Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.
(1/338)
We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals. (+info)
Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia.
(2/338)
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder expressed in infancy and characterized by isolated thrombocytopenia and megakaryocytopenia with no physical anomalies. Our previous hematological analysis indicated similarities between human CAMT and murine c-mpl (thrombopoietin receptor) deficiency. Because the c-mpl gene was considered as one of the candidate genes for this disorder, we analyzed the genomic sequence of the c-mpl gene of a 10-year-old Japanese girl with CAMT. We detected two heterozygous point mutations: a C-to-T transition at the cDNA nucleotide position 556 (Q186X) in exon 4 and a single nucleotide deletion of thymine at position 1,499 (1,499 delT) in exon 10. Both mutations were predicted to result in a prematurely terminated c-Mpl protein, which, if translated, lacks all intracellular domains essential for signal transduction. Each of the mutations was segregated from the patient's parents. Accordingly, the patient was a compound heterozygote for two mutations of the c-mpl gene, each derived from one of the parents. The present study suggests that at least a certain type of CAMT is caused by the c-mpl mutation, which disrupts the function of thrombopoietin receptor. (+info)
Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism.
(3/338)
In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis. (+info)
Apoptosis of erythroid precursors under stimulation with thrombopoietin: contribution to megakaryocytic lineage choice.
(4/338)
Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34+ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34-/CD41- cells showed the labeling pattern of anti-CD42b and anti-CD9 antibodies closer to that of the CD34+/CD41- cells than the CD34+/CD41+ cells. Replating experiments revealed that approximately 40% of the CD34-/CD41- cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34+/CD41+ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34-/CD41- cells may result from the apparently low expression of c-MpI, as determined by flow cytometric analysis and reverse transcription-polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO. (+info)
High-level expression of Mpl in platelets and megakaryocytes is independent of thrombopoietin.
(5/338)
Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels of c-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism. (+info)
Consequences of GATA-1 deficiency in megakaryocytes and platelets.
(6/338)
In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1-deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIbalpha, GPIbbeta, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1-deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation. (+info)
Deletion of the extracellular membrane-distal cytokine receptor homology module of Mpl results in constitutive cell growth and loss of thrombopoietin binding.
(7/338)
The thrombopoietin receptor, Mpl, is a member of the cytokine receptor superfamily. The extracellular domain of Mpl contains two copies of the cytokine receptor homology module (CRM). Mpl is encoded by c-mpl, the cellular homologue of the oncogene v-mpl. The oncogenic potential of v-mpl may arise from deletion of all but the 43 most membrane-proximal amino acids of the extracellular domain of the wild-type receptor. To test the hypothesis that the extracellular domain of Mpl plays a role in controlling receptor activity, we created mutants of murine Mpl in which the membrane-distal CRM was either deleted or replaced by the membrane-proximal CRM. Introduction of these mutant receptors into factor-dependent BaF3 cells led to constitutive cell growth in the absence of growth factor. Both mutant receptors failed to bind 125I-Tpo. These results suggest that the membrane-distal CRM of Mpl acts as a brake on cell proliferation and that this region is required for ligand binding. (+info)
Developmental expression of plasminogen activator inhibitor-1 associated with thrombopoietin-dependent megakaryocytic differentiation.
(8/338)
Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet alpha-granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34(+)/CD41(-) cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-type c-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34(+)/CD41(-) cells to CD34(-)/CD41(+) cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO. (+info)