcription. To our surprise, of the various core components in the NuRD complex with which Nanog and Oct4 interact, Mta1 was preferred, whereas Mbd3 and Rbbp7 were either absent or present at sub-stoichiometric levels. We named this unique Hdac1/2- and Mta1/2-containing complex NODE (for Nanog and Oct4 associated deacetylase). Interestingly, NODE contained histone deacetylase (HDAC) activity that seemed to be comparable to NuRD, and retained its association with Nanog and Oct4 in Mbd3(-/-) ES cells. In contrast to Mbd3 loss-of-function, knockdown of NODE subunits led to increased expression of developmentally regulated genes and ES-cell differentiation. Our data collectively suggest that Nanog and Oct4 associate with unique repressor complexes on their target genes to control ES cell fate.