A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis

Homozygous mutant (Shp-2Delta46-110) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2Delta46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory factor (LIF)-stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2Delta46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and hemangioblasts was lower in Shp-2Delta46-110 cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2Delta46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Delta46-110 ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.     

The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.     

Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals

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.     

SHP-2 phosphatase is required for hematopoietic cell transformation by Bcr-Abl

SHP-2 phosphatase forms a stable protein complex with and is heavily tyrosine-phosphorylated by the oncogenic tyrosine kinase Bcr-Abl. However, the role of SHP-2 in Bcr-Abl-mediated leukemogenesis is unclear. In the present report, we provide evidence that SHP-2 is required for hematopoietic cell transformation by Bcr-Abl. In vitro biological effects of Bcr-Abl transduction were diminished in SHP-2Delta/Delta hematopoietic cells, and the leukemic potential of Bcr-Abl-transduced SHP-2Delta/Delta cells in recipient animals was compromised. Further analyses showed that Bcr-Abl protein (p210) was degraded, and its oncogenic signaling was greatly decreased in SHP-2Delta/Delta cells. Treatment with proteasome inhibitors or reintroduction of SHP-2 restored p210 level in Bcr-Abl-transduced SHP-2Delta/Delta cells. Subsequent investigation revealed that SHP-2 interacted with heat shock protein 90, an important chaperone protein protecting p210 from proteasome-mediated degradation. The role of SHP-2 in the stability of p210 is independent of its catalytic activity. Blockade of SHP-2 expression in p210-expressing cells by antisense or small-interfering RNA approaches decreased p210 level, causing cell death. Inhibition of SHP-2 enzymatic activity by overexpression of catalytically inactive SHP-2 mutant did not destabilize p210 but enhanced serum starvation-induced apoptosis, suggesting that SHP-2 also plays an important role in downstream signaling of p210 kinase. These studies identified a novel function of SHP-2 and suggest that SHP-2 might be a useful target for controlling Bcr-Abl-positive leukemias.     

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal

OBJECTIVE: Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. METHODS: Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. RESULTS: Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. CONCLUSION: These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.     

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.     

Cloning and characterization of fetal liver phosphatase 1, a nuclear protein tyrosine phosphatase isolated from hematopoietic stem cells

We report the isolation of cDNAs encoding protein tyrosine phosphatases (PTPs) from highly purified hematopoietic stem cell populations. One such cDNA encodes a novel PTP, designated fetal liver phosphatase 1 (FLP1), which consists of one PTP domain followed by a carboxy terminal domain of 160 amino acids. Northern blot and in situ hybridization analysis showed that expression of FLP1 mRNA is restricted to thymus in 15.5-day-old and 17.5-day-old mouse embryos and to kidney and hematopoietic tissues in adult mice. Furthermore, polymerase chain reaction-based analysis shows that FLP1 is expressed in hematopoietic stem cells as well as in more mature hematopoietic cells. Peptide antisera against FLP1 immunoprecipitated a 48-kD protein that is localized in the nuclei of Ba/F3 lymphoid cells. We have analyzed the effects of overexpressing either wild-type FLP1 or a functionally inactive mutant of FLP1 in hematopoietic cells. In the progenitor K562 cell line, cells ectopically expressing functional FLP1 differentiated normally to megakaryocytes after induction with tetradecanoyl phorbol acetate (TPA). In contrast, when K562 transfectants expressing an inactive mutant FLP1 protein were treated with TPA, the characteristic cell spreading and substrate adhesion that accompany megakaryocytic differentiation did not occur. We show that, in these cells, the induction of the differentiation marker alphaIIb beta3 is not affected. However, both constitutive and TPA-induced expression of alpha2 integrin, a late megakaryocytic marker, are inhibited. These results suggest that the expression of an inactive form of FLP1 affects late signaling events of K562 megakaryocytic differentiation.     

蛋白酪氨酸磷酸酶-1B与2型糖尿病

研究证实,蛋白酪氨酸磷酸酶-1B(PTP-1B)主要通过以下几方面参与2型糖尿病的发病(1)可与胰岛素受体及其底物相作用,减弱胰岛素信号转导,引起胰岛素抵抗。(2)参与对胰岛β细胞数量的调节。(3)与瘦素抵抗及脂代谢异常关系密切,由此引发并加重2型糖尿病。目前已合成各种类型的PTP-1B抑制剂,有良好的降糖等疗效,临床应用前景广阔。     

Requirement of Gab2 for mast cell development and KitL/c-Kit signaling

Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BMMCs) from the Gab2-deficient mice grew poorly in response to KitL. KitL-induced ERK MAP kinase and Akt activation were impaired in Gab2-deficient BMMCs. These data indicate that Gab2 is required for mast cell development and KitL/c-Kit signaling.     

Protein tyrosine phosphatase RQ is a phosphatidylinositol phosphatase that can regulate cell survival and proliferation

Protein tyrosine phosphatase RQ (PTPRQ) was initially identified as a protein tyrosine phosphatase (PTPase)-like protein that is upregulated in a model of renal injury. Here we present evidence that, like PTEN, the biologically important enzymatic activity of PTPRQ is as a phosphatidylinositol phosphatase (PIPase). The PIPase specificity of PTPRQ is broader than that of PTEN and depends on different amino acid residues in the catalytic domain. In vitro, the recombinant catalytic domain of PTPRQ has low PTPase activity against tyrosine-phosphorylated peptide and protein substrates but can dephosphorylate a broad range of phosphatidylinositol phosphates, including phosphatidylinositol 3,4,5-trisphosphate and most phosphatidylinositol monophosphates and diphosphates. Phosphate can be hydrolyzed from the D3 and D5 positions in the inositol ring. PTPRQ does not have either of the basic amino acids in the catalytic domain that are important for the PIPase activity of PTEN or the sequence motifs that are characteristic of type II phosphatidylinositol 5-phosphatases. Instead, the PIPase activity depends on the WPE sequence present in the catalytic cleft of PTPRQ, and in the "inactive" D2 domains of many dual-domain PTPases, in place of the WPD motif present in standard active PTPases. Overexpression of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. An E2171D mutation that retains or increases PTPase activity but eliminates PIPase activity, eliminates the inhibitory effects on proliferation and apoptosis. These results indicate that PTPRQ represents a subtype of the PTPases whose biological activities result from its PIPase activity rather than its PTPase activity.     

The tyrosine phosphatase SHP-1 dampens murine Th17 development

Th17 cells represent a subset of CD4+ T helper cells that secrete the proinflammatory cytokine IL-17. Th17 cells have been ascribed both a beneficial role in promoting clearance of pathogenic fungi and bacteria, and a pathogenic role in autoimmune diseases. Here we identify the tyrosine phosphatase SHP-1 as a critical regulator of Th17 development, using 3 complementary approaches. Impaired SHP-1 activity through genetic deletion of SHP-1, transgenic expression of an inducible dominant negative SHP-1, or pharmacologic inhibition of SHP-1 strongly promotes the development of Th17. Ex vivo Th17 skewing assays demonstrate that genetic or pharmacologic disruption of SHP-1 activity in T cells results in a hyper-response to stimulation via IL-6 and IL-21, 2 cytokines that promote Th17 development. Mechanistically, we find that SHP-1 decreases the overall cytokine-induced phosphorylation of STAT3 in primary CD4+ T cells. These data identify SHP-1 as a key modifier of IL-6-and IL-21-driven Th17 development via regulation of STAT3 signaling and suggest SHP-1 as a potential new therapeutic target for manipulating Th17 differentiation in vivo.     

Allogeneic hematopoietic cell transplantation in patients with myelodysplastic syndrome and concurrent lymphoid malignancy

Allogeneic hematopoietic cell transplantation (HCT) can be curative for both myelodysplastic syndromes (MDS) and lymphoid malignancies. Little is known about the efficacy of allogeneic HCT in patients in whom both myeloid and lymphoid disorders are present at the time of HCT. We analyzed the outcomes in 21 patients with MDS and concurrent lymphoid malignancy when undergoing allogeneic HCT. A total of 17 patients had previously received extensive cytotoxic chemotherapy, including autologous HCT in 7, for non-Hodgkin lymphoma (NHL, n=7), Hodgkin lymphoma (HL, n=2), CLL (n=5), NHL plus HL (n=1), multiple myeloma (n=1) or T-cell ALL (n=1), and had presumably developed MDS as a consequence of therapy. Four previously untreated patients had CLL. A total of 19 patients were conditioned with high-dose (n=14) or reduced-intensity regimens (n=5), and were transplanted from HLA-matched or one Ag/allele mismatched related (n=10) or unrelated (n=9) donors; two patients received HLA-haploidentical related transplants, following a modified conditioning regimen. Currently, 2 of 4 previously untreated and 2 of 17 previously treated patients are surviving in remission of both MDS and lymphoid malignancies. However, the high non-relapse mortality among previously treated patients, even with reduced-intensity conditioning regimens, indicates that new transplant strategies need to be developed.     

Polypeptides controlling hematopoietic cell development and activation

Summary Recombinant DNA technology has been central in answering some of the most relevant questions in the research of regulation of the functional status of hematopoietic progenitor cells and their progeny. This leading article will focuse on recent results that have emerged from studies utilizing recombinant molecules that control hematopoietic blood cell development and activation. The following features will be detailed: The molecular and biological characteristics and biochemistry of hematopoietic growth factors, synergizing factors and releasing factors, their role in the regulation of hematopoiesis and activation of normal and leukemic cells, their cellular sources, and regulation of production.     

Requirement of c-myb in T cell development and in mature T cell function

Previous reports have suggested that the protooncogene c-myb participates in T cell development in the thymus and mature T cell proliferation. We have generated two T cell-specific c-myb knockout mouse models, myb/LckCre and myb/CD4Cre. We have demonstrated that c-myb is required for the development of thymocytes at the DN3 stage, for survival and proliferation of double-positive thymocytes, for differentiation of single-positive CD4 and CD8 T cells, and for the proliferative responses of mature T cells. In addition, our data show that c-myb is directly involved in the formation of double-positive CD4+CD8+CD25+, CD4+CD25+, and CD8+CD25+ T cells, developmental processes that may imply a role for c-myb in autoimmune dysfunction.     

Erythropoietin-induced tyrosine phosphorylations in a high erythropoietin receptor-expressing lymphoid cell line.

Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross-linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.     

Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease

Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2(PTPN2) with the onset of inflammatory bowel disease(IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation.