ABSTRACT
The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.
Subject(s)
Erythroid Precursor Cells/enzymology , Erythropoietin/physiology , Phosphatidylinositol 3-Kinases/physiology , Protein Serine-Threonine Kinases , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Cell Cycle Proteins/metabolism , Cell Division , Cell Survival , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Chromones/pharmacology , Cyclin-Dependent Kinase Inhibitor p27 , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/drug effects , Erythropoietin/pharmacology , Fetal Blood/cytology , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors , Humans , Infant, Newborn , Insulin Receptor Substrate Proteins , Intracellular Signaling Peptides and Proteins , Ligases/metabolism , MAP Kinase Signaling System , Mice , Morpholines/pharmacology , Multienzyme Complexes/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoinositide-3 Kinase Inhibitors , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptors, Erythropoietin/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein LigasesABSTRACT
Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization. In the present study, we have investigated the relationship between shape change and PAK activation. We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-Fc(gamma)RIIA IV.3 induces an activation of Cdc42 and Rac1. The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1. Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK. Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading. The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation. Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin. These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.
Subject(s)
Blood Platelets/ultrastructure , Lectins, C-Type , Microfilament Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Pseudopodia/physiology , cdc42 GTP-Binding Protein/physiology , rac1 GTP-Binding Protein/physiology , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Adult , Antibodies, Monoclonal/pharmacology , Antigens, CD/immunology , Biopolymers , Blood Platelets/drug effects , Blood Platelets/enzymology , Cell Size , Cortactin , Crotalid Venoms/pharmacology , Enzyme Activation , Guanosine Triphosphate/physiology , Humans , Kinetics , Phosphorylation/drug effects , Platelet Activation/drug effects , Protein Binding , Protein Processing, Post-Translational/drug effects , Protein Structure, Tertiary , Proteins/pharmacology , Receptors, IgG/immunology , Receptors, Thrombin , Recombinant Fusion Proteins/physiology , Signal Transduction , Thrombin/pharmacology , p21-Activated KinasesABSTRACT
Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.
