Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes vErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal dierentiation under appropriate conditions, were established from fetal livers of p537/7 mice. Expression and activation of the EGF-receptor (HER-1/ c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal dierentiation. Upon inhibition of ErbB function, dierentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5-and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms. Oncogene (2001) 20, 3651 ± 3664.
The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP-and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.
Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.
Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCγ1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)–receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.
Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoi- IntroductionErythropoietin (EPO) is required for the survival, proliferation, and differentiation of erythroblasts. It acts with other growth factors and hormones to balance the expansion and terminal differentiation of the erythroid compartment. For instance, stem cell factor (SCF), a ligand of c-Kit, cooperates with EPO to delay differentiation and to enhance progenitor numbers. 1,2 The EPO receptor (EpoR) is a homodimer constitutively associated with Janus tyrosine kinase 2 (Jak2). Ligand binding induces a conformational change of the EpoR dimer, 3,4 resulting in the activation of Jak2, the phosphorylation of the EpoR on 8 tyrosine residues, and the recruitment of multiple signaling molecules. [5][6][7] Mice deficient in EPO, EpoR, or Jak2 lack definitive erythropoiesis. [8][9][10][11] Although this demonstrates a crucial role for EPO-induced Jak2 activation in erythropoiesis, the contribution of individual downstream signaling pathways to erythroblast proliferation, differentiation, and survival is less clear. Notably, mice expressing a truncated EpoR, lacking all tyrosine residues, are not anemic. 12 This may implicate that additional proteins, such as docking molecules, can form a complex with the EpoR to create a signaling platform with sufficient specificity to regulate the balance of erythroid expansion and differentiation.Candidates for this function are the Grb2-associated binder (Gab) family members. At present, 3 Gab family members have been identified-Gab1, Gab2, and Gab3. 13,14 Gab1 is ubiquitously expressed, whereas Gab2 and Gab3 expression are tissue specific. In the hematopoietic system, Gab3 is expressed in B cells and T cells and in the myeloid compartment, whereas Gab2 is expressed in all hematopoietic cells except T cells. Gab1 deficiency is embryonically lethal because of placental defects; Gab1Ϫ/Ϫ embryos are also characterized by reduced liver size and migration defects of muscle precursor cells. 15,16 In contrast, Gab2 deficiency results only in mild defects in mast cell and macrophage responses, 17,18 and Gab3-deficient mice show normal hematopoiesis. 19 Gab2 and Gab3 lack the c-Met binding sequence (MBS) of Gab1 and, therefore, may not complement Gab1 in c-Met signaling. Furthermore, the high homology between family members suggests that complementation by the ubiquitously expressed Gab1 may occur in Gab2/3 knockouts. On tyrosine phosphorylation, Gab proteins recruit signaling intermediates, among them Shc (Src homology-containing protein), Shp2 (Src-homology domain containing phosphatase), p85 subunit of phosphatidylinositol 3-kinase (PI3K), and phosp...
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