ABSTRACT
OBJECTIVES: CD4+ T-cell decline and increasing virus levels are considered hallmarks of HIV/AIDS pathogenesis but we previously demonstrated in rhesus macaques that tissue macrophage destruction by simian immunodeficiency virus (SIV) infection associated with increased monocyte turnover also appear to impact pathogenesis. It remains unclear, however, which factors best predict onset of terminal disease progression and survival time. The objective of this study, therefore, was to directly compare these co-variates of infection for predicting survival times in retrospective studies of SIV/simian-HIV (SHIV)-infected adult rhesus macaques. METHODS: Rhesus macaques were infected with various strains of SIV/SHIV and evaluated longitudinally for monocyte turnover, CD4+ T-cell loss, plasma viral load, and SIV/SHIV strain. Correlation analyses and machine learning algorithm modeling were applied to compare relative contributions of each of the co-variates to survival time. RESULTS: All animals with AIDS-related clinical signs requiring euthanasia exhibited increased monocyte turnover regardless of CD4+ T-cell level, viral strain, or plasma viral load. Regression analyses and machine learning algorithms indicated a stronger correlation and contribution between increased monocyte turnover and reduced survival time than between CD4+ T-cell decline, plasma viral load, or virus strain and reduced survival time. Decision tree modeling categorized monocyte turnover of 13.2% as the initial significant threshold that best predicted decreased survival time. CONCLUSION: These results demonstrate that monocytes/macrophages significantly affect HIV/SIV pathogenesis outcomes. Monocyte turnover analyses are not currently feasible in humans, so there is a need to identify surrogate biomarkers reflecting tissue macrophage damage that predict HIV infection disease progression.
Subject(s)
HIV Infections , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Disease Progression , HIV Infections/complications , Humans , Macaca mulatta , Retrospective Studies , Viral LoadABSTRACT
Wilm's tumor 1 interacting protein (Wtip) was identified as an interacting partner of Wilm's tumor protein (WT1) in a yeast two-hybrid screen. WT1 is expressed in the proepicardial organ (PE) of the heart, and mouse and zebrafish wt1 knockout models appear to lack the PE. Wtip's role in the heart remains unexplored. In the present study, we demonstrate that wtip expression is identical in wt1a, tcf21, and tbx18positive PE cells, and that Wtip protein localizes to the basal body of PE cells. We present the first genetic evidence that Wtip signaling in conjunction with WT1 is essential for PE specification in the zebrafish heart. By overexpressing wtip mRNA, we observed ectopic expression of PE markers in the cardiac and pharyngeal arch regions. Furthermore, wtip knockdown embryos showed perturbed cardiac looping and lacked the atrioventricular (AV) boundary. However, the chamberspecific markers amhc and vmhc were unaffected. Interestingly, knockdown of wtip disrupts early leftright (LR) asymmetry. Our studies uncover new roles for Wtip regulating PE cell specification and early LR asymmetry, and suggest that the PE may exert nonautonomous effects on heart looping and AV morphogenesis. The presence of cilia in the PE, and localization of Wtip in the basal body of ciliated cells, raises the possibility of cilia-mediated PE signaling in the embryonic heart.
Subject(s)
Heart/embryology , Morphogenesis/genetics , Organogenesis/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Animals , Gene Expression , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Myocardium/metabolism , Phenotype , Protein Binding , Signal Transduction , WT1 Proteins/metabolism , Zebrafish Proteins/metabolismABSTRACT
Monocyte and dendritic cell (DC) development was evaluated using in vivo BrdU pulse-chase analyses in rhesus macaques, and phenotype analyses of these cells in blood also were assessed by immunostaining and flow cytometry for comparisons among rhesus, cynomolgus, and pigtail macaques, as well as African green monkeys and humans. The nonhuman primate species and humans have three subsets of monocytes, CD14(+)CD16(-), CD14(+)CD16(+), and CD14(-)CD16(+) cells, which correspond to classical, intermediate, and nonclassical monocytes, respectively. In addition, there exist presently two subsets of DC, BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC, that were first confirmed in rhesus macaque blood. Following BrdU inoculation, labeled cells first appeared in CD14(+)CD16(-) monocytes, then in CD14(+)CD16(+) cells, and finally in CD14(-)CD16(+) cells, thus defining different stages of monocyte maturation. A fraction of the classical CD14(+)CD16(-) monocytes gradually expressed CD16(+) to become CD16(+)CD14(+) cells and subsequently matured into the nonclassical CD14(-)CD16(+) cell subset. The differentiation kinetics of BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC were distinct from the monocyte subsets, indicating differences in their myeloid cell origins. Results from studies utilizing nonhuman primates provide valuable information about the turnover, kinetics, and maturation of the different subsets of monocytes and DC using approaches that cannot readily be performed in humans and support further analyses to continue examining the unique myeloid cell origins that may be applied to address disease pathogenesis mechanisms and intervention strategies in humans.
Subject(s)
Cell Differentiation , Dendritic Cells/cytology , Dendritic Cells/metabolism , Monocytes/cytology , Monocytes/metabolism , Animals , Antigens, CD/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , CD11c Antigen/metabolism , Humans , Immunophenotyping , Macaca , Myeloid Cells/cytology , Myeloid Cells/metabolism , PhenotypeABSTRACT
The slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and is crucial in the formation of the filtration barrier in the renal glomeruli. Zebrafish Nephrin and Podocin are important in the formation of the podocyte SD and mutations in NEPHRIN and PODOCIN genes cause human nephrotic syndrome. In the present study, the zebrafish Podocin protein was observed to be predominantly localized in the pronephric glomerular podocytes, as previously reported for Nephrin. To understand the function of Podocin and Nephrin in zebrafish, spliceblocking morpholino antisense oligonucleotides were used. Knockdown of Podocin or Nephrin by this method induced pronephric glomerular hypoplasia with pericardial edema. Human Nephrin and Podocin mRNA rescued this glomerular phenotype, however, the efficacy of the rescues was greatly reduced when mRNAencoding human diseasecausing NEPHRINR1109X and PODOCINR138Q were used. Furthermore, an association between zebrafish Nephrin and Podocin proteins was observed. Notably, PodocinR150Q, corresponding to human PODOCINR138Q, markedly interacted with Nephrin compared with wildtype Podocin, suggesting that this strong binding capacity of mutated Podocin impairs the transport of Nephrin and Podocin out of the endoplasmic reticulum. The results suggest that the functions of Nephrin and Podocin are highly conserved between the zebrafish pronephros and mammalian metanephros. Accordingly, the zebrafish pronephros may provide a useful tool for analyzing diseasecausing gene mutations in human kidney disorders.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Kidney Diseases/genetics , Membrane Proteins/metabolism , Pronephros/metabolism , Animals , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kidney Diseases/metabolism , Kidney Diseases/pathology , Kidney Glomerulus/metabolism , Mammals , Membrane Proteins/genetics , Mutation , Podocytes/metabolism , Pronephros/pathology , Protein Binding , ZebrafishABSTRACT
Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants.
Subject(s)
Membrane Proteins/analysis , Oryzias/metabolism , Podocytes/chemistry , Zebrafish/metabolism , Animals , Podocytes/cytologyABSTRACT
Glioblastoma is the most common form of primary adult brain tumors. A majority of glioblastomas grow invasively into distant brain tissue, leading to tumor recurrence, which is ultimately incurable. It is, therefore, essential to discover master regulators that control glioblastoma invasiveness and target them therapeutically. We show here that the transcriptional regulator Id-1 plays a critical role in modulating the invasiveness of glioblastoma cell lines and primary glioblastoma cells. Id-1 expression levels positively correlate with glioma cell invasiveness in culture and with histopathologic grades in patient biopsies. Id-1 knockdown dramatically reduces glioblastoma cell invasion that is accompanied by profound morphologic changes and robust reduction in expression levels of "mesenchymal" markers, as well as inhibition of self-renewal potential and downregulation of glioma stem cell markers. Importantly, genetic knockdown of Id-1 leads to a significant increase in survival in an orthotopic model of human glioblastoma. Furthermore, we show that a nontoxic compound, cannabidiol, significantly downregulates Id-1 gene expression and associated glioma cell invasiveness and self-renewal. In addition, cannabidiol significantly inhibits the invasion of glioblastoma cells through an organotypic brain slice and glioma progression in vivo. Our results suggest that Id-1 regulates multiple tumor-promoting pathways in glioblastoma and that drugs targeting Id-1 represent a novel and promising strategy for improving the therapy and outcome of patients with glioblastoma.
Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Inhibitor of Differentiation Protein 1/physiology , Neoplasm Invasiveness/genetics , Animals , Brain Neoplasms/pathology , Cannabidiol/pharmacology , Cell Line, Tumor , Female , Glioblastoma/pathology , Humans , Inhibitor of Differentiation Protein 1/antagonists & inhibitors , Inhibitor of Differentiation Protein 1/metabolism , Mice , Mice, Nude , Neurospora , RNA Interference , Transplantation, Heterologous , Up-RegulationABSTRACT
Defects in cilia and basal bodies function are linked to ciliopathies, which result in kidney cyst formation. Recently, cell division defects have been observed in cystic kidneys, but the underlying mechanisms of such defects remain unclear. Wtip is an LIM domain protein of the Ajuba/Zyxin family, but its role in ciliogenesis during embryonic development has not been previously described. We report Wtip is enriched in the basal body and knockdown of wtip leads to pronephric cyst formation, cloaca malformation, hydrocephalus, body curvature, and pericardial edema. We additionally show that wtip knockdown embryos display segment-specific defects in the pronephros: mitotic spindle orientation defects are observed only in the anterior and middle pronephros; cloaca malformation is accompanied by a reduced number of ciliated cells; and ciliated cells lack the striated rootlet that originates from basal bodies, which results in a lack of cilia motility. Our data suggest that loss of Wtip function phenocopies Vangl2 loss of function, a core planar cell polarity (PCP) protein located in the basal body protein. Furthermore, we demonstrate that wtip and vangl2 interact genetically. Taken together, our results indicate that in zebrafish, Wtip is required for mitotic spindle orientation in the anterior and middle of the pronephros, cloaca morphogenesis, and PCP, which may underlie the molecular etiology of ciliopathies.
ABSTRACT
BACKGROUND: The podocyte slit diaphragm (SD) is an essential component of the selective filtration barrier in the glomerulus. Several structural proteins required for formation and maintenance of SD have been identified; however, molecular mechanisms regulating these proteins are still limited. RESULTS: Here, we demonstrate that MAGUK p55 subfamily member 5a (Mpp5a)/Nagie oko, a component of the Crb multi-protein complex, was colocalized with an SD-associated protein ZO-1 in the zebrafish pronephric glomerulus. To characterize the function of Mpp5a, zebrafish mpp5a(m520) mutant embryos, which are known to have defects in cardiac and neuronal morphogenesis, were analyzed. These mutants failed to merge the bilateral glomerular primordia and to form the glomerular capillary and mesangium, but the foot processes and SD showed normal appearance. The structural disorganization in the mpp5a(m520) mutant glomerulus was quite similar to that of a cardiac troponin T2a/tnnt2a/silent heart knockdown zebrafish, which exhibited circulatory failure due to lack of heart beating. CONCLUSIONS: Mpp5a is not prerequisite to form podocyte slit diaphragm in the pronephric glomerular development in zebrafish. The structural disorganization of the pronephric glomerulus in the mpp5a(m520) mutant is likely to result from circulatory failure, rather than the anomaly of Mpp5a protein in the glomerulus.
Subject(s)
Glomerular Mesangium/embryology , Guanylate Cyclase/metabolism , Mutation , Podocytes/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Gene Knockdown Techniques , Guanylate Cyclase/genetics , Heart/embryology , Troponin T/genetics , Troponin T/metabolism , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.
Subject(s)
Fish Proteins/genetics , Kidney Glomerulus/anatomy & histology , Oryzias/anatomy & histology , Pronephros/anatomy & histology , Renin/genetics , Zebrafish/anatomy & histology , Animals , Cytoplasmic Granules/physiology , Cytoplasmic Granules/ultrastructure , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Kidney Glomerulus/embryology , Kidney Glomerulus/metabolism , Mice , Morphogenesis/physiology , Oryzias/embryology , Oryzias/genetics , Pronephros/embryology , Pronephros/metabolism , Rats , Species Specificity , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.
Subject(s)
DNA/chemistry , YY1 Transcription Factor/metabolism , Base Sequence , Binding Sites , Consensus Sequence , HeLa Cells , Humans , Molecular Dynamics Simulation , Plasminogen/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Protein BindingABSTRACT
Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16(INK4A) and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16(INK4A) and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16(INK4A), but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16(INK4A)-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16(INK4A) and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating PML stability.
Subject(s)
Cellular Senescence/physiology , Cyclin-Dependent Kinase Inhibitor p16/physiology , DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Retinoblastoma Protein/physiology , Transcription Factors/physiology , Tumor Suppressor Proteins/physiology , Cell Line, Tumor , Cells, Cultured , Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p16/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Down-Regulation , Fibroblasts , Gene Expression Regulation , Humans , Intranuclear Inclusion Bodies/metabolism , MAP Kinase Signaling System , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Protein Isoforms , Protein Stability , RNA Interference , Retinoblastoma Protein/antagonists & inhibitors , Retinoblastoma Protein/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Tumor Suppressor Protein p53/physiology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitination , ras Proteins/metabolismABSTRACT
E2FBP1/hDRIL1, a DNA-binding A/T-rich interaction domain (ARID) family transcription factor, is expressed ubiquitously in human tissues and plays an essential role in maintaining the proliferation potential of passage-limited human fibroblasts by dissociating promyelocytic leukemia nuclear bodies (PML-NBs). This effect on PML-NBs is similar to that of viral immediate-early gene products, such as infected cellular protein 0 (ICP0) from human herpes simplex virus 1 (HSV-1), which also disrupts PML-NBs to override the intrinsic cellular defense. Here we report that E2FBP1 inhibits accumulation of ICP0 RNA and, at the same time, is degraded via ICP0's herpes ubiquitin ligase 2 (HUL-2) activity upon HSV-1 infection. These reciprocal regulatory roles of ICP0 and E2FBP1 are linked in an ARID-dependent fashion. Our results suggest that E2FBP1 functions as an intrinsic cellular defense factor in spite of its PML-NB dissociation function.
Subject(s)
DNA-Binding Proteins/metabolism , Herpesvirus 1, Human/pathogenicity , Host-Pathogen Interactions , Immediate-Early Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Virulence Factors/metabolism , Cell Line , Humans , Repressor Proteins/geneticsABSTRACT
We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma (PPARG). Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming.
Subject(s)
Stem Cells/cytology , Stem Cells/radiation effects , Adipose Tissue/cytology , Animals , Cell Differentiation , Computer Simulation , Cytoplasm/metabolism , DNA/genetics , Gene Expression Regulation , Mesoderm/metabolism , Mice , Oligonucleotide Array Sequence Analysis , PPAR gamma/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Terahertz RadiationABSTRACT
We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding-DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely 'transcription factor-centric' view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation.
Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic , Computer Simulation , DNA/chemistry , HeLa Cells , Humans , MutationABSTRACT
Geldanamycin is a benzoquinone ansamycin antibiotic that manifests anti-cancer activity through the inhibition of HSP90-chaperone function. The HSP90 molecular chaperone is expressed at high levels in a wide variety of human cancers including melanoma, leukemia, and cancers in colon, prostate, lung, and breast. In cancer cells dependent upon mutated and/or over-expressed oncogene proteins, HSP90 is thought to have a critical role in regulating the stability, folding, and activity of HSP90-associated proteins, so-called "client proteins". These client proteins include the growth-stimulating proteins and kinases that support malignant transformation. Recently, oncogenic activating BRAF mutants have been identified in variety of cancers where constitutive activation of the MEK/ERK MAPK signaling pathway is the key for tumorigenesis, and they have been shown to be client proteins for HSP90. Accordingly, HSP90 inhibition can suppress certain cancer-causing client proteins and therefore represents an important therapeutic target. The molecular mechanism underlying the anti-cancer effect of HSP90 inhibition is complicated. Geldanamycin and its derivatives have been shown to induce the depletion of mutationally-activated BRAF through several mechanisms. In this review, we will describe the HSP90-inhibitory mechanism, focusing on recent progress in understanding HSP90 chaperone structure-function relationships, the identification of new HSP90 client proteins and the development of HSP90 inhibitors for clinical applications.
Subject(s)
Antineoplastic Agents/pharmacology , Benzoquinones/pharmacology , Lactams, Macrocyclic/pharmacology , Neoplasms/drug therapy , Signal Transduction/drug effects , Animals , HSP90 Heat-Shock Proteins/drug effects , HSP90 Heat-Shock Proteins/metabolism , HumansABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) is a key regulatory kinase in the proapoptotic response to various stresses. ASK1 phosphorylation of Daxx, an ASK1 activator protein, increases Daxx accumulation in cells and further enhances ASK1 activity through a positive feedback mechanism. Here, we show that ASK1-dependent phosphorylation of Daxx induces Lys(63) (K63)-linked polyubiquitination on Lys(122) of Daxx. Polyubiquitination is dispensable for Daxx accumulation or Daxx interaction with ASK1 because mutant Daxx deficient in polyubiquitin still exhibits ASK1-dependent accumulation and interaction with cellular ASK1. However, K63-linked Daxx polyubiquitination is required for tumor necrosis factor-alpha (TNF-alpha)-induced activation of ASK1. Therefore, K63-linked polyubiquitination of Daxx functions as a molecular switch to initiate and amplify the stress kinase response in the TNF-alpha signaling pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Nuclear Proteins/metabolism , Polyubiquitin/metabolism , Tumor Necrosis Factor-alpha/metabolism , Cell Line, Tumor , Co-Repressor Proteins , Enzyme Activation , HeLa Cells , Humans , Lysine/metabolism , MAP Kinase Kinase Kinase 5/biosynthesis , MAP Kinase Kinase Kinase 5/genetics , Molecular Chaperones , Phosphorylation , Transfection , UbiquitinationABSTRACT
Daxx is a regulatory protein for apoptosis signal-regulating kinase 1 (ASK1) which activates c-Jun NH2-terminal kinase (JNK) and p38 pathways in response to stressors such as tumor necrosis factor-alpha (TNFalpha). Here, we show that TNFalpha treatment induces the accumulation of Daxx protein through ASK1 activation by preventing its proteasome-dependent degradation. ASK1 directly phosphorylates Daxx at Ser(176) and Ser(184) and Daxx is required for the sustained activation of JNK. Tumorigenic mutant p53, which binds to Daxx and inhibits Daxx-dependent activation of ASK1, prevents Daxx phosphorylation and stabilization. When mutant p53 was depleted in cancer cells, Daxx was accumulated and the cell-killing effect of TNFalpha was restored. Our results indicate that Daxx not only activates ASK1 but also is a downstream target of ASK1 and that accumulated Daxx further activates ASK1. Thus, the Daxx-ASK1 positive feedback loop amplifying JNK/p38 signaling plays an important role in the cell-killing effects of stressors, such as TNFalpha. Tumorigenic mutant p53 disrupts this circuit and makes cells more tolerable to stresses, as its gain-of-function mechanism.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/physiology , Nuclear Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Cell Line, Tumor , Co-Repressor Proteins , Enzyme Activation , HT29 Cells , HeLa Cells , Humans , MAP Kinase Kinase 4/metabolism , MAP Kinase Signaling System/drug effects , Molecular Chaperones , Phosphorylation/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Tumor Suppressor Protein p53/geneticsABSTRACT
The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E).
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Benzoquinones/pharmacology , Lactams, Macrocyclic/pharmacology , Mutation , Oxidative Stress , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Acetylcysteine/pharmacology , Cell Line, Tumor , Enzyme Activation , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/geneticsABSTRACT
Interactions between pathogens and host induce human disorders including periodontitis, disintegration of the tooth supporting tissues. Tannerella forsythia has been linked to the periodontitis and several cytopathic reagents have been found in the bacterium; however, its contribution to the disease remains unclear. Biochemical approach to explore the cytopathic effect revealed two distinct activities in T. forsythia (ATCC 43037) extract; one detaches adherent cells from substratum and another arrests cells at G2. An executor of former activity, forsythia detaching factor (FDF) was identified; its genomic sequence and peptidase activity revealed that FDF is a substantial form of putative PrtH; prtH gene was hypothetically identified directly from a DNA fragment of the bacterium and its native product has never been shown. Since FDF was found in the bacterial culture supernatant, its activity implies a contribution to the disintegration of tissues although the mechanism how FDF disturbs cellular anchors remains elusive.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/pharmacology , Carcinoma/pathology , Cell Extracts/chemistry , Cell Extracts/pharmacology , Cell Survival/drug effects , Porphyromonas/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Cell Extracts/isolation & purification , Cell Line , Humans , Molecular Sequence DataABSTRACT
Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.