Early events in erythropoietin-induced signaling

The cloning of the erythropoietin receptor (EpoR) in 1989 has allowed very rapid progress in our understanding of the early intracellular events that may be triggered by erythropoietin (Epo) in erythroid progenitor cells. From studies carried out primarily with cell lines expressing exogenous wild-type and mutant EpoRs, it appears that the activated EpoR is capable of triggering many of the same cascades that are utilized by receptors possessing endogenous tyrosine kinase domains. The major challenge over the next decade lies in seeing if these same signaling pathways are also utilized by normal Epo-responsive erythroid progenitors, discriminating between proliferation and differentiation inducing events in these cells, and determining whether various hematologic disorders can be attributed to aberrations in these signaling pathways.     

The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood 89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.     

Physical and functional interaction between p72(syk) and erythropoietin receptor

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid cells through interaction with a cell surface receptor (EpoR) that belongs to the cytokine receptor family. The Jak2 tyrosine kinase was previously shown to bind to the EpoR, to be activated upon Epo stimulation, and to play a critical role in Epo-induced proliferation. However, little is known about the role of other tyrosine kinases in Epo signaling. In this paper, we examined whether Syk was involved in EpoR activation. Coimmunoprecipitation experiments showed that the phosphorylated EpoR was associated with the Syk kinase in activated UT7 cells. The interaction of Epo with its receptor led to an increased kinase activity. The use of recombinant Syk Src homology 2 (SH2) domains expressed in tandem or individually revealed that both N- and C-SH2 domains of Syk participated in EpoR binding with a major contribution of the C-terminal SH2 domain. Far Western blotting further indicated that Syk directly binds to the EpoR and that the interaction of Syk with EpoR only occurred after Epo activation. These data suggest that phosphorylation of EpoR on tyrosine residues may mediate Syk binding to the receptor through interaction between the two SH2 domains of Syk and tyrosines of the receptor. We propose that in addition to Jak2, Syk protein kinase may be a component of EpoR signaling.     

The response of IL-3 dependent B6SUtA bone marrow cells to both erythropoietin and chemical inducers of differentiation

To develop cell lines which respond to both a physiological cytokine and chemical agents by the induction of differentiation pathway, factor dependent B6SUtA murine bone marrow cells were transfected with the erythropoietin receptor (EpoR). Clones were obtained that exhibited different sensitivities to erythopoietin (Epo), with one clone exhibiting erythroid differentiation in response to Epo, while in another Epo acted as a proliferation stimulus. Moreover, parental B6SUtA cells were sensitive to the initiation of differentiation by butyrate, diazepam and hemin. Thus, B6SUtA cells appear to represent a unique model to dissect the signaling molecules involved in the growth and differentiation pathways employed by Epo and non-physiological chemicals.     

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The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.     

STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.     

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.     

Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.     

Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells

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.     

Recombinant human c-Mpl ligand (thrombopoietin) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro

To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma-containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU-Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst-promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.     

Characterization of the domains of the erythropoietin receptor necessary for induction of cell growth and differentiation

To define the cytoplasmic region(s) of the erythropoietin receptor (EpoR) necessary for promotion of growth and induction of differentiation, mutated EpoR cDNAs containing truncations and conversions of tyrosine residues to phenylalanines were generated. Constructs were introduced into IL-3 dependent Ba/F3 cells by electroporation, and individual transfectants were propagated in methylcellulose-containing medium in the presence of erythropoietin (Epo). Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the receptor lacking the box 2 region was not capable of inducing cellular growth. When a large number of cells were screened in Epo-containing liquid medium, clones arose in which Epo-dependent growth was due to activation of the endogenous EpoR gene. Analysis of the degree of Epo-dependent differentiation of the transfectants, based on the steady-state levels of beta major-globin mRNA, showed that the carboxyl terminal 133 amino acids and tyrosyl residues located at positions 429/431 and 460/464 were not necessary for the induction of differentiation. Examination of clones from diverse origins demonstrated that an inverse relationship existed between the rate of Epo-induced cellular replication and the degree of Epo-induced differentiation.     

Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.     

Expression of an activated erythropoietin or a colony-stimulating factor 1 receptor by pluripotent progenitors enhances colony formation but does not induce differentiation

Whether the presence of specific receptors on the surface of developing cells is the cause or consequence of lineage restriction is not known. If activation of specific receptors is the driving event in differentiation, the premature expression of specific receptors would promote differentiation along that pathway. In this study pluripotent progenitors, obtained from blast cell colonies (pooled or individual) of 5-fluorouracil-treated mice, were infected with retroviral vectors containing either an activated receptor for erythropoietin (EPO), an erythroid progenitor growth factor, or the receptor for colony-stimulating factor 1 (CSF-1), a macrophage growth factor. These receptors exhibit expression patterns restricted to committed progenitors. The developmental potential of infected pluripotent progenitors was not changed, although they expressed the exogenous genes, suggesting that in these cells activation of lineage-specific receptors does not induce differentiation. Acquisition of a constitutively activated EPO receptor allowed erythroid development in mixed colonies in the absence of EPO, as expected. Infection of progenitors with a virus containing the CSF-1 receptor promoted the development of granulocyte/macrophage (GM) colonies but did not alter the differentiation potential of either colony-forming unit (CFU)-GM or CFU-mix.