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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, Lothar Hennighausen, Barbara Maurer, Reinhard Grausenburger, Eva Grundschober, Wolfgang Warsch, Leo Edlinger, Jani Huuhtanen, Sabine Lagger, 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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sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A b concentration
Nucleic Acids Research, 2013Co-Authors: Daisuke Yamaji, Gertraud W Robinson, Keunsoo Kang, 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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the transcription factors signal transducer and activator of transcription 5a STAT5A and stat5b negatively regulate cell proliferation through the activation of cyclin dependent kinase inhibitor 2b cdkn2b and cdkn1a expression
Hepatology, 2010Co-Authors: Ji Hoon Yu, Atsushi Hosui, Gertraud W Robinson, Mark Wickre, Gregory Riedlinger, Weiping Chen, Lothar HennighausenAbstract:Although the cytokine-inducible transcription factor signal transducer and activator of transcription 5 (STAT5) promotes proliferation of a wide range of cell types, there are cell-specific and context-specific cases in which loss of STAT5 results in enhanced cell proliferation. Here, we report that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15INK4B and p21CIP1. We further demonstrate that growth hormone, through the transcription factor STAT5, enhances expression of the Cdkn2b (cyclin-dependent kinase inhibitor 2B) gene and that STAT5A binds to interferon-gamma–activated sequence sites within the promoter. We recently demonstrated that ablation of STAT5 from liver results in hepatocellular carcinoma upon CCl4 treatment. We now establish that STAT5, like in MEFs, activates expression of the Cdkn2b gene in liver tissue. Loss of STAT5 led to diminished p15INK4B and increased hepatocyte proliferation. Conclusion: This study for the first time demonstrates that cytokines, through STAT5, induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context-specific role of STAT5 as tumor suppressor. (HEPATOLOGY 2010;52:1808-1818)
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Essential Role for STAT5A/b in Myeloproliferative Neoplasms Induced by BCR-ABL1 and Jak2 V617F.
Blood, 2009Co-Authors: C Walz, Lothar Hennighausen, Katherine Lazarides, Nihal Patel, Virginia M Zaleskas, Richard A Van EttenAbstract:Abstract Abstract 312 The Signal Transducer and Activator of Transcription 5 (STAT5A/b) proteins are latent transcription factors that are constitutively activated in malignant cells from patients with a variety of hematologic malignancies, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but the role of STAT5A/b in the pathogenesis of these diseases has not been fully elucidated. While therapy with tyrosine kinase inhibitors like imatinib mesylate has revolutionized the treatment of CML, the emergence of clinical resistance to imatinib has rekindled interest in STAT5A/b as a promising therapeutic target downstream of dysregulated tyrosine kinases such as BCR-ABL1 and Jak2V617F. In this study, we used mice with a conditional (floxed, fl) null mutation in the STAT5A/b gene locus (Cui et al., Mol. Cell. Biol. 2004;24:8037) to determine the requirement for STAT5A/b in MPNs induced by BCR-ABL1 and Jak2V617F in retroviral bone marrow (BM) transduction/transplantation models of CML and PV, respectively. Two distinct strategies were used to express Cre recombinase in the mutant hematopoietic cells: retroviral expression of a GFP-Cre fusion protein, and interferon-inducible expression from an Mx-Cre transgene. In recipients of BCR-ABL1-transduced BM, loss of one STAT5A/b allele resulted in attenuation of CML-like MPN and the appearance of acute B-lymphoblastic leukemia (B-ALL). Complete deletion of the STAT5A/b gene resulted in the absence of any myeloid or lymphoid leukemia in recipients, with normal spleen weight and organ histopathology. However, these recipients had significant populations of GFP+ myeloid cells in peripheral blood and marrow with expression of BCR-ABL1 and phosphorylation of CrkL, indicative of engraftment with BCR-ABL1+ stem cells. Serial transplantation revealed that the BCR-ABL1-expressing STAT5A/b-deficient HSC can self-renew and progress to acute lymphoid leukemia in secondary recipients. These results demonstrate that STAT5A/b signaling is required for BCR-ABL1 to induce the massive expansion of myeloid progenitors that is characteristic of CML. However, our results also argue that inhibition of STAT5A/b signaling may be insufficient for eradication of leukemic stem cells in CML patients, or for preventing progression to blast crisis. To address the role of STAT5A/b in the pathophysiology of PV, we utilized a similar model of PV induced by retroviral transduction of Jak2V617F into mouse BM, followed by transplantation into recipient mice. Such recipients develop nonfatal polycythemia, reticulocytosis, and leukocytosis, and eventually progress to myelofibrosis (Zaleskas et al., PLoS One 2006;1:e18). Recipients of STAT5A/bfl/− donor transduced with Jak2V617F retrovirus that exhibited efficient Cre-mediated STAT5A/b deletion failed to develop polycythemia despite engraftment with transduced donor-derived stem cells and evidence of circulating GFP+ cells. Importantly, recipients of Jak2V617F-transduced STAT5A/bfl/− BM who engrafted with Jak2V617F+ cells had evidence of subclinical MPN, with infiltration of spleen and liver with myeloerythroid cells and substantial myelofibrosis in the BM. These results suggest that inhibition of STAT5A/b signaling in PV patients may normalize their red cell mass, but might be less effective at preventing progression to myelofibrosis. Together, these results demonstrate that STAT5A/b is necessary for the pathogenesis of CML-like disease induced by BCR-ABL1 and of polycythemia by Jak2V617F, and validate the STAT5A/b pathway as a target for therapy in MPNs associated with dysregulated TKs. However, targeting Stat5 alone may not eradicate CML stem cells or prevent Jak2V617F-induced myelofibrosis. Disclosures: No relevant conflicts of interest to declare.
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essential role for STAT5A b in myeloproliferative neoplasms induced by bcr abl1 and jak2 v617f in mice
Blood, 2009Co-Authors: C Walz, Lothar Hennighausen, Wesam Ahmed, Katherine Lazarides, Monica Betancur, Nihal Patel, Virginia M Zaleskas, Richard A Van EttenAbstract:STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the STAT5A/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2V617F in retroviral transplantation models of CML and PV. Loss of one STAT5A/b allele resulted in a decrease in BCR-ABL1–induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of STAT5A/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2V617F failed to induce polycythemia in recipients after deletion of STAT5A/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5A/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2V617F, and validate STAT5A/b and the genes they regulate as targets for therapy in these MPNs.
David J Waxman - One of the best experts on this subject based on the ideXlab platform.
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loss of sexually dimorphic liver gene expression upon hepatocyte specific deletion of STAT5A stat5b locus
Endocrinology, 2007Co-Authors: Minita G Holloway, Atsushi Hosui, Lothar Hennighausen, David J WaxmanAbstract:Hepatocyte-specific, albumin-Cre recombinase-mediated deletion of the entire mouse STAT5A-Stat5b locus was carried out to evaluate the role of signal transducer and activator of transcription 5a and 5b (STAT5Ab) in the sex-dependent transcriptional actions of GH in the liver. The resultant hepatocyte STAT5Ab-deficient mice were fertile, and unlike global STAT5b-deficient male mice, postnatal body weight gain was normal, despite a 50% decrease in serum IGF-I. Whole-liver STAT5Ab RNA decreased by approximately 65–85%, and residual STAT5 immunostaining was observed in a minority of the hepatocytes, indicating incomplete excision by Cre-recombinase. Quantitative PCR analysis of 20 sexually dimorphic, liver-expressed genes revealed significant down-regulation of 10 of 11 male-specific genes in livers of male hepatocyte STAT5Ab-deficient mice. Class I female-specific liver genes were markedly up-regulated (de-repressed), whereas the expression of class II female genes, belonging to the Cyp3a subfamily, was unaf...
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serine phosphorylation of gh activated signal transducer and activator of transcription 5a STAT5A and stat5b impact on stat5 transcriptional activity
Molecular Endocrinology, 2001Co-Authors: Soohee Park, Hiroko Yamashita, David J WaxmanAbstract:Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity...
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growth hormone but not prolactin maintains low level activation of STAT5A and stat5b in female rat liver
Endocrinology, 1999Co-Authors: Hee K Choi, David J WaxmanAbstract:STAT5b, a member of the signal transducers and activators of transcription family, is activated in rat liver in response to the intermittent (pulsatile) plasma pattern of GH that is characteristic of adult males. Previous studies have shown that the near-continuous plasma GH pattern of adult female rats is associated with a dramatic down-regulation of the STAT5 activation pathway. The present study demonstrates the presence of a low-level STAT5 DNA-binding activity in adult female rat liver and investigates the hormonal factors required for its maintenance. PRL is not responsible for this low-level STAT5 activity, as demonstrated in experiments involving estrus cycle monitoring (to investigate a possible role of the proestrus PRL surge), implantation of bromocriptine pellets (to eliminate PRL release by the pituitary), and direct injection of purified PRL. Rather, the low-level STAT5 activity is shown to result from chronic plasma GH stimulation, as demonstrated by GH infusion studies carried out in hypophysectomized rats. Furthermore, gel mobility supershift experiments demonstrate that the same STAT5-containing DNA-binding complexes are formed by both male and female adult rat liver extracts, albeit at approximately 10- to 20-fold lower levels by the female extracts. This DNA-binding activity is primarily comprised of STAT5b but also contains STAT5A, which is shown to be preferentially activated by the male plasma GH pattern in a manner similar to STAT5b. Thus, the dominance of activated STAT5b, compared with STAT5A, in the strong DNA-binding complexes formed in adult male rat liver nuclear extracts, is a reflection of the relative abundance in liver of the two STAT5 forms and is not attributable to an intrinsic, preferential activation of STAT5b by plasma GH pulses. The physiological significance of the low-level activated STAT5A and STAT5b seen in female rat liver and its effects on liver gene expression are uncertain but may involve the activation of femaleexpressed cytochromes P450 and other liver genes. (Endocrinology 140: 5126 ‐5135, 1999)
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distinctive roles of STAT5A and stat5b in sexual dimorphism of hepatic p450 gene expression impact of STAT5A gene disruption
Journal of Biological Chemistry, 1999Co-Authors: Soohee Park, Lothar Hennighausen, Helen W Davey, David J WaxmanAbstract:Abstract Stat5b gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239–7244). In the present study, disruption of the mouse STAT5A gene, whose coding sequence is ∼90% identical to the Stat5b gene, resulted in no loss of expression in male mice of several sex-dependent, GH-regulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5b feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16α-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6β-hydroxylase). Since STAT5A is thus nonessential for these male GH responses, STAT5b homodimers, but not STAT5A-STAT5b heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either STAT5A or Stat5bled to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. STAT5A or Stat5b gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5A, which is much less abundant in liver than STAT5b, and STAT5b are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.
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distinctive roles of STAT5A and stat5b in sexual dimorphism of hepatic p450 gene expression
1999Co-Authors: Soohee Park, Lothar Hennighausen, Helen W Davey, David J WaxmanAbstract:Stat5b gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239 ‐7244). In the present study, disruption of the mouse STAT5A gene, whose coding sequence is ;90% identical to the Stat5b gene, resulted in no loss of expression in male mice of several sex-dependent, GHregulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5b feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16a-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6b-hydroxylase). Since STAT5A is thus nonessential for these male GH responses, STAT5b homodimers, but not STAT5ASTAT5b heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either STAT5A or Stat5b led to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. STAT5A or Stat5b gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5A, which is much less abundant in liver than STAT5b, and STAT5b are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and genespecific effects that GH has on the liver.
Robert A Kirken - One of the best experts on this subject based on the ideXlab platform.
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specific inhibition of STAT5A b promotes apoptosis of il 2 responsive primary and tumor derived lymphoid cells
Journal of Immunology, 2003Co-Authors: Fariba Behbod, Zsuzsanna S Nagy, Stanislaw M Stepkowski, James G Karras, Charlene R Johnson, David W Jarvis, Robert A KirkenAbstract:STAT5A/b exhibits 96% homology and are required for normal immune function. The present studies examined STAT5A/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2′- O -methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in ≥55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of STAT5A (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for STAT5A was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of STAT5A/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from STAT5A/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with STAT5A/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate STAT5A and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.
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role of serine phosphorylation of STAT5A in prolactin stimulated β casein gene expression
Molecular and Cellular Endocrinology, 2001Co-Authors: Hiroko Yamashita, Lothar Hennighausen, Jun Xu, Rebecca A Erwin, Marja T. Nevalainen, Matthew J Lebaron, Kay Uwe Wagner, Jeffrey M Harmon, Robert A KirkenAbstract:Abstract Milk production remains suppressed in mammals during late pregnancy despite high levels of lactogenic polypeptide hormones. At parturition, associated with a precipitous fall in circulating progesterone, rising glucocorticoid levels synergize with prolactin to initiate copious milk production. This synergy is mediated at least in part through the coordinated activation of glucocorticoid receptors and transcription factor Stat5, particularly STAT5A. Here we show that two proline-juxtaposed serine residues within the transactivation domain of STAT5A are phosphorylated in the mammary gland during late gestation and lactation, and that these phosphorylation sites inhibit the transcriptional activity of STAT5A in the absence of glucocorticoid receptor costimulation. Specifically, transfection assays revealed that phosphorylation of residues S725 and S779 of STAT5A cooperatively suppressed prolactin-stimulated transcription from the β-casein promoter in both COS-7 kidney and MCF-7 mammary cells. This suppression was associated with shortened duration and reduced amplitude of nuclear DNA binding activity of wild type STAT5A relative to that of the serine phosphorylation-defective Stat5 mutant. However, costimulation of glucocorticoid receptors completely reversed the suppressive effect of STAT5A serine phosphorylation on β-casein gene transcription. We propose that serine phosphorylation within the transactivation domain may limit the activity of STAT5A in the absence of proper coactivation by glucocorticoid receptors.
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functional uncoupling of the janus kinase 3 stat5 pathway in malignant growth of human t cell leukemia virus type 1 transformed human t cells
Journal of Immunology, 2000Co-Authors: Rebecca A Erwin, Robert A Kirken, Lihua Wang, Yuling Wang, William L FarrarAbstract:Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors STAT5A and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/STAT5A/b signaling with AG-490 (50 μM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, STAT5A (Tyr 694 ), and Stat5b (Tyr 699 ); 2) serine phosphorylation of STAT5A (Ser 726 ) as determined by a novel phosphospecific Ab; and 3) STAT5A/b DNA binding to the Stat5-responsive β-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-κB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.
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differential control of the phosphorylation state of proline juxtaposed serine residues ser725 of STAT5A and ser730 of stat5b in prolactin sensitive cells
Journal of Biological Chemistry, 1998Co-Authors: Hiroko Yamashita, Jun Xu, Rebecca A Erwin, William L Farrar, Robert A KirkenAbstract:Abstract Transcription factors of the Stat family are controlled by protein kinases. Phosphorylation of a positionally conserved tyrosine residue is obligatory for Stat dimerization, nuclear translocation, and specific DNA binding. Studies of Stat1 and Stat3 have suggested that serine phosphorylation may also regulate function. We now identify serine residues located in a conserved PSP motif of STAT5A (Ser725) and Stat5b (Ser730) as major phosphorylation sites, using mutagenesis, phosphoamino acid analysis, and site-specific anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation control of this PSP motif differed between the highly homologous STAT5A and Stat5b proteins. Whereas Ser725 of STAT5A was constitutively phosphorylated both in COS-7 cells and Nb2 lymphocytes, phosphorylation of Ser730of Stat5b was markedly stimulated by prolactin. The data also suggested the existence of a second major serine phosphorylation site in STAT5A. Interestingly, constitutive phosphorylation of the PSP motif was suppressed by PD98059 but not by staurosporine under conditions in which both agents inhibited mitogen-activated protein kinases. Furthermore, pretreatment of cells with staurosporine, PD98059, H7, or wortmannin did not prevent either STAT5A or Stat5b from becoming maximally serine-phosphorylated after prolactin exposure. We propose that two pathways regulate Stat5 serine phosphorylation, one that is prolactin-activated and PD98059-resistant and one that is constitutively active and PD98059-sensitive and preferentially targets STAT5A. Finally, phosphorylation of the PSP motif of STAT5A or Stat5b was not essential for DNA binding or transcriptional activation of a β-casein reporter gene in COS-7 cells, suggesting that serine kinase control of Stat5 activity differs from that of Stat1 and Stat3.
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two discrete regions of interleukin 2 il2 receptor beta independently mediate il2 activation of a pd98059 rapamycin wortmannin insensitive STAT5A b serine kinase
Journal of Biological Chemistry, 1997Co-Authors: Robert A Kirken, L Hennighausen, Jun Xu, Rebecca A Erwin, Maria Grazia Malabarba, L Dasilva, William L FarrarAbstract:Abstract Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous STAT5A and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether STAT5A and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both STAT5A and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No STAT5A/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that STAT5A/b phosphotyrosine levels were maximized within 1–5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of STAT5A differed quantitatively and temporally from that of Stat5b with STAT5A serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor β (IL2Rβ) that could independently restore the ability of a truncated IL2Rβ mutant to mediate STAT5A/b phosphorylation and DNA binding to the γ-activated site of the β-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rβ region for Stat5 phosphorylation. Finally, we established that the IL2-activated STAT5A/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates STAT5A/b activation.
Gertraud W Robinson - One of the best experts on this subject based on the ideXlab platform.
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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, Gertraud W Robinson, Keunsoo Kang, 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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the transcription factors signal transducer and activator of transcription 5a STAT5A and stat5b negatively regulate cell proliferation through the activation of cyclin dependent kinase inhibitor 2b cdkn2b and cdkn1a expression
Hepatology, 2010Co-Authors: Ji Hoon Yu, Atsushi Hosui, Gertraud W Robinson, Mark Wickre, Gregory Riedlinger, Weiping Chen, Lothar HennighausenAbstract:Although the cytokine-inducible transcription factor signal transducer and activator of transcription 5 (STAT5) promotes proliferation of a wide range of cell types, there are cell-specific and context-specific cases in which loss of STAT5 results in enhanced cell proliferation. Here, we report that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15INK4B and p21CIP1. We further demonstrate that growth hormone, through the transcription factor STAT5, enhances expression of the Cdkn2b (cyclin-dependent kinase inhibitor 2B) gene and that STAT5A binds to interferon-gamma–activated sequence sites within the promoter. We recently demonstrated that ablation of STAT5 from liver results in hepatocellular carcinoma upon CCl4 treatment. We now establish that STAT5, like in MEFs, activates expression of the Cdkn2b gene in liver tissue. Loss of STAT5 led to diminished p15INK4B and increased hepatocyte proliferation. Conclusion: This study for the first time demonstrates that cytokines, through STAT5, induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context-specific role of STAT5 as tumor suppressor. (HEPATOLOGY 2010;52:1808-1818)
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gm csf controls proliferation and survival of the granulocyte lineage through the transcription factors STAT5A b
Blood, 2008Co-Authors: Akiko Kimura, Gertraud W Robinson, Weiping Chen, Michael A Rieger, James M Simmon, John J Oshea, Timm Schroeder, Lothar HennighausenAbstract:Neutrophils, one kind of granulocytes, are the most abundant type of white blood cells in human peripheral blood and form an integral part of the immune system. In addition, the majority of acute myelogeneous leukemia (AML) cells are from the granulocyte lineage. Granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) control migration, proliferation and survival of granulocytes. G-CSF and GM-CSF activate the transcription factors STAT5A/B (STAT5), which are essential for the development of T and B cells and the erythroid lineage. However, it is not clear to what extent G-CSF or GM-CSF signaling through STAT5 controls the differentiation, proliferation, survival in granulocyte lineage. STAT5 is not only essential for normal development and its constitutive activation has been linked to AML patients with Flt3 mutations. The objective of this study was to explore the contribution of STAT5 in G-CSF- and GM-CSF-induced granulopoiesis and to elucidate the underlying molecular mechanisms. Towards this goal, the STAT5A/b genes were deleted in mouse hematopoietic stem cells in vivo using Cre-loxP-mediated recombination (mutant mice). Injection of 5-FU resulted in a cytokine storm, which in controls, but not in mutant mice, led to a 10-fold elevation of neutrophils. Strikingly, the distribution of myeloid progenitor populations in bone marrow was not altered in STAT5-null animals in homeostasis. Colony assays were performed to address which cytokine controls granulopoiesis from these progenitors. While common multipotent progenitor cells (CMPs) and granulocyte macrophage progenitor cells (GMPs) from control mice formed large colonies in the presence of GM-CSF, mutant cells responded poorly. No difference between control and mutant colonies was observed in the presence of G-CSF. To investigate GM-CSF-mediated survival, apoptosis-assays were performed with peritoneal neutrophils. Greatly elevated apoptosis was observed with STAT5-null neutrophils. To further dissect the contribution of apoptosis and/or proliferation in the observed defects, long-term time-lapse imaging and single cell tracking was applied. Control and STAT5-null GMPs were cultured with GM-CSF and individual cells and all their progeny were continuously observed for 5 generations. Despite an equal number of initial GMPs responding to GM-CSF, the generation time of STAT5-null GMP-derived progeny was significantly prolonged in each generation and the number of cell death events increased dramatically from generation to generation. Therefore, GM-CSF-mediated STAT5 signaling is necessary to generate high numbers of granulocytic cells from GMPs by providing pro-survival and pro-proliferation signals. To identify GM-CSF-mediated and STAT5-dependent genetic cascades that control proliferation and survival of the granulocyte lineage, we performed gene expression profiling and ChIP-seq of control and STAT5-null CMPs, GMPs and neutrophils. STAT5 target genes specific to CMPs, GMPs and neutrophils were identified and their contribution to normal granulopoiesis is currently being investigated.
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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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the transcription factors STAT5A b are not required for islet development but modulate pancreatic β cell physiology upon aging
Biochimica et Biophysica Acta, 2007Co-Authors: Oksana Gavrilova, Gertraud W Robinson, Behrous Davani, Risu Na, Lothar HennighausenAbstract:Abstract In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors STAT5A and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of β-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in β-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic β-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed STAT5Alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate β-cell function.
Hiroko Yamashita - One of the best experts on this subject based on the ideXlab platform.
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serine phosphorylation of gh activated signal transducer and activator of transcription 5a STAT5A and stat5b impact on stat5 transcriptional activity
Molecular Endocrinology, 2001Co-Authors: Soohee Park, Hiroko Yamashita, David J WaxmanAbstract:Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity...
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role of serine phosphorylation of STAT5A in prolactin stimulated β casein gene expression
Molecular and Cellular Endocrinology, 2001Co-Authors: Hiroko Yamashita, Lothar Hennighausen, Jun Xu, Rebecca A Erwin, Marja T. Nevalainen, Matthew J Lebaron, Kay Uwe Wagner, Jeffrey M Harmon, Robert A KirkenAbstract:Abstract Milk production remains suppressed in mammals during late pregnancy despite high levels of lactogenic polypeptide hormones. At parturition, associated with a precipitous fall in circulating progesterone, rising glucocorticoid levels synergize with prolactin to initiate copious milk production. This synergy is mediated at least in part through the coordinated activation of glucocorticoid receptors and transcription factor Stat5, particularly STAT5A. Here we show that two proline-juxtaposed serine residues within the transactivation domain of STAT5A are phosphorylated in the mammary gland during late gestation and lactation, and that these phosphorylation sites inhibit the transcriptional activity of STAT5A in the absence of glucocorticoid receptor costimulation. Specifically, transfection assays revealed that phosphorylation of residues S725 and S779 of STAT5A cooperatively suppressed prolactin-stimulated transcription from the β-casein promoter in both COS-7 kidney and MCF-7 mammary cells. This suppression was associated with shortened duration and reduced amplitude of nuclear DNA binding activity of wild type STAT5A relative to that of the serine phosphorylation-defective Stat5 mutant. However, costimulation of glucocorticoid receptors completely reversed the suppressive effect of STAT5A serine phosphorylation on β-casein gene transcription. We propose that serine phosphorylation within the transactivation domain may limit the activity of STAT5A in the absence of proper coactivation by glucocorticoid receptors.
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epithelial defect in prostates of STAT5A null mice
Laboratory Investigation, 2000Co-Authors: Marja T. Nevalainen, Philip M Grimley, Hiroko Yamashita, Gertraud W Robinson, Tommi J Ahonen, Varadaraj Chandrashekar, Andrzej Bartke, Lothar HennighausenAbstract:The transcription factor STAT5A critically mediates prolactin (PRL)-induced mammary gland development and lactogenesis. PRL also stimulates growth and differentiation of prostate tissue. Specifically, hyperprolactinemia gives rise to prostate hyperplasia, and prostate size is reduced in PRL-deficient mice. We therefore investigated the importance of STAT5A for prostate development and function by examining STAT5A-null mice. The absence of STAT5A in mice was associated with a distinct prostate morphology characterized by an increased prevalence of local disorganization within acinar epithelium of ventral prostates. Affected acini were typically filled with desquamated, granular epithelial cells that had become embedded in dense, coagulated secretory material. These features were reminiscent of acinar cyst formation and degeneration frequently observed in human benign prostate hyperplasia, however, cystic changes in prostate acini of STAT5A-deficient mice were not associated with increased prostate size or morphologic hallmarks of epithelial hyperplasia. Instead, immunohistochemistry of the prostate-specific secretory marker, probasin, suggested that hypersecretory function of the epithelium could underlie local congestion and cyst formation in prostates of STAT5A-null mice. Serum testosterone and PRL levels were normal in STAT5A knockout mice, but prostate PRL receptor expression was reduced as determined by immunohistochemistry. Expression levels or activation states of other PRL signal transduction proteins, including Stat5b, Stat3, Stat1, ERK1, and ERK2 were not altered. The present study offers the first evidence for a direct role of STAT5A in the maintenance of normal tissue architecture and function of the mouse prostate.
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differential control of the phosphorylation state of proline juxtaposed serine residues ser725 of STAT5A and ser730 of stat5b in prolactin sensitive cells
Journal of Biological Chemistry, 1998Co-Authors: Hiroko Yamashita, Jun Xu, Rebecca A Erwin, William L Farrar, Robert A KirkenAbstract:Abstract Transcription factors of the Stat family are controlled by protein kinases. Phosphorylation of a positionally conserved tyrosine residue is obligatory for Stat dimerization, nuclear translocation, and specific DNA binding. Studies of Stat1 and Stat3 have suggested that serine phosphorylation may also regulate function. We now identify serine residues located in a conserved PSP motif of STAT5A (Ser725) and Stat5b (Ser730) as major phosphorylation sites, using mutagenesis, phosphoamino acid analysis, and site-specific anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation control of this PSP motif differed between the highly homologous STAT5A and Stat5b proteins. Whereas Ser725 of STAT5A was constitutively phosphorylated both in COS-7 cells and Nb2 lymphocytes, phosphorylation of Ser730of Stat5b was markedly stimulated by prolactin. The data also suggested the existence of a second major serine phosphorylation site in STAT5A. Interestingly, constitutive phosphorylation of the PSP motif was suppressed by PD98059 but not by staurosporine under conditions in which both agents inhibited mitogen-activated protein kinases. Furthermore, pretreatment of cells with staurosporine, PD98059, H7, or wortmannin did not prevent either STAT5A or Stat5b from becoming maximally serine-phosphorylated after prolactin exposure. We propose that two pathways regulate Stat5 serine phosphorylation, one that is prolactin-activated and PD98059-resistant and one that is constitutively active and PD98059-sensitive and preferentially targets STAT5A. Finally, phosphorylation of the PSP motif of STAT5A or Stat5b was not essential for DNA binding or transcriptional activation of a β-casein reporter gene in COS-7 cells, suggesting that serine kinase control of Stat5 activity differs from that of Stat1 and Stat3.
