Behrmann Iris | Université du Luxembourg (original) (raw)
Papers by Behrmann Iris
Journal of Bacteriology, Sep 1, 1990
Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a pote... more Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PVT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of ginli encoding a 37,3-kilodalton protein of 343 amino acids. A threeto fourfold increase in oy-glutamyltransferase activity could be observed in S. lividans transformants carrying the ginlI gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by ginli was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, ginA and glnlI.
Journal of Bacteriology
Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a pote... more Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PVT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of ginli encoding a 37,3-kilodalton protein of 343 amino acids. A threeto fourfold increase in oy-glutamyltransferase activity could be observed in S. lividans transformants carrying the ginlI gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by ginli was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, ginA and glnlI.
European cytokine network
Biochemical Journal
The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncost... more The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogenactivated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed. Key words: cytokine signalling, glycoprotein 130 (gp 130) interleukin-6 (IL-6), Janus kinase (JAK), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT).
Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kina... more Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.
... MH FcrWc,3 G. Gcrrc/n,1 C. K/as,1 E. Knipping,] K.-M. Lücking-Famira,] S. Matzku* A. Oehm,] S... more ... MH FcrWc,3 G. Gcrrc/n,1 C. K/as,1 E. Knipping,] K.-M. Lücking-Famira,] S. Matzku* A. Oehm,] S. Richards,] BC Trauth,] GW Bornkamm,3 W. Falk,] P. Möller* and K.-M. Debatin2 institute for Immunology and Genetics, German Cancer Research Center, Heidelberg, Germany ...
Biochemical Journal
The family of cytokines signalling through the common receptor subunit gp130 comprises interleuki... more The family of cytokines signalling through the common receptor subunit gp130 comprises interleukin (IL)-6, IL-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. These so-called IL-6-type cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation. The current knowledge on the signal-transduction mechanisms of these cytokines from the plasma membrane to the nucleus is reviewed. In particular, we focus on the assembly of receptor complexes after ligand binding, the activation of receptor-associated kinases of the Janus family, and the recruitment and phosphorylation of transcription factors of the STAT family, which dimerize, translocate to the nucleus, and bind to enhancer elements of respective target genes leading to transcriptional activation. The important players in the signalling pathway, namely the cytokines and the receptor components, the Janus kinases...
Oncotarget, Jan 26, 2015
MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are prof... more MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in seru...
Signal Transducers and Activators of Transcription (STATs), 2003
Advances in experimental medicine and biology, 2001
Biochemical Society transactions, 1994
JAK-STAT, 2013
Furthermore both pathways control erythroid colony formation and act synergistically on cell prol... more Furthermore both pathways control erythroid colony formation and act synergistically on cell proliferation of JAK2-V617Fpositive patient cells. Results JAK2 and the MAPK cascade act synergistically on the proliferation of JAK2-V617F positive patient cells. It is known that JAK2-V617F activates the JAK-STAT and the MAPK pathway, both of which are involved in the regulation of cell proliferation. 5,27-30 In order to investigate whether inhibitors of the MAPK cascade influence the proliferation of JAK2-V617F-positive cells we incubated in vitro differentiated erythroid cells (amplified from CD34 + cells from JAK2-V617F-positive patients) with the MEK1 inhibitor U0126 and the JAK inhibitor 1 (Fig. 1A). JAK inhibitor 1 (2 μM) strongly suppressed cell proliferation. U0126 suppressed cell proliferation already at 5 μM. Inhibition was even more pronounced at higher concentrations (10 and 20 μM) of U0126. Additionally, we investigated whether signaling through JAK and MEK/ERK exerts any synergistic, additive or antagonistic activity on JAK2-V617F-positive primary cell growth. Thus, we performed growth assays in the presence of both JAK inhibitor 1 and U0126 (ratio of concentrations was kept constant at 4:1). Dose-effect analyses of the drug combinations were performed according to Chou and Talalay using the CompuSyn software. 31-34 All combination index (CI) values measured for cells from three different patients varied between 0.257 and 0.618 indicating strong synergistic to moderate synergistic effects (see also Materials and Methods) (Fig. 1B for one of three patients). These results indicate that in JAK2-V617F-expressing primary cells activity of JAK2 and of the MAPK cascade cooperate to drive proliferation. JAK2-V617F-specific gene induction. To identify MAPKdependent mechanisms involved in JAK2-V617F-mediated deregulation of cell signaling and proliferation we screened for JAK2-V617F-specific MAPK-dependent gene induction. For this aim we utilized HEK293 cells expressing JAK2 wildtype or JAK2-V617F under control of a doxycycline-inducible promoter. 27 Doxycycline-dependent expression of JAK2 and JAK2-V617F is shown in Figure 1C. First, we investigated JAK2-V617F-specific gene induction by gene array analyses in the absence and presence of the MEK inhibitor U0126 (data not shown). These analyses identified the matrix metalloprotease (MMP)-10 and the serine protease inhibitor Serpin B2 as JAK2-V617F-inducible and MAPK-dependent genes. Serpins and MMPs are involved in fibrin and matrix remodeling, as well as in blood clotting. Plasminogen activator inhibitors (PAI-1 and PAI-2 [also known as Serpin B2]) inhibit plasminogen activation, fibrinolysis and ECM degradation. The activity or upregulation of PAIs is correlated with bleeding or thrombosis in patients with PV, ET, or other MPNs who often suffer from hemostasis disorders as a result of disordered fibrinolytic activity. 35-37 Increased Serpin B2 levels were also reported in atypical myeloproliferative diseases. 38 Importantly, Serpin B2 exerts cytoprotective activities by inhibiting retinoblastoma protein degradation and inhibition of procaspase 3 activation. 39,40 Matrix metalloproteases
The FASEB Journal, 2000
Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP-1 relative t... more Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP-1 relative to matrix metalloproteinases (MMPs) promotes progression of fibrosis in liver, lung, and kidney. As to therapeutic strategies, it would be advantageous to antagonize TIMP-1. MMP-9 binds TIMP-1 with high affinity (K i < 50 pM). Three histidine residues constitute the active site of the enzyme by binding the catalytic zinc. MMP-9 mutants were generated in which the histidine residues at position 401, 405 or 411, respectively, were replaced by alanine. All mutants were enzymatically inactive as demonstrated by gelatin zymography. In immuno-precipitation experiments, the MMP-9 mutant H401A bound 2-fold more efficiently to TIMP-1 than H405A, whereas H411A did not bind at all. The mutant H401A was approximately 3-fold less active in TIMP-1 binding than wild-type MMP-9. Recently, we established cell lines secreting TIMP-1 constitutively (HepG2-TIMP-1 cells). In this cell line, increased expression of TIMP-1 resulted in suppressed migration and enhanced cell-cell contact. In co-cultures of HepG2-TIMP-1 cells with HepG2 cells overexpressing the H401A mutant of MMP-9, the TIMP-1-associated phenotype was completely neutralized. These results suggest that a catalytically inactive metalloproteinase (e.g., MMP-9-H401A) that still binds TIMP-1 can be used as a specific antagonist of TIMP-1 activity in vivo. Key words: catalytic zinc • fibrosis • hepatoma cells • liver he extracellular matrix (ECM) modulates cell shape and polarity, and influences cell function and cell-cell interactions. Pathological changes of ECM are associated with many diseases such as rheumatoid arthritis, tumor invasion, fibrosis, and sclerosis (1, 2). In healthy tissue, steady-state levels of ECM proteins are regulated by the rates of their synthesis and degradation. Degradation is performed by matrix metalloproteinases (MMPs), which are inhibited by their specific inhibitors (TIMPs) (3). High levels of TIMPs lead to matrix accumulation by reducing matrix degradation and seem to be involved in the pathogenesis of fibrosis in liver, kidney, and other organs. Tissue inhibitors of metalloproteinases belong to the family of collagenase inhibitors. Four TIMPs have been identified so far. In general, these extracellular proteins are able to inhibit T
Molecular Cancer Research, 2007
Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signalin... more Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and sustained activation of STATs, especially STAT3, is a trait of many different cancers and their derived cell lines. Constitutively active STAT3 has been reported to prevent programmed cell death and enhance cell proliferation, whereas the disruption of STAT3 signaling can inhibit tumor growth. The physiologic activation of STAT3 by cytokines has been well established; however, little is known about altered, stimulationindependent STAT3 activation. Here, we show that, in most but not all melanoma cell lines, STAT3 phosphorylation increased substantially with cell density and that this STAT3 was able to bind to DNA and to activate transcription. Inhibitor studies showed that the cell density-dependent STAT3 activation relies on Janus kinases (JAK) rather than Src kinases. Using a specific JAK inhibitor, sustained STAT3 activation was completely abrogated in all tested melanoma lines, whereas inhibition of Src or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 had no effect on constitutively tyrosine-phosphorylated STAT3 levels. Although STAT3 activation was completely blocked with JAK inhibitor I and to a lesser extent with the common JAK inhibitor AG490, only the latter compound markedly decreased proliferation and induced apoptosis. Taken together, variations in cell density can profoundly modify the extent of JAK-mediated persistent STAT3 phosphorylation; however, STAT3 activation was not sufficient to provide critical growth and survival signals in melanoma cell lines.
Journal of Investigative Dermatology, 2004
Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for ... more Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-gamma. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-gamma-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-gamma receptor 1. In contrast, dose-response studies for interferon-gamma revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-gamma (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-gamma receptor, which may counteract the anti-proliferative function of STAT1.
Journal of Cellular and Molecular Medicine, 2008
IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interes... more IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL-20R2), it is therefore unlikely that IL-24 has classical immune-modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL-24 and related cytokines such as IL-19,-20, and-22. However, the initial interest in IL-24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL-24-induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL-24, we were unable to detect any apoptosis-inducing properties of this cytokine in melanoma cells. In the present 'Point of view' we will briefly summarizse these findings and put them in context of published reports stating that IL-24 might be a long sought after treatment for several types of cancer.
Journal of Biological Chemistry, 2005
Transcription factors of the STAT (signal transducer and activator of transcription) family are i... more Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by phosphorylation on tyrosine and serine residues. Recent evidence suggested that STATs are methylated on a conserved arginine residue within the N-terminal region. STAT arginine methylation has been described to be important for STAT function and loss of arginine methylation was discussed to be involved in interferon resistance of cancer cells. Here we provide several independent lines of evidence indicating that the issue of arginine methylation of STATs has to be reassessed. First, we show that treatment of melanoma and fibrosarcoma cells with inhibitors used to suppress methylation (N-methyl-2-deoxyadenosine, adenosine, DL-homocysteine) had profound and rapid effects on phosphorylation of STAT1 and STAT3 but also on p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Second, we show that anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Third, we show that mutation of Arg 31 to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg 31. Finally, purified catalytically active protein arginine methyltransferases (PRMT1,-2,-3,-4, and-6) did not methylate STAT proteins, and cotransfection with PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, our data suggest the absence of arginine methylation of STAT1 and STAT3.
Journal of Biological Chemistry, 2000
The common use of the cytokine receptor gp130 has served as an explanation for the extremely redu... more The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr 861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr 861 is essential for activation of ERK1/2 and for full activation of the ␣ 2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.
FEBS Letters, 2001
The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin... more The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1^19, 1^18 and 1^8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/ receptor orientation to sustain function.
FEBS Letters, 2000
The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signa... more The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signal transducer gp130. Binding of IL-6 to its receptor results in dimerization of gp130, activation of the Jak/STAT pathway, and in a down-regulation of IL-6 binding sites by endocytosis. The STAT activation after stimulation is transient, being maximal after 15^30 min and disappearing after 60^90 min. The mechanism which leads to the termination of the signal is still unknown. In this paper we have studied whether the down-modulation of the STAT signal requires the endocytosis of the receptor complex. Our results suggest that the desensitization of the IL-6 signal is not due to internalization of the receptor complex but requires de novo protein synthesis.
Journal of Bacteriology, Sep 1, 1990
Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a pote... more Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PVT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of ginli encoding a 37,3-kilodalton protein of 343 amino acids. A threeto fourfold increase in oy-glutamyltransferase activity could be observed in S. lividans transformants carrying the ginlI gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by ginli was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, ginA and glnlI.
Journal of Bacteriology
Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a pote... more Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PVT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of ginli encoding a 37,3-kilodalton protein of 343 amino acids. A threeto fourfold increase in oy-glutamyltransferase activity could be observed in S. lividans transformants carrying the ginlI gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by ginli was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, ginA and glnlI.
European cytokine network
Biochemical Journal
The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncost... more The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogenactivated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed. Key words: cytokine signalling, glycoprotein 130 (gp 130) interleukin-6 (IL-6), Janus kinase (JAK), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT).
Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kina... more Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.
... MH FcrWc,3 G. Gcrrc/n,1 C. K/as,1 E. Knipping,] K.-M. Lücking-Famira,] S. Matzku* A. Oehm,] S... more ... MH FcrWc,3 G. Gcrrc/n,1 C. K/as,1 E. Knipping,] K.-M. Lücking-Famira,] S. Matzku* A. Oehm,] S. Richards,] BC Trauth,] GW Bornkamm,3 W. Falk,] P. Möller* and K.-M. Debatin2 institute for Immunology and Genetics, German Cancer Research Center, Heidelberg, Germany ...
Biochemical Journal
The family of cytokines signalling through the common receptor subunit gp130 comprises interleuki... more The family of cytokines signalling through the common receptor subunit gp130 comprises interleukin (IL)-6, IL-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. These so-called IL-6-type cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation. The current knowledge on the signal-transduction mechanisms of these cytokines from the plasma membrane to the nucleus is reviewed. In particular, we focus on the assembly of receptor complexes after ligand binding, the activation of receptor-associated kinases of the Janus family, and the recruitment and phosphorylation of transcription factors of the STAT family, which dimerize, translocate to the nucleus, and bind to enhancer elements of respective target genes leading to transcriptional activation. The important players in the signalling pathway, namely the cytokines and the receptor components, the Janus kinases...
Oncotarget, Jan 26, 2015
MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are prof... more MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in seru...
Signal Transducers and Activators of Transcription (STATs), 2003
Advances in experimental medicine and biology, 2001
Biochemical Society transactions, 1994
JAK-STAT, 2013
Furthermore both pathways control erythroid colony formation and act synergistically on cell prol... more Furthermore both pathways control erythroid colony formation and act synergistically on cell proliferation of JAK2-V617Fpositive patient cells. Results JAK2 and the MAPK cascade act synergistically on the proliferation of JAK2-V617F positive patient cells. It is known that JAK2-V617F activates the JAK-STAT and the MAPK pathway, both of which are involved in the regulation of cell proliferation. 5,27-30 In order to investigate whether inhibitors of the MAPK cascade influence the proliferation of JAK2-V617F-positive cells we incubated in vitro differentiated erythroid cells (amplified from CD34 + cells from JAK2-V617F-positive patients) with the MEK1 inhibitor U0126 and the JAK inhibitor 1 (Fig. 1A). JAK inhibitor 1 (2 μM) strongly suppressed cell proliferation. U0126 suppressed cell proliferation already at 5 μM. Inhibition was even more pronounced at higher concentrations (10 and 20 μM) of U0126. Additionally, we investigated whether signaling through JAK and MEK/ERK exerts any synergistic, additive or antagonistic activity on JAK2-V617F-positive primary cell growth. Thus, we performed growth assays in the presence of both JAK inhibitor 1 and U0126 (ratio of concentrations was kept constant at 4:1). Dose-effect analyses of the drug combinations were performed according to Chou and Talalay using the CompuSyn software. 31-34 All combination index (CI) values measured for cells from three different patients varied between 0.257 and 0.618 indicating strong synergistic to moderate synergistic effects (see also Materials and Methods) (Fig. 1B for one of three patients). These results indicate that in JAK2-V617F-expressing primary cells activity of JAK2 and of the MAPK cascade cooperate to drive proliferation. JAK2-V617F-specific gene induction. To identify MAPKdependent mechanisms involved in JAK2-V617F-mediated deregulation of cell signaling and proliferation we screened for JAK2-V617F-specific MAPK-dependent gene induction. For this aim we utilized HEK293 cells expressing JAK2 wildtype or JAK2-V617F under control of a doxycycline-inducible promoter. 27 Doxycycline-dependent expression of JAK2 and JAK2-V617F is shown in Figure 1C. First, we investigated JAK2-V617F-specific gene induction by gene array analyses in the absence and presence of the MEK inhibitor U0126 (data not shown). These analyses identified the matrix metalloprotease (MMP)-10 and the serine protease inhibitor Serpin B2 as JAK2-V617F-inducible and MAPK-dependent genes. Serpins and MMPs are involved in fibrin and matrix remodeling, as well as in blood clotting. Plasminogen activator inhibitors (PAI-1 and PAI-2 [also known as Serpin B2]) inhibit plasminogen activation, fibrinolysis and ECM degradation. The activity or upregulation of PAIs is correlated with bleeding or thrombosis in patients with PV, ET, or other MPNs who often suffer from hemostasis disorders as a result of disordered fibrinolytic activity. 35-37 Increased Serpin B2 levels were also reported in atypical myeloproliferative diseases. 38 Importantly, Serpin B2 exerts cytoprotective activities by inhibiting retinoblastoma protein degradation and inhibition of procaspase 3 activation. 39,40 Matrix metalloproteases
The FASEB Journal, 2000
Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP-1 relative t... more Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP-1 relative to matrix metalloproteinases (MMPs) promotes progression of fibrosis in liver, lung, and kidney. As to therapeutic strategies, it would be advantageous to antagonize TIMP-1. MMP-9 binds TIMP-1 with high affinity (K i < 50 pM). Three histidine residues constitute the active site of the enzyme by binding the catalytic zinc. MMP-9 mutants were generated in which the histidine residues at position 401, 405 or 411, respectively, were replaced by alanine. All mutants were enzymatically inactive as demonstrated by gelatin zymography. In immuno-precipitation experiments, the MMP-9 mutant H401A bound 2-fold more efficiently to TIMP-1 than H405A, whereas H411A did not bind at all. The mutant H401A was approximately 3-fold less active in TIMP-1 binding than wild-type MMP-9. Recently, we established cell lines secreting TIMP-1 constitutively (HepG2-TIMP-1 cells). In this cell line, increased expression of TIMP-1 resulted in suppressed migration and enhanced cell-cell contact. In co-cultures of HepG2-TIMP-1 cells with HepG2 cells overexpressing the H401A mutant of MMP-9, the TIMP-1-associated phenotype was completely neutralized. These results suggest that a catalytically inactive metalloproteinase (e.g., MMP-9-H401A) that still binds TIMP-1 can be used as a specific antagonist of TIMP-1 activity in vivo. Key words: catalytic zinc • fibrosis • hepatoma cells • liver he extracellular matrix (ECM) modulates cell shape and polarity, and influences cell function and cell-cell interactions. Pathological changes of ECM are associated with many diseases such as rheumatoid arthritis, tumor invasion, fibrosis, and sclerosis (1, 2). In healthy tissue, steady-state levels of ECM proteins are regulated by the rates of their synthesis and degradation. Degradation is performed by matrix metalloproteinases (MMPs), which are inhibited by their specific inhibitors (TIMPs) (3). High levels of TIMPs lead to matrix accumulation by reducing matrix degradation and seem to be involved in the pathogenesis of fibrosis in liver, kidney, and other organs. Tissue inhibitors of metalloproteinases belong to the family of collagenase inhibitors. Four TIMPs have been identified so far. In general, these extracellular proteins are able to inhibit T
Molecular Cancer Research, 2007
Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signalin... more Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and sustained activation of STATs, especially STAT3, is a trait of many different cancers and their derived cell lines. Constitutively active STAT3 has been reported to prevent programmed cell death and enhance cell proliferation, whereas the disruption of STAT3 signaling can inhibit tumor growth. The physiologic activation of STAT3 by cytokines has been well established; however, little is known about altered, stimulationindependent STAT3 activation. Here, we show that, in most but not all melanoma cell lines, STAT3 phosphorylation increased substantially with cell density and that this STAT3 was able to bind to DNA and to activate transcription. Inhibitor studies showed that the cell density-dependent STAT3 activation relies on Janus kinases (JAK) rather than Src kinases. Using a specific JAK inhibitor, sustained STAT3 activation was completely abrogated in all tested melanoma lines, whereas inhibition of Src or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 had no effect on constitutively tyrosine-phosphorylated STAT3 levels. Although STAT3 activation was completely blocked with JAK inhibitor I and to a lesser extent with the common JAK inhibitor AG490, only the latter compound markedly decreased proliferation and induced apoptosis. Taken together, variations in cell density can profoundly modify the extent of JAK-mediated persistent STAT3 phosphorylation; however, STAT3 activation was not sufficient to provide critical growth and survival signals in melanoma cell lines.
Journal of Investigative Dermatology, 2004
Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for ... more Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-gamma. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-gamma-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-gamma receptor 1. In contrast, dose-response studies for interferon-gamma revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-gamma (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-gamma receptor, which may counteract the anti-proliferative function of STAT1.
Journal of Cellular and Molecular Medicine, 2008
IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interes... more IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL-20R2), it is therefore unlikely that IL-24 has classical immune-modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL-24 and related cytokines such as IL-19,-20, and-22. However, the initial interest in IL-24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL-24-induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL-24, we were unable to detect any apoptosis-inducing properties of this cytokine in melanoma cells. In the present 'Point of view' we will briefly summarizse these findings and put them in context of published reports stating that IL-24 might be a long sought after treatment for several types of cancer.
Journal of Biological Chemistry, 2005
Transcription factors of the STAT (signal transducer and activator of transcription) family are i... more Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by phosphorylation on tyrosine and serine residues. Recent evidence suggested that STATs are methylated on a conserved arginine residue within the N-terminal region. STAT arginine methylation has been described to be important for STAT function and loss of arginine methylation was discussed to be involved in interferon resistance of cancer cells. Here we provide several independent lines of evidence indicating that the issue of arginine methylation of STATs has to be reassessed. First, we show that treatment of melanoma and fibrosarcoma cells with inhibitors used to suppress methylation (N-methyl-2-deoxyadenosine, adenosine, DL-homocysteine) had profound and rapid effects on phosphorylation of STAT1 and STAT3 but also on p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Second, we show that anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Third, we show that mutation of Arg 31 to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg 31. Finally, purified catalytically active protein arginine methyltransferases (PRMT1,-2,-3,-4, and-6) did not methylate STAT proteins, and cotransfection with PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, our data suggest the absence of arginine methylation of STAT1 and STAT3.
Journal of Biological Chemistry, 2000
The common use of the cytokine receptor gp130 has served as an explanation for the extremely redu... more The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr 861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr 861 is essential for activation of ERK1/2 and for full activation of the ␣ 2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.
FEBS Letters, 2001
The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin... more The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1^19, 1^18 and 1^8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/ receptor orientation to sustain function.
FEBS Letters, 2000
The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signa... more The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signal transducer gp130. Binding of IL-6 to its receptor results in dimerization of gp130, activation of the Jak/STAT pathway, and in a down-regulation of IL-6 binding sites by endocytosis. The STAT activation after stimulation is transient, being maximal after 15^30 min and disappearing after 60^90 min. The mechanism which leads to the termination of the signal is still unknown. In this paper we have studied whether the down-modulation of the STAT signal requires the endocytosis of the receptor complex. Our results suggest that the desensitization of the IL-6 signal is not due to internalization of the receptor complex but requires de novo protein synthesis.