ABSTRACT
Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.
Subject(s)
Connexin 43/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Peptide Fragments/metabolism , Psoriasis/immunology , Th17 Cells/immunology , Animals , Cell Line , Connexin 43/genetics , Connexin 43/immunology , Disease Models, Animal , Genetic Predisposition to Disease , Humans , Interleukin-17/metabolism , Mice , Mice, Knockout , Molecular Chaperones/genetics , Mutation/genetics , Peptide Fragments/genetics , Peptide Fragments/immunology , Polymorphism, Genetic , Protein Binding/genetics , Protein Binding/immunology , Psoriasis/genetics , Signal TransductionABSTRACT
Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.
Subject(s)
Hematopoiesis , STAT5 Transcription Factor , Mice , Animals , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Mice, Knockout , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Bone Marrow/metabolism , Mice, Transgenic , Stem Cell Niche/physiologyABSTRACT
Leucine carboxyl methyltransferase-1 (LCMT-1) methylates the C-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In this study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down â¼30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and usually death by embryonic day 16.5. Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit+Lin-Sca1+ cells. The percent cycling cells and mitotic indices of WT and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, noncompetitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multilineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis.
Subject(s)
Embryo, Mammalian/pathology , Fetus/pathology , Hematopoiesis , Liver/pathology , Protein O-Methyltransferase/physiology , Animals , Apoptosis , Cell Proliferation , DNA Methylation , Embryo, Mammalian/enzymology , Fetus/enzymology , Liver/enzymology , Mice , Mice, Knockout , Protein Phosphatase 2/metabolismABSTRACT
The difficulty in maintaining the reconstituting capabilities of hematopoietic stem cells (HSCs) in culture outside of the bone marrow microenvironment has severely limited their utilization for clinical therapy. This hurdle is largely due to the differentiation of long-term stem cells. Emerging evidence suggests that energy metabolism plays an important role in coordinating HSC self-renewal and differentiation. Here, we show that treatment with alexidine dihydrochloride, an antibiotic and a selective inhibitor of the mitochondrial phosphatase Ptpmt1, which is crucial for the differentiation of HSCs, reprogrammed cellular metabolism from mitochondrial aerobic metabolism to glycolysis, resulting in a remarkable preservation of long-term HSCs ex vivo in part through hyperactivation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In addition, inhibition of mitochondrial metabolism and activation of AMPK by metformin, a diabetes drug, also decreased differentiation and helped maintain stem cells in culture. Thus, manipulating metabolic pathways represents an effective new strategy for ex vivo maintenance of HSCs.
Subject(s)
Biguanides/pharmacology , Cellular Reprogramming/drug effects , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , PTEN Phosphohydrolase/antagonists & inhibitors , AMP-Activated Protein Kinases/metabolism , Aerobiosis/drug effects , Animals , Cell Differentiation/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Glycolysis/drug effects , Hematopoietic Stem Cells/cytology , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Mice , Oxygen Consumption/drug effectsABSTRACT
IL-25 is a member of the IL-17 family of cytokines that promotes Th2 cell-mediated inflammatory responses. IL-25 signals through a heterodimeric receptor (IL-25R) composed of IL-17RA and IL-17RB, which recruits the adaptor molecule Act1 for downstream signaling. Although the role of IL-25 in potentiating type 2 inflammation is well characterized by its ability to activate the epithelium as well as T cells, the components of its signaling cascade remain largely unknown. In this study, we found that IL-25 can directly activate STAT5 independently of Act1. Furthermore, conditional STAT5 deletion in T cells or epithelial cells led to a defective IL-25-initiated Th2 polarization as well as defective IL-25 enhancement of Th2 responses. Finally, we found that STAT5 is recruited to the IL-25R in a ligand-dependent manner through unique tyrosine residues on IL-17RB. Together, these findings reveal a novel Act1-independent IL-25 signaling pathway through STAT5 activation.
Subject(s)
Interleukins/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Animals , Cell Line , Connexin 43/metabolism , Cytokines/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Ligands , Mice , Mice, Transgenic , Models, Biological , Peptide Fragments/metabolism , Protein Binding , Receptors, Interleukin/metabolism , Receptors, Interleukin-17/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
DNA repair is essential for hematopoietic stem cell (HSC) maintenance. Ku70 is a key component of the nonhomologous end-joining pathway, which is the major pathway for DNA double-strand break repair. We find that HSCs from Ku70-deficient mice are severely defective in self-renewal, competitive repopulation, and bone marrow (BM) hematopoietic niche occupancy and that loss of quiescence results in a dramatic defect in the maintenance of Ku70-deficient HSCs. Interestingly, although overexpression of Bcl2 does not rescue the severe combined immunodeficiency phenotype in Ku70-deficient mice, overexpression of Bcl2 in Ku70-deficient HSCs almost completely rescued the impaired HSC quiescence, repopulation, and BM hematopoietic niche occupancy capacities. Together, our data indicate that the HSC maintenance defect of Ku70-deficient mice is due to the loss of HSC quiescent populations, whereas overexpression of Bcl2 rescues the HSC defect in Ku70-deficient mice by restoration of quiescence. Our study uncovers a novel role of Bcl2 in HSC quiescence regulation.
Subject(s)
Antigens, Nuclear/genetics , Cell Cycle Checkpoints/genetics , DNA-Binding Proteins/genetics , Genes, bcl-2/physiology , Hematopoietic Stem Cells/physiology , Animals , Antigens, Nuclear/metabolism , Bone Marrow Cells/physiology , Cell Proliferation , Cells, Cultured , DNA-Binding Proteins/metabolism , Immunophenotyping , Ku Autoantigen , Mice , Mice, Inbred C57BL , Mice, Knockout , Stem Cell Niche/genetics , Up-Regulation/geneticsABSTRACT
We previously demonstrated that TGF-ß1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high-affinity IgE receptor, FcεRI. The in vivo effects of TGF-ß1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-ß1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGF-ß1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGF-ß1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-ß1, because Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward stem cell factor, and that TGF-ß1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-ß1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. Furthermore, TGF-ß1 did not suppress IgE-induced cytokine release and did increase c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGF-ß1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-ß1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGF-ß1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.
Subject(s)
Immunoglobulin E/immunology , Mast Cells/metabolism , Receptors, IgE/immunology , STAT5 Transcription Factor/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Cell Movement/immunology , Cells, Cultured , Cytokines/metabolism , Humans , Immunoglobulin E/metabolism , Mast Cells/immunology , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-fyn/metabolism , Proto-Oncogene Proteins c-kit/metabolism , RNA Interference , RNA, Small Interfering , Receptors, IgE/biosynthesis , Receptors, IgE/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/immunology , Signal Transduction/immunology , Transforming Growth Factor beta1/immunology , Tumor Necrosis Factors/biosynthesisABSTRACT
BACKGROUND: Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations. METHODS: The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target--eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1). RESULTS: Assays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible. CONCLUSION: Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Immunoassay/methods , Leukemia, Myeloid, Acute/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Cell Cycle Proteins , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/pathology , PhosphorylationABSTRACT
Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.
Subject(s)
Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Repressor Proteins/deficiency , Trans-Activators/deficiency , Animals , Apoptosis/physiology , Blotting, Western , Flow Cytometry , Gene Expression Profiling , Hematopoietic Stem Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Repressor Proteins/genetics , Trans-Activators/geneticsABSTRACT
Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.
Subject(s)
Cell Transformation, Neoplastic/pathology , GRB2 Adaptor Protein/physiology , Mutation/genetics , Myeloproliferative Disorders/etiology , Myeloproliferative Disorders/prevention & control , Protein Tyrosine Phosphatase, Non-Receptor Type 11/physiology , Proto-Oncogene Proteins c-kit/genetics , Animals , Apoptosis , Blotting, Western , Bone Marrow Transplantation , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Immunoprecipitation , Integrases/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Myeloproliferative Disorders/mortality , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/metabolism , Signal Transduction/drug effects , Survival Rate , Tyrosine/metabolismABSTRACT
Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcεRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcεRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Bruton's tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcεRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.
Subject(s)
Cytokines/biosynthesis , Mast Cells/metabolism , Receptors, IgE/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , Cholesterol/genetics , Cholesterol/metabolism , Cytokines/genetics , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mast Cells/cytology , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Receptors, IgE/genetics , STAT5 Transcription Factor/genetics , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Leukemia/metabolism , Milk Proteins/chemistry , Milk Proteins/metabolism , Protein Structure, Quaternary , Trans-Activators/chemistry , Trans-Activators/metabolism , Animals , Biomarkers , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Bone Marrow Transplantation , Cell Lineage , Cell Transformation, Neoplastic , Cells, Cultured , DNA-Binding Proteins/genetics , Female , Genetic Complementation Test , Growth Substances/metabolism , Humans , Leukemia/genetics , Leukemia/pathology , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Knockout , Milk Proteins/genetics , Mutation , Nuclear Proteins , Oncogenes , STAT5 Transcription Factor , Spleen/metabolism , Spleen/pathology , Trans-Activators/genetics , Transfection , Tumor Suppressor ProteinsABSTRACT
The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.
Subject(s)
Acute-Phase Proteins/metabolism , Apoptosis/physiology , Blood Cells/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Lipocalins/metabolism , Oncogene Proteins/metabolism , Acute-Phase Proteins/genetics , Animals , Anti-Inflammatory Agents/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Dexamethasone/pharmacology , Hematopoiesis/drug effects , Lipocalin-2 , Lipocalins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Oncogene Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolismABSTRACT
Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5a(S711F)) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5ab(null/null) primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aDeltaN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aDeltaN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5ab(null/null) fetal liver cells and transplantation, persistently active STAT5a(S711F) lacking the N-domain (STAT5aDeltaN(S711F)) was insufficient to protect c-Kit(+)Lin(-)Sca-1(+) (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5a(S711F) caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aDeltaN(S711F) was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain-dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain-mediated regulation of bcl-2 family members.
Subject(s)
MicroRNAs/metabolism , Myeloproliferative Disorders/physiopathology , Proto-Oncogene Proteins c-bcl-2/genetics , STAT5 Transcription Factor/genetics , Animals , Cell Survival/physiology , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/physiology , Introns/physiology , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Morphinans/metabolism , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Protein Structure, Tertiary , RNA Processing, Post-Transcriptional/physiology , STAT5 Transcription Factor/chemistry , STAT5 Transcription Factor/metabolism , Signal Transduction/physiologyABSTRACT
Germline and somatic gain-of-function mutations in tyrosine phosphatase PTPN11 (SHP-2) are associated with juvenile myelomonocytic leukemia (JMML), a myeloproliferative disease (MPD) of early childhood. The mechanism by which PTPN11 mutations induce this disease is not fully understood. Signaling partners that mediate the pathogenic effects of PTPN11 mutations have not been explored. Here we report that germ line mutation Ptpn11(D61G) in mice aberrantly accelerates hematopoietic stem cell (HSC) cycling, increases the stem cell pool, and elevates short-term and long-term repopulating capabilities, leading to the development of MPD. MPD is reproduced in primary and secondary recipient mice transplanted with Ptpn11(D61G/+) whole bone marrow cells or purified Lineage(-)Sca-1(+)c-Kit(+) cells, but not lineage committed progenitors. The deleterious effects of Ptpn11(D61G) mutation on HSCs are attributable to enhancing cytokine/growth factor signaling. The aberrant HSC activities caused by Ptpn11(D61G) mutation are largely corrected by deletion of Gab2, a prominent interacting protein and target of Shp-2 in cell signaling. As a result, MPD phenotypes are markedly ameliorated in Ptpn11(D61G/+)/Gab2(-/-) double mutant mice. Collectively, our data suggest that oncogenic Ptpn11 induces MPD by aberrant activation of HSCs. This study also identifies Gab2 as an important mediator for the pathogenic effects of Ptpn11 mutations.
Subject(s)
Hematopoietic Stem Cells/pathology , Mutation , Myeloproliferative Disorders/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Bone Marrow Cells/cytology , Bone Marrow Cells/enzymology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/metabolism , Interleukin-3/immunology , Mice , Mice, Inbred C57BL , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Phosphoproteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolismABSTRACT
Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.
Subject(s)
Cell Transformation, Neoplastic , Hematopoiesis/physiology , Leukemia/pathology , STAT5 Transcription Factor/metabolism , Serine/metabolism , Tumor Suppressor Proteins/metabolism , Adult , Aged , Animals , Blotting, Western , Bone Marrow Transplantation , Cell Lineage , Cell Proliferation , Cells, Cultured , Female , Fetus , Flow Cytometry , Humans , Immunoenzyme Techniques , Leukemia/genetics , Leukemia/metabolism , Liver Transplantation , Male , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Middle Aged , Phosphorylation , Precursor Cells, B-Lymphoid/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/genetics , Serine/genetics , T-Lymphocytes/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1ß, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1ß receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1ß signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.
Subject(s)
Hematopoietic Stem Cells , Inflammation , Interleukin-1beta , Leukemia, Myelomonocytic, Juvenile , Animals , Bone Marrow/pathology , Hematopoietic Stem Cells/pathology , Humans , Interleukin-1beta/biosynthesis , Interleukin-1beta/metabolism , Leukemia, Myelomonocytic, Juvenile/immunology , Leukemia, Myelomonocytic, Juvenile/metabolism , Leukemia, Myelomonocytic, Juvenile/pathology , Mice , Myeloproliferative Disorders/immunology , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Receptors, Interleukin-1/deficiency , Tumor MicroenvironmentABSTRACT
MLLT11, an MLL fusion partner, is a poor prognostic biomarker for paediatric acute myeloid leukaemia (AML), adult normal cytogenetics AML, and adult myelodysplastic syndrome. MLLT11 is highly regulated during haematopoietic progenitor differentiation and development but its regulatory mechanisms have not been defined. In this study, we demonstrate by transfection experiments that MIR29B directly regulates MLLT11 expression in vitro. MIR29B expression level was also inversely related to MLLT11 expression in a cohort of 56 AML patients (P<0·05). AML patients with low MIR29B/elevated MLLT11 expression had poor overall survival (P=0·038). Therefore, MIR29B may be a potential prognostic biomarker for AML patients.
Subject(s)
Biomarkers, Tumor/physiology , Leukemia, Myeloid, Acute/metabolism , MicroRNAs/physiology , Neoplasm Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Biomarkers, Tumor/biosynthesis , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/genetics , Humans , Leukemia, Myeloid, Acute/genetics , MicroRNAs/biosynthesis , MicroRNAs/genetics , Neoplasm Proteins/genetics , Prognosis , Proto-Oncogene Proteins/genetics , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Survival Analysis , Tumor Cells, CulturedABSTRACT
Currently, there is a major need in hematopoietic stem cell (HSC) transplantation to develop reduced-intensity regimens that do not cause DNA damage and associated toxicities and that allow a wider range of patients to receive therapy. Cytokine receptor signals through c-Kit and c-Mpl can modulate HSC quiescence and engraftment, but the intracellular signals and transcription factors that mediate these effects during transplantation have not been defined. Here we show that loss of one allele of signal transducer and activator of transcription 5 (STAT5) in nonablated adult mutant mice permitted engraftment with wild-type HSC. Conditional deletion of STAT5 using Mx1-Cre caused maximal reduction in STAT5 mRNA (> 97%) and rapidly decreased quiescence-associated c-Mpl downstream targets (Tie-2, p57), increased HSC cycling, and gradually reduced survival and depleted the long-term HSC pool. Host deletion of STAT5 was persistent and permitted efficient donor long-term HSC engraftment in primary and secondary hosts in the absence of ablative conditioning. Overall, these studies establish proof of principle for targeting of STAT5 as novel transplantation conditioning and demonstrate, for the first time, that STAT5, a mitogenic factor in most cell types, including hematopoietic progenitors, is a key transcriptional regulator that maintains quiescence of HSC during steady-state hematopoiesis.
Subject(s)
Adult Stem Cells/physiology , Cell Proliferation , Gene Deletion , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/physiology , STAT5 Transcription Factor/genetics , Adaptor Proteins, Signal Transducing , Adult Stem Cells/metabolism , Animals , Gene Dosage/physiology , Hematopoiesis/genetics , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cells/metabolism , Immune Tolerance/genetics , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphoproteins/genetics , Transplantation ConditioningABSTRACT
Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.