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Fabrice Gouilleux - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological Inhibition of Oncogenic STAT3 and STAT5 Signaling in Hematopoietic Cancers
Cancers, 2020Co-Authors: Marie Brachet-botineau, Marion Polomski, Heidi Neubauer, Ludovic Juen, Damien Hédou, Marie-claude Viaud-massuard, Gildas Prié, Fabrice GouilleuxAbstract:Signal Transducer and Activator of Transcription (STAT) 3 and 5 are important effectors of cellular transformation, and aberrant STAT3 and STAT5 signaling have been demonstrated in hematopoietic cancers. STAT3 and STAT5 are common targets for different tyrosine kinase oncogenes (TKOs). In addition, STAT3 and STAT5 proteins were shown to contain activating mutations in some rare but aggressive leukemias/lymphomas. Both proteins also contribute to drug resistance in hematopoietic malignancies and are now well recognized as major targets in cancer treatment. The development of inhibitors targeting STAT3 and STAT5 has been the subject of intense investigations during the last decade. This review summarizes the current knowledge of oncogenic STAT3 and STAT5 functions in hematopoietic cancers as well as advances in preclinical and clinical development of pharmacological inhibitors.
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Interleukin-7 induces apoptosis of 697 pre-B cells expressing dominant-negative forms of STAT5: evidence for caspase-dependent and -independent mechanisms
Oncogene, 2004Co-Authors: Olivia Lanvin, Fabrice Gouilleux, Catherine Mullié, Cécile Mazière, Vincent Fuentes, Eliane Bissac, Françoise Dantin, Jean-claude Maziere, Aline Regnier, Kaiss LassouedAbstract:The transcription factors STAT5A and STAT5B (STAT: signal transducer and activator of transcription) play a major role in the signaling events elicited by a number of growth factor and cytokine receptors. In this work, we aimed to investigate the role of STAT5 in human precursor B cell survival by introducing dominant-negative (DN) forms of STAT5A or STAT5B in the 697 pre-B cell line. All clones expressing DN forms of either transcription factor exhibited a higher spontaneous apoptotic rate that was massively enhanced upon interleukin-7 (IL-7) stimulation. This was associated with caspase 8 cleavage, mitochondrial transmembrane potential disruption and caspase 3 activation. However, the DN forms of STAT5 did not alter the expression of Bcl-2, Bax, Bcl-x, Bim, A1 and Mcl1 proteins in IL-7-stimulated cells. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromylmethyl ketone partially suppressed IL-7-mediated mitochondrial transmembrane potential disruption and cell death, suggesting that IL-7 induced the death of DN STAT5 expressing 697 cells through caspase-dependent and -independent mechanisms that both require mitochondrial activation.
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Constitutively active STAT5 variants induce growth and survival of hematopoietic cells through a PI 3-kinase/Akt dependent pathway
Oncogene, 2001Co-Authors: Susana Constantino Rosa Santos, Sylvie Gisselbrecht, Virginie Lacronique, Isabelle Bouchaert, Richard Monni, Olivier Bernard, Fabrice GouilleuxAbstract:Signal Transducer and Activator of Transcription (STATs) are important mediators of cytokine and growth factor-induced signal transduction. STAT5A and STAT5B have been shown to play a role in survival and proliferation of hematopoietic cells both in vitro and in vivo and to contribute to the growth and viability of cells transformed by the TEL-JAK2 oncoprotein. In this study, we investigated the molecular mechanisms by which constitutively active STAT5 proteins induce cell proliferation and survival of Ba/F3 cell lines expressing either dominant positive STAT5A or STAT5B variants or TEL-JAK2 or TEL-ABL fusion proteins. Our results showed that active STAT5 constitutively interacted with p85, the regulatory subunit of the PI 3-kinase. A constitutive activity of the PI 3-kinase/Akt pathway was observed in these cells and required for their cell cycle progression. In contrast, while activity of the PI 3-kinase/Akt pathway was required for survival of Ba/F3 cells expressing the constitutively active forms of STAT5A or STAT5B, it was dispensable for cells transformed by TEL-JAK2 or TEL-ABL fusion proteins, suggesting that additional survival pathways take place in these transformed cells.
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Transforming properties of chimeric TEL-JAK proteins in Ba/F3 cells.
Blood, 2000Co-Authors: V Lacronique, Sylvie Gisselbrecht, Fabrice Gouilleux, A. Boureux, R Monni, S Dumon, M Mauchauffé, P. Mayeux, R. Berger, J GhysdaelAbstract:The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated STAT5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the STAT5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of STAT5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate STAT5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)
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IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line
Oncogene, 1999Co-Authors: S Dumon, Christine Boucheron, Sylvie Gisselbrecht, V Gouilleux-gruart, L Cocault, S Constantino Rosa Santos, F Debierre-grockiego, P Mollat, Fabrice GouilleuxAbstract:Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.
Lothar Hennighausen - One of the best experts on this subject based on the ideXlab platform.
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twins with different personalities STAT5b but not STAT5a has a key role in bcr abl induced leukemia
Leukemia, 2019Co-Authors: Sebastian Kollmann, Reinhard Grausenburger, Eva Grundschober, Wolfgang Warsch, Leo Edlinger, Jani Huuhtanen, Sabine Lagger, Barbara Maurer, Lothar Hennighausen, Peter ValentAbstract:Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B, but not STAT5A, have been frequently described in hematopoietic tumors, we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving STAT5b−/− cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line, STAT5b−/− leukemic cells induced leukemia with a significantly prolonged disease onset, whereas STAT5a−/− cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-α and IFN-γ signatures in STAT5b−/− cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of STAT5b−/−-deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-α/γ, thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors.
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janus kinase 1 is essential for inflammatory cytokine signaling and mammary gland remodeling
Molecular and Cellular Biology, 2016Co-Authors: Kazuhito Sakamoto, Barbara L Wehde, Patrick D Radler, Ha Youn Shin, Aleata A. Triplett, Keunsoo Kang, Fabian Schuler, Nirakar Rajbhandari, Andreas Villunger, Lothar HennighausenAbstract:Despite a wealth of knowledge about the significance of individual signal transducers and activators of transcription (STATs), essential functions of their upstream Janus kinases (JAKs) during postnatal development are less well defined. Using a novel mammary gland-specific JAK1 knockout model, we demonstrate here that this tyrosine kinase is essential for the activation of STAT1, STAT3, and STAT6 in the mammary epithelium. The loss of JAK1 uncouples interleukin-6-class ligands from their downstream effector, STAT3, which leads to the decreased expression of STAT3 target genes that are associated with the acute-phase response, inflammation, and wound healing. Consequently, JAK1-deficient mice exhibit impaired apoptosis and a significant delay in mammary gland remodeling. Using RNA sequencing, we identified several new JAK1 target genes that are upregulated during involution. These include Bmf and Bim, which are known regulators of programmed cell death. Using a BMF/BIM-double-knockout epithelial transplant model, we further validated that the synergistic action of these proapoptotic JAK1 targets is obligatory for the remodeling of the mammary epithelium. The collective results of this study suggest that JAK1 has nonredundant roles in the activation of particular STAT proteins and this tyrosine kinase is essential for coupling inflammatory cytokine signals to the cell death machinery in the differentiated mammary epithelium.
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sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5a b concentration
Nucleic Acids Research, 2013Co-Authors: Daisuke Yamaji, Keunsoo Kang, Gertraud W Robinson, Lothar HennighausenAbstract:The transcription factors Signal Transducer and Activator of Transcription (STAT) 5A/B mediate prolactin-induced mammary development during pregnancy. However, it is not clear how the different processes, expansion and maturation of alveolar precursor cells and the differential induction of milk protein genes are regulated on a molecular level. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts mammary epithelial development during pregnancy and the regulation of target genes. The presence of only a single STAT5a or STAT5b allele was sufficient for the establishment of histologically undifferentiated alveolar units and two alleles permitted the execution of a differentiation program similar to that found with all four alleles. While one copy of STAT5 induced limited expression of target genes, two copies activated a lactation-like gene signature. Using ChIP-seq analyses on intact tissue under physiological conditions, we found that highly expressed and regulated genes were bound by STAT5 in their promoter proximal regions, whereas upstream binding had minor biological consequences. Remarkably, 80% of the genes bound by STAT5 in vivo were not under STAT5 control. RNA polymerase II intensity was directly proportional to STAT5 concentration only on STAT5 regulated genes providing mechanistic insight by which STAT5 activates mammary specific genes.
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interpretation of cytokine signaling through the transcription factors STAT5a and STAT5b
Genes & Development, 2008Co-Authors: Lothar Hennighausen, Gertraud W RobinsonAbstract:Transcription factors from the family of Signal Transducers and Activators of Transcription (STAT) are activated by numerous cytokines. Two members of this family, STAT5A and STAT5B (collectively called STAT5), have gained prominence in that they are activated by a wide variety of cytokines such as interleukins, erythropoietin, growth hormone, and prolactin. Furthermore, constitutive STAT5 activation is observed in the majority of leukemias and many solid tumors. Inactivation studies in mice as well as human mutations have provided insight into many of STAT5’s functions. Disruption of cytokine signaling through STAT5 results in a variety of cell-specific effects, ranging from a defective immune system and impaired erythropoiesis, the complete absence of mammary development during pregnancy, to aberrant liver function. On a molecular level, STAT5 has been linked to cell specification, proliferation, differentiation, and survival. Evidence is growing that the diverse outcomes of STAT5 signaling are not only determined by the expression of specific receptors but also by the interaction of STAT5 with cofactors and the cell-specific activity of members of the SOCS family, which negatively regulate STAT function. In this review, we focus on emerging concepts and challenges in the field of Janus kinase (JAK)–STAT5 signaling. First, we discuss unique functions of STAT5 in three distinct systems: mammary epithelial cells, hepatocytes, and regulatory T cells. Second, we present an example of how STAT5 can achieve cell specificity in hepatocytes through a physical and functional interaction with the glucocorticoid receptor. Third, we focus on the relevance of STAT5 in the development and progression of leukemia. Next, we discuss lessons derived from human mutations and disease. Finally, we address an emerging issue that the interpretation of experiments from STAT5-deficient mice and cells might be compromised as these cells might reroute and reprogram cytokine signals to the “wrong” STATs and thus acquire inappropriate cues. We propose that mice with mutations in various components of the JAK–STAT signaling pathway are living laboratories, which will provide insight into the versatility of signaling hardware and the adaptability of the software.
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nonredundant roles for STAT5a b in directly regulating foxp3
Blood, 2007Co-Authors: Yuka Kanno, Marc A Kerenyi, Geoffrey L Stephens, Lydia Durant, Richard Moriggl, Wendy T. Watford, Gertraud W Robinson, Ethan M. Shevach, Arian Laurence, Lothar HennighausenAbstract:Stats (signal transducers and activators of transcription) regulate multiple aspects of T-cell fate. T regulatory (Treg) cells are a critical subset that limits immune responses, but the relative importance of STAT5a/b versus Stat3 for Treg cell development has been contentious. We observed that peripheral CD25+CD4+ T cells were reduced in STAT5ΔN mice; however, the levels of Foxp3, a transcription factor that is critical for Treg cells, were normal in splenic CD4+ T cells even though they were reduced in the thymus. In contrast, complete deletion of STAT5a/b (STAT5−/−) resulted in dramatic reduction in CD25- or Foxp3-expressing CD4+ T cells. An intrinsic requirement was demonstrated by reduction of STAT5a/b in CD4-expressing cells and by stem cell transplantation using STAT5−/− fetal liver cells. STAT5a/b were also required for optimal induction of Foxp3 in vitro and bound directly to the Foxp3 gene. Reduction of Stat3 in T cells did not reduce the numbers of Treg cells in the thymus or spleen; however, Stat3 was required for IL-6–dependent down-regulation of Foxp3. Therefore, we conclude that STAT5a/b have an essential, nonredundant role in regulating Treg cells, and that Stat3 and STAT5a/b appear to have opposing roles in the regulation of Foxp3.
B Groner - One of the best experts on this subject based on the ideXlab platform.
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Comparison of the transactivation domains of STAT5 and Stat6 in lymphoid cells and mammary epithelial cells.
Molecular and Cellular Biology, 1997Co-Authors: R Moriggl, Fabrice Gouilleux, S Berchtold, K Friedrich, G Standke, W. Kammer, M Heim, M Wissler, E Stöcklin, B GronerAbstract:Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. STAT5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates STAT5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of STAT5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of STAT5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.
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Deletion of the carboxyl-terminal transactivation domain of MGF-STAT5 results in sustained DNA binding and a dominant negative phenotype.
Molecular and Cellular Biology, 1996Co-Authors: R Moriggl, Lothar Hennighausen, V Gouilleux-gruart, R Jähne, S Berchtold, C Gartmann, X. Liu, A Sotiropoulos, B Groner, Fabrice GouilleuxAbstract:The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-STAT5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of STAT5, STAT5a and STAT5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type STAT5a (794 amino acids) and the carboxyl-terminal deletion mutant STAT5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; STAT5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three STAT5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the STAT5a delta 750 mutant. The carboxyl-terminal transactivation domain of STAT5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of STAT5a or STAT5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that STAT5a delta 750 and STAT5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in STAT5-responsive gene promoters.
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STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.
Blood, 1996Co-Authors: V Gouilleux-gruart, Fabrice Gouilleux, I Dusanter-fourt, R Weber-nordt, Jean-claude Capiod, Jean-franÇois Claisse, Jacques Delobel, C Desaint, F. Dreyfus, B GronerAbstract:A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.
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Cloning and expression of STAT5 and an additional homologue (STAT5b) involved in prolactin signal transduction in mouse mammary tissue.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: X. Liu, Fabrice Gouilleux, B Groner, G. Robinson, Lothar HennighausenAbstract:Prolactin (PRL) induces transcriptional activation of milk protein genes, such as the whey acidic protein (WAP), beta-casein, and beta-lactoglobulin genes, through a signaling cascade encompassing the Janus kinase Jak2 and the mammary gland factor (MGF; also called STAT5), which belongs to the family of proteins of signal transducers and activators of transcription (STAT). We isolated and sequenced from mouse mammary tissue STAT5 mRNA and a previously unreported member, which we named STAT5b (STAT5 is renamed to STAT5a). On the protein level STAT5a and STAT5b show a 96% sequence similarity. The 5' and 3' untranslated regions of the two mRNAs are not conserved. STAT5a comprises 793 amino acids and is encoded by a mRNA of 4.2 kb. The STAT5b mRNA has a size of 5.6 kb and encodes a protein of 786 amino acids. Both STAT5a and STAT5b recognized the GAS site (gamma-interferon-activating sequence; TTCNNNGAA) in vitro and mediated PRL-induced transcription in COS cells transfected with a PRL receptor. STAT5b also induced basal transcription in the absence of PRL. Similar levels of STAT5a and STAT5b mRNAs were found in most tissues of virgin and lactating mice, but a differential accumulation of the STAT5 mRNAs was found in muscle and mammary tissue. The two RNAs are present in mammary tissue of immature virgin mice, and their levels increase up to day 16 of pregnancy, followed by a decline during lactation. The increase of STAT5 expression during pregnancy coincides with the activation of the WAP gene.
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Thrombopoietin activates a STAT5-like factor in hematopoietic cells.
EMBO Journal, 1995Co-Authors: C Pallard, Sylvie Gisselbrecht, Fabrice Gouilleux, B Groner, L Bénit, L Cocault, M Souyri, D Levy, I Dusanter-fourtAbstract:Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF.
Jeffrey M Rosen - One of the best experts on this subject based on the ideXlab platform.
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differential effects of prolactin and src abl kinases on the nuclear translocation of STAT5b and STAT5a
Journal of Biological Chemistry, 1999Co-Authors: Alexander V. Kazansky, Elena B Kabotyanski, Shannon L Wyszomierski, Michael A. Mancini, Jeffrey M RosenAbstract:Abstract In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a β-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.
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Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A.
Journal of Biological Chemistry, 1999Co-Authors: Alexander V. Kazansky, Elena B Kabotyanski, Shannon L Wyszomierski, Michael A. Mancini, Jeffrey M RosenAbstract:Abstract In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a β-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.
V Gouilleux-gruart - One of the best experts on this subject based on the ideXlab platform.
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IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line
Oncogene, 1999Co-Authors: S Dumon, Christine Boucheron, Sylvie Gisselbrecht, V Gouilleux-gruart, L Cocault, S Constantino Rosa Santos, F Debierre-grockiego, P Mollat, Fabrice GouilleuxAbstract:Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.
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Activated Stat Related Transcription Factors in Acute Leukemia
Leukemia & lymphoma, 1997Co-Authors: V Gouilleux-gruart, Fabrice Gouilleux, Françoise Debierre-grockiego, Jean-claude Capiod, Jean-franÇois Claisse, Jacques Delobel, Lionel PrinAbstract:Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and STAT5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and STAT5 proteins are described. The mechanisms of such activation remain to be clarified.
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Deletion of the carboxyl-terminal transactivation domain of MGF-STAT5 results in sustained DNA binding and a dominant negative phenotype.
Molecular and Cellular Biology, 1996Co-Authors: R Moriggl, Lothar Hennighausen, V Gouilleux-gruart, R Jähne, S Berchtold, C Gartmann, X. Liu, A Sotiropoulos, B Groner, Fabrice GouilleuxAbstract:The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-STAT5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of STAT5, STAT5a and STAT5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type STAT5a (794 amino acids) and the carboxyl-terminal deletion mutant STAT5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; STAT5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three STAT5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the STAT5a delta 750 mutant. The carboxyl-terminal transactivation domain of STAT5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of STAT5a or STAT5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that STAT5a delta 750 and STAT5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in STAT5-responsive gene promoters.
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Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines.
Blood, 1996Co-Authors: R Weber-nordt, V Gouilleux-gruart, C Egen, J Wehinger, W. Ludwig, R Mertelsmann, J. FinkeAbstract:Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of STAT5 in these cells. Confocal microscopy revealed the nuclear localization of STAT5 and Western blot analyses showed tyrosine phosphorylation of STAT5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of STAT5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.
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STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.
Blood, 1996Co-Authors: V Gouilleux-gruart, Fabrice Gouilleux, I Dusanter-fourt, R Weber-nordt, Jean-claude Capiod, Jean-franÇois Claisse, Jacques Delobel, C Desaint, F. Dreyfus, B GronerAbstract:A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.
