RESUMO
Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony-stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated (T) factors (T-granulocyte-macrophage-CSF (T-GM-CSF)) and T-interleukin-3 (T-IL-3)) on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and -IL-3 had enhanced receptor binding, but decreased CSF activity, compared with their FL forms. Importantly, T-GM-CSF and -IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and -IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster-forming cells from patients with acute myeloid leukemia regardless of cytogenetic or molecular alterations and in vivo using animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared with their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Activating mutations, such as E76K and D61Y, in PTPN11 (SHP2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, are associated with 35% of patients with juvenile myelomonocytic leukemia (JMML), an aggressive childhood myeloproliferative neoplasm (MPN). Here we show that the interaction between leukemia-associated mutant Shp2 and Gab2, a scaffolding protein important for cytokine-induced PI3K/Akt signaling, was enhanced, and that the mTOR pathway was elevated in Ptpn11E76K/+ leukemic cells. Importantly, MPN induced by the Ptpn11E76K/+ mutation was markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice-overproduction of myeloid cells was alleviated, splenomegaly was diminished and myeloid cell infiltration in nonhematopoietic organs was decreased in these double mutants. Excessive myeloid differentiation of stem cells was also normalized by depletion of Gab2. Acute leukemia progression of MPN was reduced in the double mutant mice and, as such, their survival was much prolonged. Furthermore, treatment of Ptpn11E76K/+ mice with Rapamycin, a specific and potent mTOR inhibitor, mitigated MPN phenotypes. Collectively, this study reveals an important role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic signaling of the PTPN11 gain-of-function mutations and a therapeutic potential of Rapamycin for PTPN11 mutation-associated JMML.
Assuntos
Mutação , Transtornos Mieloproliferativos/etiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antibióticos Antineoplásicos/farmacologia , Camundongos , Camundongos Knockout , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/genética , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genéticaRESUMO
Shp-1 and Shp-2 are cytoplasmic phosphotyrosine phosphatases with similar structures. Mice deficient in Shp-2 die at midgestation with defects in mesodermal patterning, and a hypomorphic mutation at the Shp-1 locus results in the moth-eaten viable (me(v)) phenotype. Previously, a critical role of Shp-2 in mediating erythroid/myeloid cell development was demonstrated. By using the RAG-2-deficient blastocyst complementation, the role of Shp-2 in lymphopoiesis has been determined. Chimeric mice generated by injecting Shp-2(-/-) embryonic stem cells into Rag-2-deficient blastocysts had no detectable mature T and B cells, serum immunoglobulin M, or even Thy-1(+) and B220(+) precursor lymphocytes. Collectively, these results suggest a positive role of Shp-2 in the development of all blood cell lineages, in contrast to the negative effect of Shp-1 in this process. To determine whether Shp-1 and Shp-2 interact in hematopoiesis, Shp-2(-/-):me(v)/me(v) double-mutant embryos were generated and the hematopoietic cell development in the yolk sacs was examined. More hematopoietic stem/progenitor cells were detected in Shp-2(-/-):me(v)/me(v) embryos than in Shp-2(-/-) littermates. The partial rescue by Shp-1 deficiency of the defective hematopoiesis caused by the Shp-2 mutation suggests that Shp-1 and Shp-2 have antagonistic effects in hematopoiesis, possibly through a bidirectional modulation of the same signaling pathway(s).
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/enzimologia , Síndromes de Imunodeficiência/genética , Linfócitos/enzimologia , Animais , Linfócitos B/citologia , Linhagem da Célula , Quimera , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Feminino , Teste de Complementação Genética , Genótipo , Hematopoese/genética , Linfócitos/citologia , Camundongos , Camundongos Knockout , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Linfócitos T/citologia , Saco Vitelino/citologiaRESUMO
Cellular biological activities are tightly controlled by intracellular signaling processes initiated by extracellular signals. Protein tyrosine phosphatases, which remove phosphate groups from phosphorylated signaling molecules, play equally important tyrosine roles as protein tyrosine kinases in signal transduction. SHP-2, a cytoplasmic SH2 domain containing protein tyrosine phosphatase, is involved in the signaling pathways of a variety of growth factors and cytokines. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus, and is a critical intracellular regulator in mediating cell proliferation and differentiation.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Animais , Células-Tronco Hematopoéticas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2RESUMO
Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Quimiocinas CXC/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Células da Medula Óssea/citologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12 , Quimiotaxia/efeitos dos fármacos , Primers do DNA/genética , Ativação Enzimática/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/deficiência , Receptores de Quimiocinas/metabolismoRESUMO
By using both genetic and biochemical approaches, we have investigated the physiological role of Shp-2, a cytoplasmic tyrosine phosphatase with two Src homology 2 domains, in signaling pathways downstream of epidermal growth factor receptor (EGF-R). In previous studies, a targeted deletion mutation in the SH2-N domain of Shp-2 was introduced into the murine Shp-2 locus, which resulted in embryonic lethality of homozygous mutant (Shp-2(-/-)) mice at midgestation. By aggregating Shp-2(-/-) embryonic stem cells with wild-type embryos, we created Shp-2(-/-)/wild-type chimeric animals. Most chimeras had open eyelids at birth and abnormal skin development, a phenotype characteristic of mice with mutations in EGF-R signaling components. In genetic crosses, a heterozygous Shp-2 mutation dominantly enhanced the phenotype of a weak mutant allele of EGF-R (wa-2), resulting in distinctive growth retardation, developmental defects in the skin, lung, and intestine, and perinatal mortality that are reminiscent of EGF-R knockout mice. Biochemical analysis revealed that signal propagation proximal to the EGF-R upon EGF stimulation was significantly attenuated in wa-2 fibroblast cells, which was exacerbated by the additional Shp-2 mutation. Thus, we provide biological evidence here that protein-tyrosine phosphatase Shp-2 acts to enhance information flow from the EGF-R in mouse growth and development.
Assuntos
Receptores ErbB/genética , Proteínas Quinases Ativadas por Mitógeno , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais/genética , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Quimera/genética , Cruzamentos Genéticos , Desenvolvimento Embrionário e Fetal/genética , Ativação Enzimática , Receptores ErbB/metabolismo , Heterozigoto , Histocitoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno , Mutação , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Domínios de Homologia de src/genéticaRESUMO
Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2(-/-)) ES cells and wild-type (WT) embryos, we created Shp-2(-/-)-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2(-/-) progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2(-/-) yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.
Assuntos
Hematopoese , Proteínas Tirosina Fosfatases/fisiologia , Animais , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/embriologia , Camundongos , Camundongos Knockout , Mutação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Saco VitelinoRESUMO
Shp-2 is a ubiquitously expressed tyrosine phosphatase with two SH2 domains. Homozygous mutant mice with a targeted deletion of 65 amino acid residues in the N-terminal SH2 domain of Shp-2 die in utero at mid-gestation, with multiple defects in mesodermal patterning. To surpass the embryonic lethality in dissecting the Shp-2 function in cell growth and differentiation, we established homozygous Shp-2 mutant embryonic stem (ES) cell lines. Our previous data showed a severe suppression of hematopoietic cell differentiation from Shp-2 mutant ES cells. Here we demonstrate that development of cardiac muscle cells was dramatically delayed and impaired in embryoid bodies (EBs) of Shp-2 mutant origin. Shp-2 mutant ES cells failed to differentiate into epithelial and fibroblast cells in vitro. However, higher efficiency of secondary EB formation was observed from the mutant than the wild-type ES cells. Further, mutant ES cells were more sensitive than wild-type cells to the differentiation suppressing effect of leukemia inhibitory factor (LIF). In addition, mutant ES cells showed a reduced growth rate compared to wild-type cells. These results suggest that the Shp-2 tyrosine phosphatase is a positive regulator for both cell differentiation and proliferation, in contrast to the Src-family kinases which promote cell growth but block differentiation.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Interleucina-6 , Proteínas Tirosina Fosfatases/fisiologia , Domínios de Homologia de src/fisiologia , Animais , Linhagem Celular , Linhagem da Célula , Células Epiteliais/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fator Inibidor de Leucemia , Linfocinas/metabolismo , Linfocinas/farmacologia , Camundongos , Proteína MyoD/genética , Miocárdio/citologia , Miogenina/genética , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de SinaisRESUMO
The SH2-containing tyrosine phosphatase Shp-2 appears to function downstream of a variety of growth factor receptors and might play a positive role in cell proliferation. Here we report that expression of the beta subunit of platelet-derived growth factor receptor (PDGFR-beta) was specifically downregulated in mutant fibroblasts lacking a functional Shp-2, while the levels of PDGFR-alpha EGFR and IGFIR were not changed. PDGF-stimulated DNA synthesis and extracellular signal regulated kinase (Erk) activation was severely suppressed in mutant cells. RasGAP, that responds to activation of PDGFR-beta but not PDGFR-alpha, was not phosphorylated on tyrosine in mutant cells upon PDGF-treatment. Northern blot analysis failed to detect PDGFR-beta mRNA in mutant cells. The transcription initiation from the PDGFR-beta gene promoter was not significantly changed, but the half-life of its mRNA was shortened in Shp-2 mutant cells. These observations indicate that Shp-2 not only participates in transmission of signals from growth factor receptors but also plays a specific role in the control of the PDGFR-beta expression. We propose that this is an important mechanism for the positive control of cell proliferation by Shp-2.
Assuntos
Regulação para Baixo , Proteínas Tirosina Fosfatases/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Domínios de Homologia de src , Animais , Becaplermina , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Mitógenos/metabolismo , Mitógenos/farmacologia , Mutagênese , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de SinaisRESUMO
Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains, is believed to participate in signal relay downstream of growth factor receptors. We show here that this phosphatase also plays an important role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblast cells lacking a functional Shp-2 were impaired in their ability to spread and migrate on fibronectin compared with wild-type cells. Furthermore, Shp-2 mutant cells displayed an increased number of focal adhesions and condensed F-actin aggregation at the cell periphery, properties reminiscent of focal adhesion kinase (FAK)-deficient cells. This is consistent with our previous observations in vivo that mice homozygous for the Shp-2 mutation died at midgestation with similar phenotype to FAK and fibronectin-deficient embryos, having severe defects in mesodermal patterning, particularly the truncation of posterior structures. Biochemical analysis demonstrated that FAK dephosphorylation was significantly reduced in Shp-2 mutant cells in suspension. Furthermore, regulated association of Src SH2 domain with FAK and paxillin during cell attachment and detachment on fibronectin was disrupted in Shp-2 mutant cells. This report defines a unique role of the Shp-2 tyrosine phosphatase in cell motility, which might guide the design of a new strategy for pharmaceutical interference of tumor metastasis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Movimento Celular , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/citologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Heterozigoto , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Paxilina , Fenótipo , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2RESUMO
Shp-1 and Shp-2 are cytoplasmic protein tyrosine phosphatases that contain two Src homology 2 (SH2) domains. A negative regulatory role of Shp-1 in hematopoiesis has been strongly implicated by the phenotype of motheaten mice with a mutation in the Shp-1 locus, which is characterized by leukocyte hypersensitivity, deregulated mast cell function, and excessive erythropoiesis. A targeted deletion of 65 amino acids in the N-terminal SH2 (SH2-N) domain of Shp-2 leads to an embryonic lethality at midgestation in homozygous mutant mice. To further dissect the Shp-2 function in hematopoietic development, we have isolated homozygous Shp-2 mutant embryonic stem (ES) cells. Significantly reduced hematopoietic activity was observed when the mutant ES cells were allowed to differentiate into embryoid bodies (EBs), compared to the wild-type and heterozygous ES cells. Further analysis of ES cell differentiation in vitro showed that mutation in the Shp-2 locus severely suppressed the development of primitive and definitive erythroid progenitors and completely blocked the production of progenitor cells for granulocytes-macrophages and mast cells. Reverse transcriptase PCR analysis of the mutant EBs revealed reduced expression of several specific marker genes that are induced during blood cell differentiation. Stem cell factor induction of mitogen-activated protein kinase activity was also blocked in Shp-2 mutant cells. Taken together, these results indicate that Shp-2 is an essential component and primarily plays a positive role in signaling pathways that mediate hematopoiesis in mammals. Furthermore, stimulation of its catalytic activity is not sufficient, while interaction via the SH2 domains with the targets or regulators is necessary for its biological functions in cells. The in vitro ES cell differentiation assay can be used as a biological tool in dissecting cytoplasmic signaling pathways.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas Tirosina Fosfatases/genética , Domínios de Homologia de src , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Mutação Puntual , Reação em Cadeia da Polimerase , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Deleção de SequênciaRESUMO
Hypotensive and other ocular effects were studied for 24 hr after topical application of prostaglandin F2 alpha as the tromethamine salt (PGF2 alpha) in 45 normotensive human subjects. After baseline intraocular pressure (IOP) measurements, 62.5 micrograms, 125 micrograms and 250 micrograms of PGF2 alpha dissolved in 50 microliter of saline was applied to one eye of 15 subjects for each dose tested. Contralateral control eyes received 50 microliter of saline. As compared with the IOP of the contralateral control eyes, topical application of 62.5 micrograms PGF2 alpha caused a significant IOP reduction at 1-12 hr, with a maximal IOP reduction of 2.2 mm Hg at 2 hr. Treatment with 125 micrograms of PGF2 alpha lowered IOP significantly at 1-21 hr, with a maximal reduction of 3.1 mm Hg at 9 hr. Administration of 250 micrograms PGF2 alpha produced a significant reduction of IOP, which lasted for at least 24 hr. A maximal IOP reduction of 2.9 mm Hg occurred at 7 hr. Pupillary diameter was not altered. Aqueous flare and anterior chamber cellular response were not seen in any of the eyes of the subjects at any time after topical application of 62.5-250 micrograms PGF2 alpha. The drug caused side effects consisting of reddened skin of lower lid, ocular irritation, conjunctival hyperemia and headache.