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HEMATOPOIESIS
From the Whitehead Institute for Biomedical Research
and Massachusetts Institute of Technology, Cambridge, MA; Departments
of Pathology, Children's Hospital and Harvard Medical School, Boston,
MA; Technical University of Munich, Munich, Germany.
The receptor-associated protein tyrosine kinase janus-kinase 2 (JAK2) is essential for normal red cell development and for erythropoietin receptor (EpoR) signaling. JAK2 Erythropoietin (Epo) is essential for the
proliferation, differentiation, and survival of erythroid cells as
demonstrated definitively by the properties of Epo and erythropoietin
receptor (EpoR) Janus-kinase 2 (JAK2), a member of the JAK family of protein tyrosine
kinases, is constitutively bound to cell surface EpoR and is crucial
for EpoR signaling. JAK2
Activated JAK2 phosphorylates several tyrosine residues on the EpoR;
these phosphotyrosine residues on the EpoR provide docking sites for
SH2 (src-homology-2) domain-containing signal transduction proteins
including STAT5 that bind to the receptor and are phosphorylated by
JAK2, thereby initiating EpoR signaling cascades including activation
of STAT5, Ras, mitogen-activated protein kinase (MAPK), JNK,
P38, PI3-kinase-AKT, SHP1, SHP2, SHIP, and BCL-xL
(reviewed in Constantinescu et al7). JAK2 may also
directly phosphorylate and activate signaling proteins such as Shc in a
receptor tyrosine-independent fashion8; as another
example, an EpoR devoid of any cytosolic tyrosines activates STAT5 to a
level approximately 10% that of the wild-type receptor.9
The relative contribution of the different EpoR-activated
signaling pathways to erythroid development, specifically proliferation, differentiation, and survival, of erythroid progenitors is not well understood.
JAK2 is the major JAK associated with and activated by a number
of cytokine receptors besides the EpoR, including the growth hormone receptor, the type 2 interferon receptor, the
thrombopoietin receptor Mpl, and the Many of the signaling pathways activated by JAK2 in response to Epo are
also activated by the BCR-ABL oncoprotein (reviewed in Constantinescu
et al7 and Ghaffari et al11), the molecular hallmark of chronic myeloid leukemia (CML). P210BCR-ABL
(P210) and a related fusion protein P185BCR-ABL
(P185)12,13 are constitutively active protein tyrosine
kinases whose activity is significantly more potent than their normal c-ABL counterpart.14,15 These chimeric BCR-ABL proteins
result from the fusion of the N-terminal segment of BCR (902 amino
acids in P210 and 426 amino acids in P185) to most of the c-ABL
protein.16 Despite a mild anemia which affects most
patients at the chronic phase, erythroid progenitors are
Epo-independent in culture and their number is increased in CML
patients.17 In addition, BCR-ABL complements EpoR
signaling and supports the proliferation, differentiation, and
maturation of red cell progenitors when expressed in
EpoR Since JAK2 is crucial for EpoR signaling and red cell development, we
investigated whether JAK2 is required for BCR-ABL complementation of
EpoR signaling. Although JAK2 was constitutively tyrosine
phosphorylated in cultured and primary erythroid cells expressing
BCR-ABL, erythropoiesis proceeded normally in JAK2 In addition to the tyrosine kinase domain, several distinct
sequences within BCR-ABL, either alone or in concert, contribute to
BCR-ABL transforming potential by activating signaling pathways that
are also activated by many cytokine receptors. Signal transduction pathways generated by BCR-ABL and its individual domains (Table 1) that promote proliferation and
survival have been studied extensively both in vitro and in
vivo.19 Thus, we also expressed several BCR-ABL mutants
that exhibit impaired signaling (summarized in Table 1) in
JAK2 Cells
The erythroleukemic cell line HCD57 was maintained in Iscoves modified
Dulbecco medium (IMDM) containing 20% FCS, 0.01 M Phoenix packaging cells (kindly provided by Dr Gary Nolan, Stanford University, CA) were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% FCS. Retroviral constructs cDNAs were cloned upstream of the internal ribosomal entry site (IRES) in the bicistronic retroviral MSCV-IRES-(green fluorescent protein) GFP (MIG) vector, a gift of Dr Luk Van Parijs (Massachusetts Institute of Technology, Cambridge, MA).20 The translation of the complementary DNA (cDNA)-encoded protein and GFP is tightly linked in that the expression of GFP is proportional over a 100-fold range to the level of expression of the protein encoded by the cDNA placed upstream of the IRES.21 To construct the vectors MSCV-P210-IRES-GFP, MSCV-P210 Y177F-IRES-GFP, MSCV-P210 SH2-IRES-GFP, MSCV-P185-IRES-GFP, and MSCV-P185 176-427-IRES-GFP,
the desired inserts were flanked by EcoRI sites and cloned
into the corresponding site in the MIG vector. The BCR-ABL P185 triple
mutant (P185 TM) and JAK2 cDNA were inserted by blunt-end ligation into
the HpaI site of the MIG vector. MSCV-P210-pac was described
previously.18
Retroviral supernatant production and infection procedure High titer replication-free retroviral supernatants were generated as follows: 10 µg retroviral plasmids together with the pCL-Eco vector22 were cotransfected using calcium phosphate (Invitrogen kit) into 106 cells Phoenix packaging cells plated on 60-mm dishes 18 hours prior to transfection. The resulting retroviral supernatant was collected 48 hours later and was used to infect fetal liver cells. Titers of 2 × 106 to 8 × 106 were routinely obtained. A 1-to-5 dilution of the packaged virus was used to infect NIH 3T3 cells and protein expression was analyzed by Western blot using the appropriate antibody (anti-ABL or anti-JAK2 antibodies). JAK2 / fetal liver
cells (2 × 105 cells/mL) were resuspended in viral
supernatants at a multiplicity of infection of 5 to 10 and plated in 60 mm retronectin (Takara Biomedicals)-coated dishes in the presence of
100 ng/mL each of IL-6 and SF (PeproTech) for 36 hours. Cells were then
washed once and resuspended in  -MEM containing 15% FCS and the same
growth factors for another 24 hours. Wild-type fetal liver cells were incubated with IL-3 (6 ng/mL) (PeproTech), IL-6 (10 ng/mL), and SF (100 ng/mL) for 24 hours prior to infection. Cells were resuspended in the
appropriate viral supernatant in the presence of the same growth
factors for 48 hours. Cells were then washed and resuspended in media
containing the same growth factors for an additional 24 hours.
Flow cytometry and immunostaining Retrovirally infected cells were washed twice in phosphate buffer saline (PBS) solution containing 2% FCS. Cells were then incubated with control rat serum at room temperature for 15 minutes, followed by incubation with 1 µg/mL Ter119-PE antibody (BD PharMingen) for 30 minutes on ice. Afterward, the cells were washed once with cold PBS 2% FCS, and once with PBS 2% FCS containing 1 µg/mL propidium iodide (PI), resuspended in 500 µL PBS containing 2% FCS prior to a FACS sort (Becton Dickinson). GFP+ or GFP+ Ter119 cells were selected and
FACS-sorted for further analysis by cytospin or colony assays.
CELLQuest (Becton Dickinson) was used for FACS analysis.
Colony assays Retrovirally transduced cells were washed once in-MEM
containing 15% FCS and plated in duplicate in semisolid medium
containing 0.9% methylcellulose in IMDM containing 15% FCS, 1%
bovine serum albumine (BSA), 10 µg/mL bovine insulin, 200 µg/mL
human transferrin, 104 M 2-mercaptoethanol, 2 mM
L-glutamine (MethoCult M3234; StemCell Technologies), to measure colony
formation as previously described.18,23 CFU-E formation
was carried in methylcellulose cultures containing SF (100 ng/mL;
PeproTech) with or without Epo (3 units/mL; Amgen, Thousand Oaks, CA),
while BFU-E formation was assayed in methylcellulose cultures in the
presence or absence of IL-6 (10 ng/mL) and SF (100 ng/mL), with or
without Epo (3 u/mL). The number of CFU-E colonies was determined after
diaminobenzidine staining of hemoglobin and counted 2 days after
plating. BFU-E colonies of hemoglobinized erythroblasts were counted
after 9 days. Colonies were individually aspirated for reverse
transcriptase (RT)-PCR or cytospin, and analysis of their erythroblast
morphology determined after Wright Giemsa staining.
Cytospin and cytoplasmic staining Cells were washed in PBS with 2% FCS, and resuspended in PBS containing 1% BSA at a concentration of 3 × 105 cells/mL. Cells (100 µL per slide) were subjected to a cytospin for 2 minutes at 600 rpm (Cytospin 3; Shandon) and air dried. Cells were then stained with Wright Giemsa (Harleco) according to the manufacturer's recommendations.PolyA RT-PCR from single erythroid colonies To analyze the gene expression profile of transduced erythroid colonies, we performed RT-PCR using oligo-dT-based primers and a polyA tailing strategy as previously described.24,25 Briefly, single BFU-E colonies were aspirated (2 µL to 10 µL) from methylcellulose plates and lysed directly in a 5 M guanidinium isothyocyanate solution containing 20 mM dithiothreitol. Nucleic acids were precipitated and the entire sample was reverse transcribed using an oligo-dT primer (1 µg/µL) (5'-CAT-GTC-GTC-CAG-GCC-GCT-CTG-GAC-AAA-ATA-TGA-ATT-C[T]24-3'), tailed, and subjected to PCR25 containing the oligo-dT primer described above. Total cDNA was amplified using 5 units of Taq polymerase. One-fourth of the total amplified product from each colony was separated by electrophoresis through 1% agarose and transferred to a Zeta-probe GT membrane (Bio-rad) and probed with either -major
globin,26 GATA-1,24,27 or
L-32.24 Probes (see Figure 5D)were prepared as
previously described.24,27
Immunoprecipitation and Western blot analysis Populations of HCD57 or HCDP210 cells or of infected wild-type fetal liver cells were washed 4 times in serum-free media and starved overnight in IMDM containing 0.1% FCS. HCD57 and HCDP210 (5 × 107 cells), and fetal liver cells (2 × 106 cells) were stimulated 18 hours later with or without Epo (100 u/mL) for 5 minutes at 37°C. Cells were then washed twice with cold PBS and extracts were prepared by the addition of 1 mL lysis buffer, 50 mM Tris-HCl (pH 7.5 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 2 mM Na3VO4, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1% Brij-96, 1 mM dithiothreitol (DTT). For immunoprecipitations, cell extracts were incubated overnight at 4°C with 5 µL anti-JAK2 polyclonal anti-sera (Upstate Biotechnology, Lake Placid, NY) or with 1 µg mouse anti-c-ABL monoclonal antibody (sc-23; Santa Cruz). Immune complexes were recovered by binding to protein A-Sepharose or protein G-Sepharose beads (Roche-Boehringer). The captured immunocomplexes were washed 3 times with lysis buffer and once with PBS and were then eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer. Samples were fractionated through SDS-polyacrylamide gels, transferred electrophoretically to nitrocellulose membranes, and incubated with the indicated antiserum: (1) antiphosphotyrosine monoclonal antibodies (4G10, Upstate Biotechnology) (1:1000); (2) anti-JAK2 polyclonal antibodies (1:1000); and (3) anti-ABL monoclonal antibodies (1:1000). Bound antibodies were detected by enhanced-chemiluminescence system (DuPont-NEN).
JAK2 is constitutively phosphorylated in cultured erythroleukemia cells and primary erythroid cells expressing BCR-ABL We first examined the tyrosine phosphorylation status of JAK2 in an erythroid cell line expressing BCR-ABL. We expressed P210 in the Epo-dependent erythroleukemic HCD57 cells (Figure 2A) and derived a population that proliferated in the absence of Epo (HCDP210 cells, Figure 2B). The Western blot in Figure 2A shows that P210 is expressed in the Epo-independent HCDP210 cells (right panel) and as expected, P210 BCR-ABL but not c-ABL (left panel) was tyrosine phosphorylated in HCDP210 cells starved of all growth factors. As shown in Figure 2C, JAK2 is tyrosine phosphorylated and thus activated in the presence but not the absence of Epo in HCD57 cells. In contrast, HCDP210 cells exhibited a low level of JAK2 tyrosine phosphorylation in the absence of Epo, and Epo stimulation induced a further increase of JAK2 tyrosine phosphorylation (Figure 2C, upper panel). In HCDP210 cells, Epo addition also caused the mobility of the tyrosine phosphorylated JAK2 to decrease, suggesting that after Epo addition JAK2 becomes phosphorylated on tyrosine residues additional to those phosphorylated as a result of BCR-ABL expression, and that causes a slower gel migration.
To examine the status of JAK2 tyrosine phosphorylation in primary erythroid cells expressing P210 we chose wild-type E14 fetal liver cells since this population of cells is mostly erythroid. We used a bicistronic retroviral vector to express P210 and GFP together (MSCV-P210-IRES-GFP) or, as a control, GFP alone (MSCV-IRES-GFP), and we used GFP expression as a marker of retroviral transduction. In these bicistronic retroviral vectors, the expression of GFP and P210 is under the transcriptional control of the retroviral long-terminal repeat and the translational control of IRES of the encephalomyocarditis virus, resulting in the expression of both GFP and P210 in the same cell (see "Materials and methods"). Wild-type E14 fetal liver cells were retrovirally transduced with
either MSCV-P210-IRES-GFP or the control vector and analyzed by FACS
for GFP expression. Approximately 50% of P210-infected and 70% of
control vector-infected fetal liver cells expressed the GFP marker
(Figure 3A). To avoid cellular loss, we
subjected the totality of these populations, without further FACS
selection, to serum starvation and examined the tyrosine
phosphorylation status of JAK2 in response to Epo stimulation. A low
level of JAK2 tyrosine phosphorylation in response to Epo stimulation
was detected in primary fetal liver cells infected with control vector but, as expected, none was detected in the absence of Epo (Figure 3B,
upper panel). In contrast, tyrosine phosphorylation of JAK2 was easily
detectable in P210-transduced fetal liver cells starved in the absence
of serum and Epo, and Epo stimulation did not affect the level of JAK2
tyrosine phosphorylation (Figure 3B, upper panel, i). A shorter
exposure of the Western blot confirmed the conclusion that the level of
JAK2 tyrosine phosphorylation in BCR-ABL-transduced cells is
unaffected by addition of Epo (Figure 3B, upper panel, ii). As a
control, we showed that JAK2 protein was present at equal levels in
P210-transduced and control fetal liver cells (Figure 3B, lower panel).
Thus, JAK2 becomes constitutively tyrosine phosphorylated and presumably active, as a result of BCR-ABL expression both in HCD57 erythroleukemia and in fetal liver erythroid cells. JAK2 is not required for red cell formation by BCR-ABL To assess whether tyrosine phosphorylation of JAK2 and activation of JAK2 signaling pathways is required for BCR-ABL-induced maturation of erythroid progenitors, we examined the potential of P210 to support red cell formation in the absence of JAK2 by using primary JAK2/ fetal liver cells. To this end, E12.5
JAK2/ fetal liver cells were retrovirally transduced
with either P210, JAK2, or vector control and analyzed for GFP
expression as a measure of infection efficiency (Figure
4B; see "Materials and methods"). We
established optimum conditions (Figure 4A) for retroviral transduction of JAK2/ fetal liver cells; this also allowed for a
significant expansion of transduced erythroid progenitors. Furthermore,
this system provided reproducible conditions for direct comparison of
the effects of transduced P210 and JAK2 on JAK2/
fetal liver cells.
Ter119 is a marker of erythroid differentiation28,29
expressed on the cell surface of most (80%-90%) wild-type E14 fetal liver cells (data not shown). In contrast, Ter119 is present only on
10% to 20% of JAK2 To confirm this key point, we used the FACS to isolate all retrovirally
transduced (ie, GFP-positive) cells from JAK2 and P210-infected
populations, regardless of their Ter119 expression (Figure 4B,
populations i and ii) and analyzed their morphology (Figure 4C). A
majority of GFP-positive cells transduced with P210 were
morphologically erythroid, at different stages of differentiation, as
were a majority of JAK2-transduced cells that were cultured in the
presence of Epo (Figure 4C). In contrast, freshly isolated JAK2 Ter119 positive cells are composed of erythroblasts at different stages
of differentiation and do not give rise to BFU-E or CFU-E erythroid
colonies29 (and data not shown). Thus, they do not contain
any committed erythroid progenitors. Consequently, the population of
GFP+Ter119
Similarly, P210 expression in JAK2 In addition to Epo, for their optimum proliferation and survival
in in vitro culture assays, primitive erythroid progenitors require
cytokines and growth factors such as IL-3, IL-6, GM-CSF, and SF.
Previously, we showed that P210-transduced EpoR JAK2 is required for red cell formation by certain BCR-ABL mutants expressed in fetal liver cells We previously demonstrated that the P210 mutants P210SH2 and
P210 Y117F, whose transforming potential and capacity in activating certain cytokine signaling pathways are compromised (Table 1), still
can induce red cell differentiation to the same extent as wild-type
P210 in EpoR/ as well as wild-type fetal liver
cells.17 Here, by transducing these mutants into
JAK2/ fetal liver cells, we determined their ability to
support erythropoiesis in the absence of JAK2 (Table
2).
Depending on the BCR-ABL mutant (Table 1) studied, we used either
wild-type P210 or P185 as the control. As expected, the ability of P185
to induce CFU-E formation from JAK2 Among the 3 BCR-ABL mutants tested (Table 2), P185
Cumulative evidence in the past decade supports the notion that cell fate is determined by the cellular context in which signaling proteins are expressed.11 Tyrosine phosphorylation and activation of JAK2 is key to erythroid development. In response to Epo stimulation, JAK2 becomes tyrosine phosphorylated and activated; JAK2 then phosphorylates the EpoR on several tyrosine residues leading to the activation of multiple signaling pathways [reviewed in Constantinescu et al7]. In addition to the EpoR, JAK2 binds to other cytokine receptors and cytosolic proteins, and directly activates, in a receptor-phosphotyrosine independent fashion, signaling pathways such as STAT5 and Shc.8,9 The relative contribution of different EpoR-activated signaling pathways to erythroid development, specifically proliferation, differentiation, and survival of erythroid progenitors, is not well understood. As a first step in identifying potential EpoR signaling components sufficient to support erythropoiesis in primary erythroid cells, we activated signaling pathways downstream of the EpoR using the well-characterized BCR-ABL oncoprotein and its mutants. The constitutively active protein tyrosine kinase BCR-ABL supports
erythropoiesis in both wild-type and EpoR Our findings also indicate that the ability of BCR-ABL to activate
signaling pathways required for erythropoiesis is different in
EpoR Other examples are provided by the BCR-ABL mutants lacking either the
SH2 domain (P210 Although JAK2 is constitutively tyrosine phosphorylated in BCR-ABL-expressing erythroid (Figures 2, 3) and other myeloid cells,33,34 the mechanism of this effect is unknown. Interestingly, JAK2 immunocomplexes recovered from HCDP210 cells constantly contained P210 (data not shown), suggesting P210 and JAK2 are interacting in BCR-ABL-expressing erythroid cells and that BCR-ABL may directly phosphorylate JAK2. The tyrosine residues in JAK2 that become phosphorylated in BCR-ABL-expressing cells are unknown. Phosphorylation of certain tyrosines in JAK2 may directly activate its kinase activity. Alternatively, phosphotyrosines can provide docking sites for binding SH2 or phosphotyrosine binding domains of cytosolic proteins that subsequently become phosphorylated by JAK2. In this study we identified at least one domain of BCR-ABL that is
crucial for erythroid development in both wild-type and JAK2 Some clues to the deficient support of erythropoiesis by the P185
STAT5 activation is important for erythroid survival as demonstrated by
studies of STAT5a Through JAK2, the EpoR activates multiple signaling pathways that
function together to prevent apoptosis and to support terminal erythroid proliferation and differentiation. Here we have shown that
BCR-ABL activates similar pathways independently of JAK2, and supports
erythroid survival, proliferation and differentiation in
JAK2
We thank Luk Van Parijs (MIT) for MSCV-IRES-GFP; Norman
Iscove (Ontario Cancer Institute) and Trang Hoang (Clinical Research Institute of Montreal) for the GATA-1 and L-32 probes; Gordon Keller
(Mount Sinai School of Medicine) for the
Supported by a Clinician Scientist Award from The National Institutes of Health (National Cancer Institute) to S.G., and the following grants to H.F.L.: grant #HL-32262 from The National Institutes of Health, a grant from Amgen Corporation, and NSF grant #EEC-9843342/67983 from the Biotechnology Process Engineering Center (BPEC) at MIT.
Submitted June 18, 2001; accepted July 10, 2001.
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.
Reprints: Harvey F. Lodish, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142; e-mail: lodish{at}wi.mit.edu.
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© 2001 by The American Society of Hematology.
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S. Ghaffari, C. Kitidis, W. Zhao, D. Marinkovic, M. D. Fleming, B. Luo, J. Marszalek, and H. F. Lodish AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation Blood, March 1, 2006; 107(5): 1888 - 1891. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
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