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  • DKFZ Publication Database  (44)
  • C-JUN
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  • DKFZ Publication Database  (44)
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  • 1
    Keywords: TRANSPLANTATION ; C-JUN ; cell-cell interaction ; cell proliferation ; GM-CSF ; organotypic culture ; cell differentiation ; dermal fibroblasts ; epidermal keratinocytes ; epithelial cell culture ; dermal equivalent ; paracrine factors ; collagen matrix ; IL-1 ; KGF ; JunB
    Abstract: This chapter focuses on the role of dermal fibroblasts in epidermal keratinocyte proliferation and differentiation and proves the usefulness of an organotypic in vitro skin equivalent model to study mechanisms of tissue regeneration. Examples are given how these can be used to study the growth of keratinocytes after transplantation in vivo, alone or with a collagen matrix. This leads on to the development of in vitro organotypic models to generate skin equivalents composed of an organized epidermis on matrix-embedded dermal fibroblasts. Protocols are provided for both the in vivo transplantation models and the combined stromal/epithelial in vitro organotypic models, along with several variations of the methods. Characterization of the proliferation and differentiation of the cultures is described with an extensive discussion of recent results on the factors controlling tissue regeneration, with emphasis on the role of c-jun and junB gene products in the production of KGF and GM-CSF by fibroblasts in response to epithelium-derived IL1, and the effect of these cytokines on the proliferation and differentiation of epidermal keratinocytes.
    Type of Publication: Book chapter
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  • 2
    Keywords: RECEPTOR ; CANCER CELLS ; CELLS ; EXPRESSION ; GROWTH ; tumor ; carcinoma ; CELL ; Germany ; human ; IN-VIVO ; KINASE ; MODEL ; PATHWAY ; CDNA ; CLONES ; GENE ; PROTEIN ; transcription ; ACTIVATION ; LIGAND ; MECHANISM ; FAMILY ; TRANSCRIPTION FACTOR ; AP-1 ; BINDING ; BIOLOGY ; C-JUN ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; ACID ; GROWTH-INHIBITION ; CARCINOMA-CELLS ; BETA ; Jun ; DEGRADATION ; HeLa cells ; DIMERIZATION ; AFFINITY ; MEDIATED REPRESSION ; N-TERMINAL KINASE ; CONSTITUTIVE EXPRESSION ; CROSS-TALK ; FACTOR-ALPHA ; F ; molecular biology ; ACTIVATED PROTEIN-KINASES ; ALL-TRANS ; cervical carcinoma cells ; HELA-CELLS ; PROTEASOME INHIBITORS ; X-RECEPTOR
    Abstract: Expression of the nuclear retinoic acid receptor beta2 (RARbeta2) gene is often disturbed in cervical carcinoma cells. One important mechanism by which RARbeta2 can exert growth inhibitory function is based on its ability to repress the AP-1 transcription factor in a ligand-dependent manner. Because less is known about the biological effects of RARbeta in the absence of ligand, the corresponding cDNA was stably introduced into HPV18-positive HeLa cervical carcinoma cells. In the present study we describe a novel mechanism by which AP-1 becomes inactivated. Constitutive expression of nonliganded RARbeta abrogated both AP-1 binding affinity and activity by a selective degradation of the c-Jun protein as major dimerization partner, without substitution by other members of the Jun family. Blockage of the proteasomal pathway completely rescued c-Jun and reconstituted the AP-1 function. Moreover, HeLa RARbeta2 clones treated either with tumor necrosis factor-alpha or transfected with a constitutive active upstream mitogen-activated protein kinase (MEKK1Delta) also resulted in c-Jun phosphorylation and restoration of AP-1 affinity and functionality similar to that found in nontransfected parental HeLa cells. These data revealed an important cross-talk between trans-repression of AP-1 and nonliganded RARbeta in human papillomavirus-positive cells. Because AP-1 activity was not irreversibly disturbed, but could be switched on through activation of the Jun N-terminal kinase pathway, a model for the transient activation of AP-1 even in the presence of RARbeta as repressor is suggested
    Type of Publication: Journal article published
    PubMed ID: 15308638
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  • 3
    Keywords: CELLS ; EXPRESSION ; GROWTH-FACTOR ; proliferation ; CELL ; CELL-PROLIFERATION ; DISEASE ; GENE ; GENES ; PROTEIN ; MICE ; COMPLEX ; RESPONSES ; COMPLEXES ; AP-1 ; T-CELLS ; C-JUN ; NUCLEAR FACTOR ; REGULATOR ; INITIATION ; CYTOKINE ; BINDING-ACTIVITY ; CHONDROITIN SULFATE-E ; E-PROTEOGLYCAN ; IL-6 PROMOTER
    Abstract: The AP-1 complex is composed of c-Jun and c-Fos and is a key component in the regulation of proinflammatory genes. Mast cells play a significant role in the initiation of many inflammatory responses, such as allergy and allergy-associated diseases. In the present work, we characterized the role of c-Fos in mast cell function by investigating IL-3-dependent cell proliferation, degranulation capability, and cytokine expression in c-Fos-deficient mice. In c-Fos-deficient mast cells, we found that FcepsilonRI-mediated degranulation was significantly inhibited, which correlates with the reduced expression of SWAP-70, VAMP-7, and Synaptotagmin I genes, which are involved directly in the degranulation process. These findings show that c-Fos plays an important role in FcepsilonRI-mediated regulation of mast cell function
    Type of Publication: Journal article published
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  • 4
    Keywords: RECEPTOR ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; proliferation ; Germany ; IN-VIVO ; NETWORKS ; SYSTEM ; GENE ; GENE-EXPRESSION ; HYBRIDIZATION ; transcription ; DIFFERENTIATION ; TISSUE ; COMPLEX ; COMPLEXES ; TRANSCRIPTION FACTOR ; AP-1 ; KERATINOCYTES ; SKIN ; C-JUN ; fibroblasts ; cytokines ; antibodies ; antibody ; MOUSE ; IN-SITU ; gene expression ; REPAIR ; SIGNALING PATHWAY ; HUMAN KERATINOCYTES ; Jun ; gene expression profiling ; expression profiling ; COLONY-STIMULATING FACTOR ; FACTOR GENE-EXPRESSION ; FUNCTIONAL EXPRESSION ; in situ hybridization ; keratinocyte ; regulation ; mesenchyme ; SDF-1 ; CHEMOKINE RECEPTORS ; INFLAMMATORY CYTOKINES ; CELL-DERIVED FACTOR ; CXCL12 ; GROWTH-ASSOCIATED MOLECULE ; HB-GAM ; LARGE INDUCTION ; pleiotrophin
    Abstract: In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and differentiation. The AP-1 transcription factor subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GMCSF). We have performed gene expression profiling of wildtype and Jun(-/-) mouse embryonic fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived factor 1 (SDF-1) as novel JUN-regulated factors. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-1 exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-1-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-1 or its receptor, CXCR4. Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-1 production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration
    Type of Publication: Journal article published
    PubMed ID: 15840658
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  • 5
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; GROWTH ; INHIBITOR ; proliferation ; tumor ; Germany ; KINASE ; CDNA ; PROTEIN ; PROTEINS ; ADHESION MOLECULES ; NF-KAPPA-B ; ACTIVATION ; COMPLEX ; RESPONSES ; COMPLEXES ; MECHANISM ; ANTIGEN ; T-CELL ; T-CELLS ; C-JUN ; PHOSPHORYLATION ; signal transduction ; ALPHA ; IMMUNE-RESPONSES ; MOUSE ; LINE ; MONOCLONAL-ANTIBODIES ; RHO-ASSOCIATED KINASE ; KAPPA-B ; CROSS-LINKING ; MAP KINASES ; FAS-MEDIATED APOPTOSIS ; rodent ; T lymphocytes ; INDUCED CELL-DEATH ; STANDARD ; ABSENCE ; secretion ; HYALURONAN RECEPTOR ; DELAYED-TYPE HYPERSENSITIVITY ; JNK ; KINASES ; TRANSMEMBRANE DOMAIN ; PHOSPHOINOSITIDE 3-KINASE/AKT
    Abstract: CD44v6 is transiently expressed during T cell activation, and constitutively CD44v4-v7 expressing transgenic T cells show accelerated responses towards nominal antigens. The underlying mechanism is unknown. The mouse thymoma EL4 was transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA (EL4-s, EL4-v6). Only EL4-v6 cells proliferated at an over 10-fold higher rate than untransfected cells, displayed up-regulated expression of CD69, CD25, and IL-2, and were protected from apoptosis by CD44v6 cross-linking. In the absence of any stimulus, ERK1/2 was partly phosphorylated, and phosphorylation was significantly increased by CD44v6 cross-linking. The same accounted for JNK, c-jun, and I kappa B alpha. Moreover, NF-kappa B was partly translocated into the nucleus. Instead, CD44s cross-linking induced ERK1/2, JNK, c-jun, and I kappa B alpha phosphorylation only in the context of TCR engagement. No selectively CD44v6 associated transmembrane proteins were uncovered in EL4 cells. However, CD44v6, as opposed to CD44s, did not colocalise with the TCR/CD3 complex after CD3 cross-linking. Furthermore, a CD44-associated 85-kDa protein became hypophosphorylated only after CD44v6 cross-linking. Threonine hypophosphorylation of this protein coincided with the activation of MAP and SAP kinases, which was prohibited in the presence of a phosphatase inhibitor. Thus, CD44v6, distinct to CD44s, stimulates autonomously growth and IL-2 secretion of a thymoma line and rescues cells from apoptosis. (c) 2004 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15894169
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  • 6
    Keywords: RECEPTOR ; CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; INHIBITOR ; INVASION ; SURVIVAL ; tumor ; CELL ; ENDOTHELIAL GROWTH-FACTOR ; Germany ; IN-VIVO ; MODEL ; PATHWAY ; VIVO ; GENE ; GENE-EXPRESSION ; RNA ; TISSUE ; TUMORS ; PATIENT ; COMPLEX ; COMPLEXES ; AP-1 ; colon ; TISSUES ; BIOLOGY ; C-JUN ; STAGE ; gene expression ; PROMOTER ; MEMBRANE ; METASTASIS ; colorectal cancer ; COLORECTAL-CANCER ; REGION ; CANCER-CELLS ; UROKINASE RECEPTOR ; CANCER-PATIENTS ; CANCER PATIENTS ; REGULATOR ; PLASMINOGEN-ACTIVATOR ; FACTOR-BETA ; INHIBITORS ; ONCOLOGY ; RE ; INCREASE ; PATIENT SURVIVAL ; SOLID TUMORS ; USA ; correlation ; cancer research ; in vivo ; Mol Oncol ; KINASE INHIBITOR ; RECEPTOR GENE ; colorectal ; PROMOTES ; PROGNOSTIC RELEVANCE ; FUNCTIONAL-ANALYSIS ; CELLULAR INVASION ; PLASMINOGEN ACTIVATION ; PROTEIN-2-ALPHA-RELATED FACTOR ; UROKINASE RECEPTOR GENE
    Abstract: The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator proteln-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH2-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -1901-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser 73/63, and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential
    Type of Publication: Journal article published
    PubMed ID: 17510314
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  • 7
    Keywords: RECEPTOR ; CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; CELL ; IN-VIVO ; SITE ; SITES ; GENE ; GENE-EXPRESSION ; PROTEIN ; transcription ; MARKER ; colon ; BINDING ; BIOLOGY ; C-JUN ; PROTEIN-KINASE ; DELETION ; IDENTIFICATION ; PROGRESSION ; gene expression ; ASSAY ; PROMOTER ; ELEMENTS ; genetics ; COLORECTAL-CANCER ; REGION ; CANCER-CELLS ; REGIONS ; ONCOGENE ; UROKINASE RECEPTOR ; CELL-MIGRATION ; heredity ; POSTTRANSCRIPTIONAL REGULATION ; PLASMINOGEN-ACTIVATOR ; ONCOLOGY ; ENHANCER ; ASSAYS ; BINDING-SITE ; USA ; CANCERS ; PLASMINOGEN-ACTIVATOR RECEPTOR ; INVASIVE COLON-CANCER ; IV COLLAGENASE EXPRESSION ; TRANSCRIPTION FACTOR-BINDING
    Abstract: The transcriptionally regulated urokinase-type plasminogen activator receptor (u-PAR) contributes to cancer progression. Although previous studies have identified multiple 50 regulatory elements, these cis motifs cannot fully account for u-PAR expression prompting a search for hitherto uncharacterized regulatory elements. DNase I hypersensitivity and chromatin immunoprecipitation assays using u-PAR-expressing colon cancer cells indicated a hypersensitive region (+665/+2068) in intron 1 enriched with acetylated histone 3 (H3) and H3 methylated at lysine 4, markers of regulatory regions. The +665/+2068 region increased transcription from a u-PAR-promoter in an orientation- and distance-independent manner fulfilling the criteria of an enhancer. Optimal stimulation of the u-PAR promoter by phorbol ester required this enhancer. Systematic truncations combined with DNase I footprinting revealed two protected regions (+1060/+1099 and +1123/+1134) with deletion of the latter practically abolishing enhancer activity. The +1123/+1134 region harbored non-consensus activator protein-1 and Ets1 binding sites bound with c-Jun (and/or the related JunD/JunB) and c-Fos (and/or the related FosB/Fra-1/Fra-2) as revealed with chromatin immunoprecipitation. Further, nuclear extract from resected colon cancers showed elevated protein binding to a +1123/_1134- spanning probe coordinate with elevated u-PAR protein. Thus, we have defined a novel intragenic enhancer in the u-PAR gene required for constitutive and inducible expression
    Type of Publication: Journal article published
    PubMed ID: 17001307
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  • 8
    Keywords: IN-VIVO ; C-JUN ; PROTEIN-KINASE ; BREAST-CANCER ; CARCINOMA-CELLS ; ACTIVATING TRANSCRIPTION FACTOR-2 ; MELANOMA-CELLS ; ESOPHAGEAL ADENOCARCINOMA ; SUPEROXIDE-DISMUTASE EXPRESSION ; CYCLIN-A PROMOTER
    Abstract: Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21(WAF1) , but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21(WAF1) protein expression, suggesting p21(WAF1) transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21(WAF1) promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21(WAF1) downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies.
    Type of Publication: Journal article published
    PubMed ID: 23800081
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  • 9
    Keywords: CELLS ; GROWTH-FACTOR ; proliferation ; GENE-EXPRESSION ; SKIN ; C-JUN ; MAP KINASES ; MORPHOGENESIS ; SIGNAL-TRANSDUCTION PATHWAY ; EPIDERMAL-KERATINOCYTES
    Abstract: Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors have a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases (JNKs), JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes, we used a heterologous coculture model combining primary human keratinocytes and murine fibroblasts. Although cocultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors, but is also critically dependent on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the coculture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK has a pivotal role in the paracrine cross talk between dermal fibroblasts and epidermal keratinocytes during wound healing.
    Type of Publication: Journal article published
    PubMed ID: 24335928
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  • 10
    Keywords: IN-VITRO ; C-JUN ; CREB ; NERVOUS-SYSTEM ; HEAT-SHOCK PROTEINS ; SUPEROXIDE-DISMUTASE ; CORTICAL-NEURONS ; OXYGEN-GLUCOSE DEPRIVATION ; FACTOR-DEPENDENT MECHANISM ; EXTRACELLULAR VESICLES
    Abstract: Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell-cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen-glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance.
    Type of Publication: Journal article published
    PubMed ID: 25135971
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  • 11
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; Germany ; human ; SYSTEM ; DEATH ; SITE ; GENE ; GENE-EXPRESSION ; DRUG ; TISSUE ; NF-KAPPA-B ; LIGAND ; AP-1 ; primary ; INDUCTION ; T cells ; T-CELLS ; BINDING ; C-JUN ; SEQUENCE ; TRANSCRIPTION FACTORS ; ASSAY ; activation-induced cell death ; c-Fos ; CARCINOMA CELLS ; CD95 ligand ; CELL-DEATH ; CYCLOSPORINE-A ; FAS-LIGAND EXPRESSION ; INDUCED APOPTOSIS ; MOBILITY ; PROMOTER ; UP-REGULATION
    Abstract: The CD95 (APO-1/Fas) system plays a major role in induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 (APO- 1/Fas) ligand (CD95L) is induced in response to a variety of signals including TCR/CD3 stimulation or application of chemotherapeutic drugs. Here we report that an AP-1 site located in the 5' untranslated region of the CD95L gene is required for TCR/CD3-mediated induction of the human CD95L promoter. Electrophoretic mobility shift assays using nuclear extracts of Jurkat T cells as well as TCR/CD3-restimulated primary human T cells demonstrated specific binding of AP-1, predominantly composed of c-Jun and FosB, to this sequence. Ectopic expression of transdominant negative Jun mutants strongly reduced CD95L promoter activity and activation-induced cell death (AICD), confirming the functional significance of FosB/c-Jun binding. Thus, our results demonstrate an important novel function for FosB dimerized with c-Jun in TCR/CD3- mediated AICD in human T cells
    Type of Publication: Journal article published
    PubMed ID: 12618758
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  • 12
    Keywords: EXPRESSION ; IN-VITRO ; proliferation ; CELL ; IN-VIVO ; VITRO ; VIVO ; SUPPORT ; DISEASE ; NEW-YORK ; GENE ; DIFFERENTIATION ; MICE ; AP-1 ; C-JUN ; TRANSGENIC MICE ; NUMBER ; MARKERS ; PHENOTYPE ; BONE-FORMATION ; REGULATOR ; MOUSE DEVELOPMENT ; AP-1,conditional gene targeting,osteoblasts,osteopenia,osteopetrosis ; TARGETED DISRUPTION
    Abstract: Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. junB(Delta/Delta) mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junB(Delta/Delta) osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity
    Type of Publication: Journal article published
    PubMed ID: 14769860
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  • 13
    Keywords: CELLS ; IRRADIATION ; tumor ; Germany ; PATHWAY ; PATHWAYS ; GENE-EXPRESSION ; transcription ; NF-KAPPA-B ; ACTIVATION ; RESPONSES ; IFN-GAMMA ; MECHANISM ; TRANSCRIPTION FACTOR ; INDUCTION ; T-CELL ; T-CELLS ; C-JUN ; PHOSPHORYLATION ; PROTEIN-KINASE ; SUPPRESSION ; cytokines ; IMMUNE-RESPONSES ; TRANSCRIPTION FACTORS ; c-Fos ; UP-REGULATION ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; TISSUE FACTOR ; IMMUNE-RESPONSE ; KAPPA-B ; IL-2 ; TNF-ALPHA ; SKIN-CANCER ; signaling ; PROGRAM ; RE ; ULTRAVIOLET ; JNK ; SUPPRESSOR ; EVENTS ; ERK ; APC ; EGR-1 EXPRESSION ; JNK PATHWAY ; OZONE DEPLETION ; TRICHINELLA-SPIRALIS ; UV-IRRADIATION
    Abstract: UV irradiation is carcinogenic and immunosuppressive. Previous studies indicate that UV-mediated alteration of APCs and induction of suppressor T cells play a critical role in UV-induced immune suppression. In this study, we show that UV irradiation can directly (independently of APCs and suppressor T cells) inhibit T cell activation by blocking TCR-mediated phosphorylation of ERK and I kappa B via overactivation of the p38 and JNK pathways. These events lead to the down-modulation of c-Jun, c-Fos, Egr-1, and NF-kappa B transcription factors and thereby inhibit production of cytokines, e.g., IL-2, IL-4, IFN-gamma, and TNF-alpha, upon TCR stimulation. We also show that UV irradiation can suppress preactivated T cells, indicating that UV irradiation does not only impair T cell function in response to T cell activation, but can also have systemic effects that influence ongoing immune responses. Thus, our data provide an additional mechanism by which UV irradiation directly suppresses immune responses
    Type of Publication: Journal article published
    PubMed ID: 16081779
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  • 14
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; SURVIVAL ; KINASE ; PATHWAY ; DEATH ; NF-KAPPA-B ; LIGAND ; INDUCTION ; T cells ; T-CELLS ; C-JUN ; PHOSPHORYLATION ; SIGNAL ; TRANSCRIPTION FACTORS ; CD95 ligand ; CELL-DEATH ; UP-REGULATION ; NUMBER ; STRESS ; SIGNALING PATHWAY ; CALCIUM ; TCR ; OXIDATIVE STRESS ; OXIDATION ; OXYGEN ; reactive oxygen species ; CD95 ; signaling ; AUTOIMMUNITY ; RE ; OXIDATIVE-STRESS ; INCREASE ; ENHANCEMENT ; REACTIVE OXYGEN ; ENZYME ; JNK ; AICD ; MNSOD ; ROS ; SIGNALS ; CD95L ; Mangifera indica ; VIMANG
    Abstract: The aqueous stem bark extract of Mangifera indica L. (Vimang) has been reported to have antioxidant properties. AIDS is characterized by up-regulation of CD95 ligand (CD95L) expression and enhancement of activation-induced cell death (AICD). Recent studies demonstrate oxidative signals combined with simultaneous calcium (Ca2+) influx into the cytosol are required for induction of CD95L expression. In this study we show that M. indica extract attenuated anti-CD3-induced accumulation of reactive oxygen species (ROS) and intracellular free Ca2+ and consequently, downregulates CD95L mRNA expression and CD95-mediated AICD. In addition, TCR triggering caused an elevation in the antioxidant enzyme manganous superoxide dismutase (Mn-SOD) and the increase in c-Jun N-terminal kinase (JNK) phosphorylation, both effects being prevented by M indica extract. We provide a number of evidences regarding how M indica extract enhance T-cell survival by inhibiting AICD, a finding associated with a decrease in oxidative stress generated through the TCR signaling pathway in activated T cells. (c) 2006 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16846844
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  • 15
    Keywords: PEPTIDE ; RECEPTOR ; CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; Germany ; IN-VIVO ; KINASE ; VITRO ; VIVO ; DISEASE ; DISEASES ; PROTEIN ; transcription ; DIFFERENTIATION ; ACTIVATION ; DOMAIN ; C-JUN ; PHOSPHORYLATION ; SIGNAL ; GLYCOPROTEIN ; PLASMA ; REGION ; PLASMA-MEMBRANE ; DOMAINS ; ERYTHROPOIETIN RECEPTOR ; CONSTITUTIVE EXPRESSION ; CYTOKINE ; BETA-SUBUNIT ; stem cells ; LEVEL ; function ; BLOCKADE ; in vivo ; TRANSMISSION ; EXTRACELLULAR DOMAINS ; MOUSE EMBRYOS ; LEUKEMIA INHIBITORY FACTOR ; IL-6 RECEPTOR ; INTERLEUKIN-6 FAMILY ; LEUCINE ZIPPERS ; ONCOSTATIN-M ; SIGNAL-TRANSDUCING RECEPTOR
    Abstract: The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of Which is frequently observed in neoplastic diseases
    Type of Publication: Journal article published
    PubMed ID: 16624864
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  • 16
    Keywords: ANGIOGENESIS ; CELLS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; tumor ; CELL ; ENDOTHELIAL GROWTH-FACTOR ; Germany ; PATHWAY ; VITRO ; VIVO ; GENE ; transcription ; NF-KAPPA-B ; COMPLEX ; COMPLEXES ; MECHANISM ; AP-1 ; INDUCTION ; BINDING ; C-JUN ; SIGNAL ; TARGET ; SUBUNIT ; PHENOTYPE ; NF-kappa B ; BODY ; VESSELS ; FACTOR-I ; TUMOR ANGIOGENESIS ; REGULATOR ; VEGF ; HYPOXIA ; signaling ; MOLECULAR-MECHANISM ; RE ; EX-VIVO ; TRANSCRIPTIONAL ACTIVATION ; LOSSES ; JunB ; AP-1 ACTIVITY ; HYPOXIA-INDUCIBLE FACTOR-1-ALPHA ; INDUCED GENE-EXPRESSION ; PROTEIN ELK-1
    Abstract: Regulation of vascular endothelial growth factor ( VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP-1 transcription factor is considered as facilitator of hypoxia-induced VEGF expression through interaction with hypoxia-inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP-1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP-1 subunit junB is a target gene of hypoxia-induced signaling via NF-kappa B. Loss of JunB in various cell types results in severely impaired hypoxia-induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB-deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB(-/-) teratocarcinomas because of severely impaired paracrine-acting VEGF and the subsequent inability to efficiently recruit host-derived vessels
    Type of Publication: Journal article published
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  • 17
    Keywords: CELLS ; EXPRESSION ; GROWTH ; INHIBITOR ; tumor ; CELL ; Germany ; human ; INHIBITION ; KINASE ; PATHWAY ; PATHWAYS ; COMMON ; SYSTEM ; GENE ; GENE-EXPRESSION ; PROTEIN ; cell line ; TISSUE ; DNA ; FAMILY ; REDUCTION ; CONTRAST ; KERATINOCYTES ; BIOLOGY ; C-JUN ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; protein kinase ; PROTEIN-KINASE ; treatment ; ACID ; MOUSE ; NO ; gene expression ; MOUSE SKIN ; CELL-LINE ; LINE ; GROWTH-INHIBITION ; DISPLAY ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; CARCINOMA-CELLS ; EPITHELIAL-CELLS ; specificity ; ANTIOXIDANT ; CHROMOSOMAL LOCALIZATION ; ARACHIDONIC-ACID ; OXYGEN ; MOLECULAR-CLONING ; reactive oxygen species ; ACTIVATED RECEPTOR-GAMMA ; FUNCTIONAL EXPRESSION ; CELL-GROWTH ; signaling ; molecular biology ; molecular ; RE ; FAMILIES ; keratinocyte ; REACTIVE OXYGEN ; LEVEL ; USA ; INDUCIBLE EXPRESSION ; ROS ; INHIBIT ; CDNA CLONING ; GENOMIC STRUCTURE ; lipid ; German ; EXPRESSION SYSTEM ; linoleic acid ; cell growth ; ARACHIDONIC-ACID METABOLITES ; EPIDERMIS-TYPE LIPOXYGENASES ; mitogen-activated protein kinases ; p38
    Abstract: Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells
    Type of Publication: Journal article published
    PubMed ID: 17164225
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  • 18
    Keywords: C-JUN ; beta-catenin ; NUCLEAR-BODIES ; PML ; TRANSCRIPTIONAL ACTIVATION ; P53 ACTIVITY ; AXIN-BINDING PROTEIN ; covalent modification ; HOMEODOMAIN-INTERACTING PROTEIN-KINASE-2 ; UBIQUITIN
    Abstract: Homeodomain-interacting protein kinase 2 (HIPK2) is involved in transcriptional regulation, growth suppression, and apoptosis. Previous reports showed that HIPK2 can signal cell death via p53, and independently of p53 by activating the c-Jun NH2-terminal kinase (JNK) pathway or mediating CtBP degradation. Here we demonstrate that human HIPK2 is small ubiquitin-related modifier-1 (SUMO-1)-modified in vitro and in vivo at lysine residue 25, a SUMO consensus modification motif conserved in human and mouse HIPK family proteins. SUMO modification of HIPK2 altered neither its nuclear body localization nor its recruitment to promyelocytic leukemia-nuclear bodies. However, SUMO-1 modification inhibited HIPK2-induced JNK activation and p53-independent antiproliferative function. HIPK2 with a mutated SUMO acceptor lysine residue was refractory to inhibition of HIPK2-mediated JNK activation by SUMO-1. Furthermore, we demonstrate that SUMO protease SuPr-1 interacts with HIPK2, and both proteins predominantly colocalize in promyelocytic leukemia-nuclear bodies. SuPr-1 deconjugates SUMO-1 from HIPK2 in vitro and in vivo, which results in modestly increased HIPK2-induced JNK activity. Thus, our data demonstrate that HIPK2 effector function on JNK is modulated through dynamic SUMO-1 modification.
    Type of Publication: Journal article published
    PubMed ID: 15958389
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  • 19
    Keywords: CELLS ; EXPRESSION ; CELL ; GENE ; DIFFERENTIATION ; MICE ; KERATINOCYTES ; SKIN ; BIOLOGY ; C-JUN ; DELETION ; ASSAY ; genetics ; STEM-CELLS ; TERMINAL DIFFERENTIATION ; EFFECTOR ; MORPHOGENESIS ; WNT ; FOLLICLE ; signaling ; HOMEOSTASIS ; receptor tyrosine kinase ; MAINTENANCE ; CONSEQUENCES ; NOTCH ; CELL BIOLOGY ; WNT5a ; Genetic ; Intrinsic ; DOWNSTREAM ; epithelial-mesenchymal interactions ; FoxN1 ; ROR2 ; TRANSCRIPTIONAL PROGRAM
    Abstract: Epithelial-mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-J kappa gene, the effector of "canonical'' Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch-CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators
    Type of Publication: Journal article published
    PubMed ID: 20634318
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  • 20
    Keywords: RECEPTOR ; CANCER ; EXPRESSION ; GROWTH ; IN-VITRO ; tumor ; KINASE ; GENE-EXPRESSION ; TIME ; ACTIVATION ; MESSENGER-RNA ; BINDING ; C-JUN ; PROTEIN-KINASE ; TRANSFORMATION ; receptor tyrosine kinase ; Activator Protein 1 (AP-1) ; Axl promoter ; TIMES ; PMA
    Abstract: Background. Axl is a receptor tyrosine kinase promoting anti-apoptosis, invasion and mitogenesis, and is highly expressed in different solid cancers. Axl basal transcriptional activity is driven by Sp1/Sp3, and overexpression of MZF-1 (myeloid zinc-finger 1) induces Axl transcription and gene expression. Furthermore, Axl expression is epigenetically controlled by CpG hypermethylation; however, little is known about inducible Axl gene expression and Axl regulation in haematopoetic malignancies. Results. In the present study, we studied Axl transcriptional regulation under PMA-stimulated conditions in leukaemia cells. Luciferase analysis with sequential 5'-deletion constructs revealed that the -660/-580 region of the Axl promoter is indispensable for induced promoter activity under PMA stimulation. This region includes AP-1 (activator protein 1)/CREB [CRE (cAMP-response-element)-binding protein] motifs, five times partially overlapping TGCGTG repeats and multiple GT repeats. Mutational, supershift and ChIP (chromatin immunoprecipitation) analysis determined that AP-1 family members bind to AP-1 motifs and to the 5 X TGCGTG overlapping repeats, thus transactivating Axl promoter activity. Furthermore, specific inhibitors of PKC (protein kinase C), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 reduced Axl expression. Additionally, mithramycin treatment abolished constitutive and PMA-induced Axl expression. Conclusions. Taken together the results of the present study suggest that PMA-induced Axl gene expression in leukaemia cells is mediated by AP-1 motifs and 5 x TGCGTG repeats within the promoter region -660/-580, and through the PKC/ERK1/2/AP-1 or PKC/p-38/AP-1 signalling axis
    Type of Publication: Journal article published
    PubMed ID: 20977427
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  • 21
    Keywords: EXPRESSION ; DISEASE ; liver ; GENE-EXPRESSION ; NF-KAPPA-B ; mechanisms ; C-JUN ; DOWN-REGULATION ; TNF-ALPHA ; inflammation ; INFLAMMATORY-BOWEL-DISEASE ; FACTOR-ALPHA ; MOLECULAR-MECHANISMS ; TRANSCRIPTIONAL ACTIVATION ; STEROID-HORMONE RECEPTORS ; ENDOTOXIC-SHOCK
    Abstract: As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNF alpha, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNF alpha-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNF alpha lethality was completely abolished when it was administered after TNF alpha stimulation, indicating compromised GR function upon TNF alpha challenge. TNF alpha-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNF alpha down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNF alpha also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNF alpha-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNF alpha inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNF alpha is intimately involved
    Type of Publication: Journal article published
    PubMed ID: 21646349
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  • 22
    Keywords: CELL-PROLIFERATION ; GENE-EXPRESSION ; C-JUN ; GROWTH-FACTOR-BETA ; MOLECULAR-CLONING ; MATRIX METALLOPROTEINASES ; NULL MUTATION ; receptor tyrosine kinase ; MOUSE EMBRYO IMPLANTATION ; UROKINASE ENHANCER
    Abstract: Lack of JunB, an immediate early gene product and member of the AP-1 transcription factor family causes embryonic lethality between E8.5 and E10.0. Although mutant embryos are severely retarded in growth and development, cellular proliferation is apparently not impaired. Retardation and embryonic death are caused by the inability of JunB-deficient embryos to establish proper vascular interactions with the maternal circulation due to multiple defects in extra-embryonic tissues. The onset of the phenotypic defects correlates well with high expression of junB in wild-type extra-embryonic tissues. In trophoblasts, the lack of JunB causes a deregulation of proliferin, matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) gene expression, resulting in a defective neovascularization of the decidua. As a result of downregulation of the VEGF-receptor 1 (flt-1), blood vessels in the yolk sac mesoderm appeared dilated. Mutant embryos which escape these initial defects finally die from a non-vascularized placental labyrinth. Injection of junB-/- embryonic stem (ES) cells into tetraploid wild-type blastocysts resulted in a partial rescue, in which the ES cell-derived fetuses were no longer growth retarded and displayed a normal placental labyrinth. Therefore, JunB appears to be involved in multiple signaling pathways regulating genes involved in the establishment of a proper feto-maternal circulatory system.
    Type of Publication: Journal article published
    PubMed ID: 10022836
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  • 23
    Keywords: GENE-EXPRESSION ; TRANSCRIPTION FACTOR ; BINDING ; C-JUN ; DNA methylation ; MAMMALIAN-CELLS ; TRANSFORMATION ; ENHANCER ; FOS ; beta-galactosidase
    Abstract: The upstream regulatory region (URR) of human papillomavirus type 18 (HPV-18) harbors transcriptional promoter and enhancer elements which are thought to determine the cell-type specificity of the virus. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice carrying the bacterial lacZ gene under the control of the HPV-18 URR. Analysis of beta-galactosidase activity by histochemical staining of tissue sections of four independent transgenic mice showed that the viral promoter was specifically active in epithelial cells within a variety of organs (e.g., tongue, ovary, uterus, testis, and small intestine). Very strong staining was observed in newborn transgenic mice in contrast to a weak activity found during fetal life. Determination of beta-galactosidase activity in crude extracts from tissues of three lines of transgenic mice proved to be a useful tool for a quantitative analysis of transgene expression. In mice from two different transgenic lines treated with dexamethasone such measurements revealed a biphasic effect of the hormone on the activity of the enzyme in the stratified epithelium of the tongue (transient increase followed by a decrease). Northern (RNA) blot analysis showed similar changes in beta-galactosidase mRNA in that tissue. Treatment with tetradecanoyl phorbol acetate (TPA) led to a twofold increase in both enzymatic activity and mRNA levels. Finally, combined treatments with dexamethasone and TPA showed that both factors interfered with each other in their respective effects on transgene expression, suggesting that a cross-talk mechanism between transcription factors could be involved in the regulation of the HPV-18 URR.
    Type of Publication: Journal article published
    PubMed ID: 8411377
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  • 24
    Keywords: ANGIOGENESIS ; EXPRESSION ; IN-VITRO ; C-JUN ; PROGRESSION ; PROSTATE-CANCER ; microenvironment ; MATRIX METALLOPROTEINASES ; CARCINOMA-ASSOCIATED FIBROBLASTS ; SKIN CARCINOMA
    Abstract: Interleukin-6 (IL-6) is one of the major inflammatory interleukins that has been linked to cancer progression. In our model for human skin squamous cell carcinoma (SCC), IL-6 expression is strongly upregulated upon progression from benign tumors to highly malignant, metastasizing SCCs. We now demonstrate that IL-6 promotes malignant and invasive tumor growth in human skin SCCs by inducing cell type specific cytokine profiles in tumor keratinocytes and stromal fibroblasts, activating the latter towards a tumor associated fibroblast (TAF) phenotype. In three-dimensional organotypic cocultures in vitro invasive growth of IL-6 overexpressing tumor keratinocytes, is associated with increased expression of matrix metalloproteinase-2 (MMP-2), MMP-14 and tissue inhibitor of metalloproteinases-2, and clearly depends on IL-6 activated fibroblasts. IL-6-induced secretion of monocyte chemotactic protein-1 (MCP-1) in tumor keratinocytes and of hepatocyte growth factor in fibroblasts is crucial for regulating expression and activation of MMP-2. This functional role of IL-6 is confirmed in vivo. Here MMP-14 and MMP-2 expression occur exclusively in surface transplants of IL-6 overexpressing keratinocytes and fibroblasts are identified as important source of MMP-2. Our data indicate that tumor keratinocytes derived IL-6 activates stromal fibroblasts towards a TAF phenotype, promoting tumor invasion via enhanced expression and activation of MMP-2.
    Type of Publication: Journal article published
    PubMed ID: 23165423
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  • 25
    Keywords: INHIBITOR ; CELL ; Germany ; INHIBITION ; KINASE ; PATHWAY ; TYROSINE KINASE ; SITE ; GENOME ; PROTEIN ; transcription ; ACTIVATION ; COMPLEX ; COMPLEXES ; FAMILY ; TRANSCRIPTION FACTOR ; AP-1 ; INDUCTION ; 16-IMMORTALIZED HUMAN KERATINOCYTES ; ACTIVATED PROTEIN-KINASE ; BINDING ; BIOLOGY ; BOVINE PAPILLOMAVIRUS ; C-JUN ; cell cycle ; CELL-CYCLE ; CELL-LINES ; CYCLE ; DOWN-REGULATION ; E7 ; EARLY GENE-EXPRESSION ; FACTOR-I RECEPTOR ; fibroblasts ; GROWTH-FACTOR RECEPTOR ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; OPEN READING FRAME ; papillomavirus ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; SEQUENCE ; SEQUENCES ; signal transduction ; SUPPRESSION ; TRANSACTIVATOR ; TRANSFORMING ACTIVITY ; treatment ; TYPE-1 ; TYPE-16 E5 PROTEIN ; TYROSINE KINASE INHIBITOR
    Abstract: The tyrosine kinase inhibitor ( tyrphostin) AG 555 selectively interferes with viral transcription in bovine papillomavirus type 1 (BPV-1)-transformed fibroblasts and induces suppression of cyclin-dependent kinase activity and cell cycle arrest. Concomitant with inhibition of viral transcription, c-Jun was strongly up-regulated, which was consistent with the observation that AG 555 treatment also led to an activation of the mitogen-activated protein kinase pathway by enhancing phosphorylation of JNK and p38. Increased JNK and p38 activity resulted in higher phosphorylation of the AP-1 family members c-Jun and activating transcription factor 2. Scanning the BPV-1 genome for potential binding sequences, an intragenic AP-1 site (BAP-1) within the E7 open reading frame was detected. Enhanced dimerization of phosphorylated activating transcription factor 2 together with c-Jun and binding to BAP-1 seem to be responsible for viral dysregulation because both suppression of BPV-1 and induction of c-Jun mRNA could be almost entirely abrogated by simultaneous treatment with SB 203580, an inhibitor of p38 mitogen-activated protein kinase activity. Moreover, dissecting the complex transcriptional pattern of episomal BPV-1 with specific primer sets for reverse transcription-PCR analysis, the repressive effect could be attributed to a selective down-regulation of the mRNA encoding the E2 transactivator function in favor of the E2 repressor, whose mRNA level remained constant during AG 555 treatment. These data indicate that tyrphostin AG 555 disturbs the balance of negative and positive regulatory factors necessary to maintain the homeostasis of a virus-transformed phenotype
    Type of Publication: Journal article published
    PubMed ID: 12867421
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  • 26
    Keywords: CELLS ; IN-VITRO ; IN-VIVO ; MICROSCOPY ; PROTEIN ; PROTEINS ; ACTIVATION ; DOMAIN ; C-JUN ; CATALYTIC SUBUNIT ; SUBUNIT ; LOCALIZATION ; NUCLEUS ; CRYSTAL-STRUCTURE ; LIVING CELLS ; CALCIUM-DEPENDENT PROTEASE ; DISSOCIATION ; MYOFIBRILLAR PROTEIN-TURNOVER ; NEUTRAL PROTEASE ; NUCLEAR-LOCALIZATION ; RAT CALPAIN ; THIOL PROTEASE
    Abstract: Ubiquitously expressed calpains are Ca2+-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca2+-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca2+-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo
    Type of Publication: Journal article published
    PubMed ID: 12591934
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  • 27
    Keywords: CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; CELL ; PATHWAY ; VITRO ; GENE ; GENE-EXPRESSION ; GENES ; PROTEIN ; transcription ; cell line ; DIFFERENTIATION ; LINES ; PATIENT ; TUMOR-NECROSIS-FACTOR ; TRANSCRIPTION FACTOR ; BINDING ; C-JUN ; CELL-LINES ; SEQUENCE ; TRANSCRIPTION FACTORS ; NUCLEAR-FACTOR ; gene expression ; ASSAY ; MOBILITY ; PROMOTER ; CELL-LINE ; LINE ; STIMULI ; EPITHELIAL-CELLS ; Jun ; FACTOR-KAPPA-B ; INDIVIDUALS ; KINETICS ; HELICOBACTER-PYLORI ; protein expression ; CD44 ; CELL-GROWTH ; CONSTRUCTS ; EGR-1 ; FACTOR-ALPHA ; FINGER-ENCODING GENE ; KINASE CASCADES ; signaling
    Abstract: The early growth response 1 (Egr-1) transcription factor is rapidly induced by various stimuli and is implicated in the regulation of cell growth, differentiation, and gene expression. The aim of this study was to examine the effect of Helicobacter pylori on the expression of Egr-1 and Egr-1-regulated genes in gastric epithelial AGS cells. Egr-1 expression was assayed by immunoblotting and electrophoretic mobility shift assays using H. pylori-stimulated AGS cells. Transient transfection experiments with promoter-reporter constructs of CD44, ICAM-1, and CD95L were used for expression studies. H. pylori induced the expression of Egr-1 in gastric epithelial cell lines in a dose-dependent manner, with the rapid kinetics that are typical of this class of transcription factors. Immunohistochemical studies of biopsies revealed that Egr-1 expression is more abundant in H. pylori-positive patients than in uninfected individuals. Reporter-promoter transfection studies indicated that Egr-1 binding is required for the H. pylori-induced transcriptional promoter activity of the CD44, ICAM-1, and CD95L (APO-1/Fas) constructs. The blocking of egr-1 with an antisense sequence prevented H. pylori-induced Egr-1 and CD44 protein expression. The MEK1/2 signaling cascade participates in H. pylori-mediated Egr-1 expression, but the p38 pathway does not. The data indicate that H. pylori induces Egr-1 expression in AGS cells in vitro and that the Egr-1 protein is readily detectable in biopsies from H. pylori-positive subjects. These observations suggest that H. pylori-associated Egr-1 expression may play a role, in part, in H. pylori-induced pathology
    Type of Publication: Journal article published
    PubMed ID: 15155664
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  • 28
    Keywords: CELLS ; EXPRESSION ; carcinoma ; CELL ; GENE ; GENOME ; PROTEIN ; PROTEINS ; transcription ; ACTIVATION ; INFECTION ; MECHANISM ; MOTIFS ; TRANSCRIPTION FACTOR ; INDUCTION ; SUFFICIENT ; ACTIVATED PROTEIN-KINASE ; C-JUN ; CYCLE ; TRANSACTIVATOR ; FORM ; virus ; MUTANT ; NO ; TRANSCRIPTION FACTORS ; LYMPHOMA ; PROMOTER ; REQUIRES ; FUSION ; LINE ; EPITHELIAL-CELLS ; INFECTIONS ; EPSTEIN-BARR-VIRUS ; MAP KINASES ; Epstein-Barr virus ; RECOMBINANT ; BZLF1 ; MOTIF ; VIRAL REPLICATION ; BZLF1 GENE ; EBV INFECTION ; CRE ; EARLY PROMOTER ; LIFE-CYCLE ; PRODUCTIVE CYCLE ; RTA PROTEIN ; ZTA TRANSACTIVATOR
    Abstract: The switch from the latent to the lytic form of Epstein-Barr virus (EBV) infection is mediated by expression of the viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). An EBV early protein, BRRF1 (Na), is encoded by the opposite strand of the BRLF1 intron, but the function of this nuclear protein in the viral life cycle is unknown. Here we demonstrate that Na enhances the R-mediated induction of lytic EBV infection in 293 cells latently infected with a recombinant EBV (R-KO) defective for the expression of both R and Na. Na also enhances R-induced lytic infections in a gastric carcinoma line (AGS) carrying the R-KO virus, although it has no effect in a Burkitt lymphoma line (BL-30) stably infected with the same mutant virus. We show that Na is a transcription factor that increases the ability of R to activate Z expression from the R-KO viral genome in 293 cells and that Na by itself activates the Z promoter (Zp) in EBV-negative cells. Na activation of Zp requires a CRE motif (ZII), and a consensus CRE motif is sufficient to transfer Na responsiveness to the heterologous E1b promoter. Furthermore, we show that Na enhances the transactivator function of a Gal4-c-Jun fusion protein but does not increase the transactivator function of other transcription factors (including ATF-1, ATF-2, and CREB) known to bind CRE motifs. Na expression in cells results in increased levels of a hyperphosphorylated form of c-Jun, suggesting a mechanism by which Na activates c-Jun. Our results indicate that Na is a transcription factor that activates the EBV Zp IE promoter through its effects on c-Jun and suggest that Na cooperates with BRLF1 to induce the lytic form of EBV infection in certain cell types
    Type of Publication: Journal article published
    PubMed ID: 15113878
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  • 29
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; GROWTH-FACTOR ; IN-VITRO ; FACTOR RECEPTOR ; IN-VIVO ; VITRO ; DISEASE ; GENE ; PROTEIN ; PROTEINS ; MICE ; ARTHRITIS ; AP-1 ; tumour ; CONTRAST ; KERATINOCYTES ; SKIN ; T-CELLS ; C-JUN ; DOWN-REGULATION ; SUSCEPTIBILITY ; DELETION ; TRANSGENIC MICE ; LESIONS ; REGION ; B-CELLS ; STEM-CELLS ; Jun ; PHENOTYPE ; MOUSE MODEL ; epidermis ; NEUTROPHILS ; PSORIASIS ; molecular ; ADULT ; RE ; keratinocyte ; development ; RECRUITS ; NECROSIS-FACTOR ; signalling ; ACTIVATED KERATINOCYTES ; MICE LACKING JUNB
    Abstract: Psoriasis is a frequent, inflammatory disease of skin and joints with considerable morbidity. Here we report that in psoriatic lesions, epidermal keratinocytes have decreased expression of JunB, a gene localized in the psoriasis susceptibility region PSORS6. Likewise, inducible epidermal deletion of JunB and its functional companion c-Jun in adult mice leads ( within two weeks) to a phenotype resembling the histological and molecular hallmarks of psoriasis, including arthritic lesions. In contrast to the skin phenotype, the development of arthritic lesions requires T and B cells and signalling through tumour necrosis factor receptor 1 ( TNFR1). Prior to the disease onset, two chemotactic proteins (S100A8 and S100A9) previously mapped to the psoriasis susceptibility region PSORS4, are strongly induced in mutant keratinocytes in vivo and in vitro. We propose that the abrogation of JunB/activator protein 1 (AP-1) in keratinocytes triggers chemokine/cytokine expression, which recruits neutrophils and macrophages to the epidermis thereby contributing to the phenotypic changes observed in psoriasis. Thus, these data support the hypothesis that epidermal alterations are sufficient to initiate both skin lesions and arthritis in psoriasis
    Type of Publication: Journal article published
    PubMed ID: 16163348
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  • 30
    Keywords: EXPRESSION ; CELL ; CELL-PROLIFERATION ; COMBINATION ; Germany ; MODEL ; PATHWAY ; PATHWAYS ; SYSTEM ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; ACTIVATION ; COMPLEX ; COMPLEXES ; FAMILY ; REDUCTION ; DOMAIN ; CONTRAST ; SKIN ; BIOLOGY ; C-JUN ; MEMBER ; MEMBERS ; papillomavirus ; ALPHA ; MUTANT ; LESIONS ; ASSAY ; PROMOTER ; genetics ; MODULATION ; p53 ; PATHOGENESIS ; HPV ; ONCOGENE ; Jun ; COMPLEX-FORMATION ; heredity ; SKIN-CANCER ; ONCOLOGY ; FAMILIES ; INCREASE ; LEADS ; LEVEL ; TARGET GENES ; ASSAYS ; P63 ; AP-1 SUBUNITS ; mtp53 R248W ; p73 ; INHIBIT ; HUMAN PAPILLOMAVIRUSES ; HPV 20 ; P53 HOMOLOG ; TAP ; TAp63 alpha
    Abstract: The p63 alpha isoforms of the p53 family have been demonstrated to play a crucial role in the development and differentiation of the skin. We show that expression of the TAp63 alpha isoform leads to an upregulation of the cutaneous papillomavirus HPV 20 promoter, which is increased at least three-fold when c-Jun is co-expressed, in contrast to a minimal increase in activity in the presence of c-Jun alone. Co-expression of TAp63 alpha with JunB or JunD, respectively, and in combination, leads to a reduction in the viral promoter activation measured by the expression of TAp63 alpha alone. JunB and JunD also inhibits the additive effect exerted on the TAp63a activation by c-Jun. Co-immunoprecipitation assays demonstrate a complex formation of c-Jun, JunB and JunD with TAp 63 alpha through the SAM domain mediating protein-protein interactions, which is characteristic for p63 alpha. Co-expression of p53 mutant R248W not only downregulates the differential modulation of the viral promoter by TAp63 alpha alone and in the presence of the Jun family members, but leads to a reduction in the protein levels of the overexpressed c-Jun, JunB, JunD, as well as TAp63 alpha. This model system provides insight into yet unknown pathways through which TAp63 alpha and Jun may cooperate in the pathogenesis of HPV associated cutaneous lesions
    Type of Publication: Journal article published
    PubMed ID: 16474846
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    Keywords: CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; proliferation ; Germany ; VITRO ; SYSTEM ; transcription ; DIFFERENTIATION ; TISSUE ; MICE ; ACTIVATION ; COMPLEX ; COMPLEXES ; FAMILY ; TRANSCRIPTION FACTOR ; KERATINOCYTES ; SKIN ; C-JUN ; fibroblasts ; treatment ; SIGNAL ; TRANSGENIC MICE ; STRESS ; REPAIR ; EPITHELIAL-CELLS ; KINETICS ; REGULATOR ; COLONY-STIMULATING FACTOR ; INTERCELLULAR COMMUNICATION ; TISSUE-SPECIFIC EXPRESSION ; inflammation ; CYTOKINE ; FAMILIES ; MICE LACKING ; KERATINOCYTE DIFFERENTIATION ; IMMUNE-SYSTEM ; LEVEL ; REGULATED GENE ; macrophage ; inflammatory cells ; COLLAGEN GEL ; GROWTH-STIMULATORY ACTIVITY ; TRANSCRIPTION FACTOR AP-1
    Abstract: The cutaneous response to injury and stress comprises a temporary change in the balance between epidermal proliferation and differentiation as well as an activation of the immune system. Soluble factors play an important role in the regulation of these complex processes by coordinating the intercellular communication between keratinocytes, fibroblasts, and inflammatory cells. In this study, we demonstrate that JunB, a member of the activator protein-1 transcription factor family, is an important regulator of cytokine expression and thus critically involved in the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis, nor homeostasis. However, upon wounding and treatment with the phorbol ester 12-O-decanoyl-phorbol-13-acetate, JunB-deficiency in the skin likewise resulted in pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation. Furthermore, delayed tissue remodelling was observed during wound healing. These phenotypic skin abnormalities were associated with JunB-dependent alterations in expression levels and kinetics of important mediators of wound repair, such as granulocyte macrophage colony-stimulating factor, growth-regulated protein-1, macrophage inflammatory protein-2, and lipocalin-2 in both the dermal and epidermal compartment of the skin, and a reduced ability of wound contraction of mutant dermal fibroblasts in vitro
    Type of Publication: Journal article published
    PubMed ID: 16439969
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