The biologic hallmark of polycythemia vera (PV) is the formation of endogenous erythroid colonies (EECs) with an erythropoietin-independent differentiation. Recently, it has been shown that an activating mutation of JAK2 (V617F) was at the origin of PV. In this work, we studied whether the STAT5/Bcl-xL pathway could be responsible for EEC formation. A constitutively active form of STAT5 was transduced into human erythroid progenitors and induced an erythropoietin-independent terminal differentiation and EEC formation. Furthermore, Bcl-xL overexpression in erythroid progenitors was also able to induce erythroid colonies despite the absence of erythropoietin. Conversely, siRNA-mediated STAT5 and Bcl-xL knock-down in human erythroid progenitors inhibited colony-forming unit-erythroid (CFU-E) formation in the presence of Epo. Altogether, these results demonstrate that a sustained level of the sole Bcl-xL is capable of giving rise to Epo-independent erythroid colony formation and suggest that, in PV patients, JAK2V617F may induce EEC via the STAT5/Bcl-xL pathway.
In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite the absence of Epo,1,2 and presence of such endogenous erythroid colonies (EECs) is routinely used as a diagnostic assay.1,3,4 To this date, mechanisms implicated in EEC formation are poorly understood. Recently, an acquired mutation in the JAK2 kinase leading to its constitutive phosphorylation has been described in PV patients. This mutation leads to a constitutive activation of JAK2 that seems to play a crucial role in the onset of the disease.5-7 During erythropoiesis, JAK2 activates many transduction pathways, which can be implicated in terminal maturation. For example, recent data showed that activation of AKT was sufficient for Epo-independent colony-forming unit-erythroid (CFU-E) formation in mice.8 During erythropoiesis, one of the principal targets of JAK2 is the signal transducer and activator of transcription STAT5. After phosphorylation by JAK2, STAT5 dimerizes and translocates into the nucleus where it regulates transcription of target genes implicated in cell proliferation and survival, among which that of the antiapoptotic protein Bcl-xL.9 The JAK2/STAT5/Bcl-xL pathway is crucial during erythropoiesis9-12 : JAK2-/- mice die in utero from severe anemia,10 and inactivation of STAT5 leads to a severe defect in erythropoiesis.9,11 In the present work, using both an siRNA and an overexpression strategy, we investigated whether the STAT5/Bcl-xL pathway was implicated in EEC formation. In particular, we overexpressed Bcl-xL or a constitutively active form of STAT5 in human normal erythroid cells and observed that the transduced cells could undergo an Epo-independent erythroid differentiation and in this way mimic PV phenotype.
UT7 clone expressing Mpl (UT7 5.3) was maintained in the presence of 5 ng/mL GM-CSF and differentiated toward erythropoiesis in the presence of 2 UI/mL human recombinant erythropoietin (Epo; OrthoBiotech, Paris, France).13 After informed consent, cells obtained from peripheral blood (PB) of patients treated with G-CSF were separated over a Ficoll-metrizoate gradient, and CD34+ cells were purified and cultured in a serum-free medium in the presence of Epo, SCF, IL-3, and dexamethasone (DXM) as previously described.14 For colony assays, CD36+/GpA- cells were plated in H4100 Medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10% FCS, 25 ng/mL SCF, with or without Epo 3 UI/mL. CFU-E numbers were counted at day 7. For colony assays without Epo, 5000 cells were plated per dish, instead of 1000 in normal conditions.
Retroviral constructs, retrovirus production, and cell infection
The STAT5 cDNA with an activating mutation15 (gift from F. Gouilleux, INSERM E0351, Amiens, France) and the human Bcl-xL cDNA were cloned upstream from the IRES-GFP sequence in the MIGR plasmid. Retrovirus particle production was achieved as previously described.14 Infection of human CD34+ cells was performed at days 4 and 5 in the presence of Epo, SCF, IL-3, and DXM as described earlier.14 At day 6, CD36+/GpA-/GFP+ cells were sorted and cultured in a serum-free medium supplemented with 25 ng/mL SCF with or without Epo or plated in methylcellulose as described in “cell culture”.
Results and discussion
Recent focus on the JAK2V617F mutation in PV patients argues for a direct implication of JAK2-dependent signaling pathways in EEC formation.5,6 Because STAT5 is the principal JAK2 target in erythroid cells, we investigated whether EEC formation was dependent only on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we used a retroviral vector coding for a constitutively active form of STAT5 (STAT5CA), which is spontaneously translocated into the nucleus.15 After transduction in UT7 cells, a leukemic cell line with erythroid properties, this vector led to a spontaneous induction of GpA expression despite the absence of Epo (Figure 1A). We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified PB CD34+ cells were cultured in a serum-free medium as described in “Materials and methods.” After transduction with the STAT5CA vector, CD36+/GpA-/GFP+ cells were sorted and cultured in the presence of SCF alone. As shown previously, these cells correspond to erythroid progenitors at a CFU-E stage and are dependent on Epo for their survival and terminal differentiation.14 After STAT5CA expression, they could undergo erythroid terminal differentiation despite the absence of Epo (Figure 1B).
Because STAT5 has been shown to play a crucial role in erythropoiesis through induction of the antiapoptotic protein Bcl-xL,9,11 we next investigated whether effects of STAT5CA were dependent on Bcl-xL induction. We cloned the coding sequence of human Bcl-xL in the retroviral vector MIGR. As shown in Figure 1C, overexpression of Bcl-xL in CD36+/GpA- cells induced GpA expression despite the absence of Epo. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute for Epo to induce terminal differentiation of primary erythroid progenitor cells. Of interest, an immunostaining experiment showed that these 2 vectors increased levels of Bcl-xL protein in erythroid progenitors (data not shown). These levels, however, did not exceed those observed in physiological conditions after Epo exposure. This is in agreement with recent data showing an effect of Bcl-xL on erythroid differentiation, either in the FDCP cell line18 or in primary mouse erythroblasts.19 This effect may be partly independent of Bcl-xL antiapoptotic properties because Bcl2, another antiapoptotic factor, is not able to induce EEC in transgenic mice,20 and its overexpression induces granulocytic but not erythroid differentiation in FDCP cells.18
In order to investigate the importance of the STAT5/Bcl-xL pathway in CFU-E formation, we transduced CD36+ progenitor cells with an siRNA targeted on STAT5 or Bcl-xL, and plated them in methylcellulose for CFU-E assays. We observed with either STAT5 or Bcl-xL siRNA a drastic reduction of cloning efficiency (Figure 2A). Deregulation of these proteins could therefore be implicated in EEC formation observed in PV cells. Since a high Bcl-xL expression has been described in PV erythroid progenitors,21 we investigated whether STAT5CA or Bcl-xL overexpression could reproduce the malignant phenotype (ie, formation of EEC) as do PV cells. PB CD34+ cells were cultured in a serum-free medium and transduced with the different retroviruses at days 4 and 5. CD36+/GpA-/GFP+ cells were sorted at day 7 and plated in methylcellulose with or without Epo. In the presence of Epo, total CFU-E number was higher with the STAT5CA or the Bcl-xL vector than with the control (Figure 2B). Without Epo, Bcl-xL as well as STA5CA vectors could induce EEC (Figure 2C). However, STAT5CA expression led to a greater number of colonies, whereas Bcl-xL overexpression induced smaller ones similar to those observed in PV patients (Figure 2D). These differences could be due to recruitment of other targets activated by STAT5, especially genes implicated in the cell cycle.
Considering these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be partially due to the JAK2-dependent activation of the STAT5/Bcl-xL pathway. In agreement with this, JAK2 inhibitors have been shown to inhibit Epo-independent terminal differentiation of PV cells.22 Moreover, when JAK2V617F was expressed in the IL-3-dependent cell line BaF3, a spontaneous STAT5 phosphorylation could be detected in the absence of cytokine.6,7 Whether this mutation requires a functional Epo-R and STAT5 to induce EEC formation via an Epo-independent Bcl-xL induction in PV erythroid cells is still under investigation.
Prepublished online as Blood First Edition Paper, May 9, 2006; DOI 10.1182/blood-2005-10-009514.
Supported by grants from INSERM and the Institut Gustave Roussy.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.
We are grateful to Frederic Larbret for cell sorting experiments, and to Dr Virginie Moucadel and to Prof Nicole Casadevall for their kind assistance.