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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3757-3773
Protein Kinase B (c-Akt), Phosphatidylinositol 3-Kinase, and STAT5
Are Activated by Erythropoietin (EPO) in HCD57 Erythroid Cells But
Are Constitutively Active in an EPO-Independent,
Apoptosis-Resistant Subclone (HCD57-SREI Cells)
By
Haifeng Bao,
Sarah M. Jacobs-Helber,
Amy E. Lawson,
Kalyani Penta,
Amittha Wickrema, and
Stephen T. Sawyer
From the Department of Pharmacology and Toxicology, Medical College
of Virginia campus of Virginia Commonwealth University, Richmond, VA;
the Department of Medicine, Stanford University, San Francisco, CA; and
Hematopoietic Stem Cell Laboratory, University of Illinois, Chicago,
IL.
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ABSTRACT |
We found that erythropoietin (EPO) and stem cell factor (SCF)
activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid
cells. To better understand signals controlling proliferation and
viability, erythroid cells that resist apoptosis in the absence of EPO
were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of
PKB/Akt because the PI3-kinase inhibitor LY294002 blocked
both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in
concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the
EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIETIN (EPO) is the primary
hormone which regulates the maturation of immature erythroid
cells1-3 into erythrocytes. EPO is necessary for the
proliferation, erythroid differentiation, and prevention of apoptosis
of erythroid cells; it is not yet clear whether these three processes
are regulated by distinct signaling pathways.1-3 Some
studies suggest that EPO might act primarily to maintain viability by
protecting mature erythroid cells from programmed cell death or
apoptosis in the relatively narrow window of EPO-dependent
differentiation.1 The "knock out" of EPO or EPO
receptor (EPOR) results in EPO / or EPOR/ null mice that
allow EPO-independent proliferation and development to relatively
mature erythroid cells (CFU-E) that then undergo apoptosis.4 This is consistent with the requirement of EPO only in the later (CFU-e) differentiation events as a viability factor.
Whereas the anti-apoptotic model is supported in EPO-responsive erythroid cells (proerythroblasts and erythroblasts) generated from
mice infected with the anemia strain of Friend virus1 (where the Friend virus may potentially drive proliferation), a
strictly anti-apoptotic signal from the EPOR may be unlikely in all
erythroid cells. The finding that many cell lines require EPO for
proliferation, but only a few erythroid cell lines partially differentiate in response to EPO and do not require EPO for
proliferation, suggests that proliferation, differentiation, and
anti-apoptotic pathways may be controlled separately by EPO. Cells do
not proliferate without cell survival; therefore, establishing the
existence of separate control of proliferation and protection from
apoptosis is difficult.
EPO acts through cell-surface receptors on immature erythroid
cells.1-3,5 The EPOR does not have a kinase domain and
transmits an intracellular signal by interacting with cytoplasmic
nonreceptor tyrosine protein kinases. Two of these kinases have been
reported to become phosphorylated after EPO binding to cells: the Janus kinase, JAK2, and the c-fes kinase.6-8
JAK2 can physically associate with the EPOR.6,9,10 This
interaction is constitutive in HCD57 erythroid cells, but JAK2 appears
to be selective in interacting with a subset of highly modified EPOR
molecules.10 Known substrates of tyrosine protein kinases
activated by EPO binding to its receptor include phosphatidylinositol-3
kinase (PI3-kinase), SHC proteins, c-raf, STAT1, STAT3, STAT5A,
STAT5B, and other molecules.4,9-23
Recent studies have suggested that c-Kit, the receptor for
stem cell factor, interacts with the EPOR and transduces an
intracellular signal through the EPOR.24 Our laboratory has
shown that stem cell factor (SCF) can stimulate proliferation of HCD57
cells but cannot protect them from apoptosis.5 EPO
activates JAK2/STAT signaling and other signaling which
protects the cells from apoptosis and stimulates proliferation. Because
SCF does not activate JAK2/STAT5 signaling in this system, we have
speculated that this pathway may protect erythroid cells from apoptosis.
Constitutive STAT activation has been observed in transformed cell
lines and cancerous cells.25-35 In most cases,
constitutively activated Janus kinases seem to be responsible for
activating the STAT proteins; however, in other cells and cancers,
other kinases may phosphorylate STAT molecules. Oncogenes such as
v-abl,30 bcr/abl,35 and
v-src30 may directly phosphorylate and activate STAT proteins. These studies suggest a possible role of STAT proteins, independent of Janus kinases, in signaling the proliferation and protecting these transformed cells from apoptosis. However, the role of
STAT5 signaling in erythroid cells is controversial. STAT5 activation
may be linked to enhanced proliferation19,36; in contrast,
a mutant EPOR incapable of activating STAT5 still promotes proliferation.37 Wakao et al38 and Iwatsuki et
al39 have separately demonstrated that STAT5 is required
for erythroid differentiation; in contrast, deletion of STAT5A and/or
STAT5B results in STAT5 null mice that have no obvious reduction or
defects in erythroid cells.40-42
The involvement of PI3-kinase in cell transformation has been
implicated in several cell lines.43,44 PI3-kinase plays an important role in regulation of cell proliferation and apoptosis; in
addition, constitutive PI3-kinase activity has been observed in
cancerous cells and, therefore, may contribute to the malignant transformation of cells. The insulin receptor substrate proteins (IRS)
are substrates of many tyrosine kinases, including the JAK2 kinase
bound to the EPOR.45,46 Activated IRS-1, -2, -3 are known
to be docking sites for PI3-kinase and are implicated in the activation
of PI3-kinase either in complexes with receptors or independently of receptors.
An important downstream effector activated by the PI3- kinase signaling
pathway is protein kinase B (PKB/Akt), also known as c-Akt and RAC-PK.
PI3-kinase activity leads to accumulation of polyphosphatidylinositols
lipids in the plasma membrane. PKB/Akt is apparently targeted to
translocate to the polyphosphatidyinositol rich membrane via a
pleckstrin homology domain and is activated at the membrane by
phosphorylation at Thr308 and Ser474 from two
distinct phosphatidylinositol dependent kinases.47 PKB/Akt
activity is linked to the control of many diverse cellular functions
that included insulin-dependent glucose transport, glycolysis, glycogen
synthesis and protein translation, and viability of
cells.47 Recent work has shown that PKB/Akt is able to
phosphorylate the pro-apoptotic protein BAD. Phosphorylation of BAD
correlates with the cell survival and it is suspected that
phosphorylated BAD is ineffective in disrupting the protective action
of Bcl anti-apoptotic proteins.
In the current study, we found that EPO and SCF activated PKB/Akt in
the EPO-dependent erythroid cells but the PI3-kinase/PKB/Akt pathway
was constitutively active in the apoptosis-resistant, EPO-independent
HCD57-SREI cells. The finding of constitutive activity of STAT5 in this
apoptosis-resistant subclone of HCD57 cells suggests that STAT5
activity which arises through a signal independent of Janus kinase
activity and the EPOR may also act in concert with PKB/Akt activity to
prevent cells from undergoing apoptosis induced by growth factor withdrawal.
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MATERIALS AND METHODS |
Cell culture.
HCD57 cells, obtained from Sandra Ruscetti (National Cancer Institute,
Frederick, MD),6,31 were cultured in the presence of 1.0 U
EPO/mL in Iscove's modified Dulbecco's medium and 25% fetal calf
serum. In experiments to test the action of signaling by EPO, the cells
were further cultured overnight in the same medium devoid of EPO. As
previously reported,6 this deprivation of EPO increased
cell-surface EPOR but was an insufficient time without EPO for
apoptosis to begin. HCD57-SREI cells were subcloned by transferring
HCD57 cells from medium containing EPO to medium free of EPO but
containing 100 ng SCF/mL. A subclone of cells that survived and
proliferated was then cultured in the same medium indicated above
except that both EPO and SCF were omitted.
Apoptosis studies.
HCD57 and HCD57-SREI cells were cultured either in the absence of
cytokines or in the presence of the indicated factors for 72 hours. The
cells were then harvested and genomic DNA was isolated.5 Ten micrograms of genomic DNA was resolved on a 2.25% agarose/1X TAE/300 ng EtBr/mL gel. DNA laddering indicative of apoptosis was
visualized using UV light.
Western blot analysis.
Tyr(P)-containing proteins and STAT5 tyrosine phosphorylation and
nuclear translocation were detected by either Western blotting alone or
the immunoprecipitation and Western blotting method described previously.5,10 Polyclonal anti-Tyr(P) antibodies from
Zymed (San Francisco, CA) were used to immunoprecipitate
and concentrate the Tyr(P)-containing proteins before they were
analyzed by Western blot. The blots were stripped of bound antibody and
reprobed with anti-JAK2 (Upstate Biotechnology, Inc [UBI], Lake
Placid, NY) antisera. The blot was stripped as described by the
Amersham literature with the ECL kit (Amersham, Arlington Heights,
IL). Briefly, the bound antibody was released in the
presence of 2% sodium dodeyl sulfate (SDS) and reducing agents at
50°C for 30 minutes. STAT5 tyrosine phosphorylation and nuclear
translocation were detected by Western blot analysis of 10 µg of
nuclear extracts probed with the anti-Tyr(P) antisera described above
or anti-STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) antisera. For
the Bcl-Xl and Bcl-2 Western blots, 10 µg of total cellular lysate
were probed with anti-Bcl-Xl and anti-Bcl-2 antisera (Santa Cruz
Biotechnology). For PKB/Akt assay, 100,000 cells were
lysed in SDS sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 50 mmol/L dithiothreitol [DTT], and 0.1% bromphenol blue).
The blots were probed with anti-phospho Akt (Ser473) antibody (New
England Biolabs, Beverly, MA) followed by detection using the
Phototope-HRP Western detection system (New England
Biolabs). After stripping of phosphospecific antibody, the blot was
reprobed with a general anti-PKB/Akt antibody (New
England Biolabs) that recognizes both phosphorylated and unphosphorylated PKB/Akt.
Gel mobility shift assay of STAT5 binding.
Nuclear STAT5 binding to radiolabeled DNA (prolactin inducible element)
(PIE) was done as described previously.11 For the supershift assays to verify the presence or absence of STAT protein in
the DNA binding complex, 2 µg of anti-STAT1, anti-STAT3, anti-STAT5A or anti-STAT5B antibodies (Zymed) were preincubated with 20 µg of
nuclear extract for 15 minutes on ice before addition of the radiolabeled DNA.
Detection of cell-surface c-Kit expression.
Surface c-Kit expression was detected using flow cytometry analysis.
For each sample, 1 × 105 HCD57 or HCD57-SREI cells were
washed in FACS buffer (Tris-buffered saline/4% fetal calf serum).
After preincubation of the cells in a monoclonal antibody (MoAb) that
blocks nonspecific binding of fluorescein isothiocyanate
(FITC)-conjugated antibodies to IgG (rat MoAb 2.4G2; a
generous gift from Dr William Paul, National Institutes of Health,
Bethesda, MD), the cells were incubated with FITC anti-cKit or FITC
anti-CD4 (Becton Dickinson, San Diego, CA) (to control for background
antibody staining) at 10 mg/mL for 30 minutes at 4°C. The cells were
then washed in fluorescence-activated cell sorter (FACS) buffer before
analysis using a FACScan flow cytometer (Becton Dickinson).
Sequencing of the EPOR in HCD57 and HCD57-SREI cells.
cDNA encoding the EPOR was cloned using reverse
transcriptase-polymerase chain reaction (RT-PCR) techniques. Briefly,
mRNA was isolated from HCD57 and HCD57-SREI cells using the Quick Prep Micro mRNA kit (Pharmacia, Piscataway, NJ). cDNA was generated from
mRNA using the First Strand cDNA Synthesis kit (Pharmacia). The cDNA
was subjected to PCR using Taq polymerase (GIBCO-BRL) and
oligonucleotides corresponding to the 5' and 3' end of the murine EPOR
gene. PCR fragments were isolated using the Geneclean procedure (BIO
101, Vista, CA) and cloned into the pCrII vector (Invitrogen, Carlsbad,
CA). Multiple clones of EPOR cDNA from both HCD57 and HCD57-SREI cells
were sequenced using the dideoxy method described by Sanger et
al48 using the Sequenase version 2.0 kit (Amersham,
Piscataway, NJ).
Immunoprecipitation, ERK-1, PI3-kinase, and PKB/Akt assay.
Immunoprecipitation of ERK-1 and quantitation of activity as determined
by incorporation of 32P from [ -32P]
adenosine triphosphate (ATP) into myelin basic protein was performed as
described previously.5 Immunoprecipitations and PI3-kinase
assays were conducted as described previously49 with slight
modifications. Briefly, 3 × 106 cells were washed twice
with ice-cold phosphate-buffered saline (PBS) and disrupted in a
standard lysis buffer (1% Nonidet P-40 [Pierce,
Rockford, IL]; 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L
EDTA, 100 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl
fluoride [PMSF], 10 mg/mL aprotinin). Lysates were centrifuged at
14,000g for 10 minutes at 4°C, and then were subjected to
immunoprecipitation with specific antibodies to EPOR, p85 domain of
PI3-kinase, IRS-2, JAK2, STAT5, and Vav by incubation at 4°C for 1 hour. Protein A-agarose beads (UBI, Lake Placid, NY) were added and
incubated for another 1 hour at 4°C. The immune complexes
were washed three times with lysis buffer, once with PBS, once with 0.5 mol/L LiCl in 0.1 mol/L Tris-HCl (pH 7.5), once with distilled water,
and once with a buffer containing 100 mmol/L NaCl, 0.5 mmol/L EGTA, and
20 mmol/L Tris-HCl, pH 7.5. The beads were suspended in 40 µL of
reaction buffer (100 mmol/L NaCl, 0.5 mmol/L EGTA, 20 mmol/L Tris-HCl,
pH 7.5, and 0.2 mmol/L adenosine). L-a-phosphatidylinositol was added
to reaction mixture at a final concentration of 0.3 mg/mL. The reaction
was initiated by addition of 10 µCi [-32P]ATP, and
cold ATP and MgCl2 to final concentrations of 135 mmol/L and 20 mmol/L, respectively. After 10 minutes at 25°C, the reaction was terminated by addition of 20 µL of 6N HCl. Phospholipids were extracted with 160 µL of CHC13:CH3OH (1:1), and separated by
thin-layer chromatography (TLC). Spots corresponding to PI3-phosphate
were visualized by a PhosphoImager (Molecular Dynamics). For PKB/Akt assay, cells were lysed in ice-cold buffer A (50 mmol/L Tris, pH 7.5, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L
Na3VO4, 0.1% 2-mercaptoethanol, 0.1%
Triton X-100 [Pierce], 50 mmol/L sodium
fluoride, 5 mmol/L sodium pyrophosphate, 10 mmol/L sodium
 -glycerophosphate). Lysates were centrifuged at 14,000g for
10 minutes at 4°C, and the supernatants were equalized for protein
concentration (1 mg/mL) by addition of buffer A. PKB/Akt was
immunoprecipitated from the supernatants containing 1 mg of protein
with an anti-PKB/Akt antibody (New England Biolabs Inc)
and incubated at 4°C for 1.5 hour. Protein A-agarose
beads (UBI) were added and incubated for another 1 hour at 4°C. The
beads were washed three times with buffer A containing 0.5 mol/L NaCl, twice with buffer B (50 mmol/L Tris-HCl, pH 7.5, 0.03% Brij-35, 0.1 mmol/L EGTA, and 0.1% 2-mercaptoethanol), and twice with assay dilution buffer (20 mmol/L MOPS, pH 7.2, 25 mmol/L b-glycerophosphate, 5 mmol/L EGTA, 1 mmol/L
Na3VO4, and 1 mmol/L DTT). The beads were
suspended in 30 µL of assay dilution buffer containing 10 mmol/L PKA
inhibitor peptide (Sigma, St Louis, MO). PKB/Akt-specific substrate
peptide (UBI) was added to reaction mixture at a final concentration of
100 mmol/L. The reaction was initiated by addition of 10 mCi
[-32P] ATP. After 10 minutes at 25°C, the
supernatants were mixed with 40% trichloroacetic acid
(TCA) and spotted onto P81 phosphocellulose paper. The
paper was washed three times with 0.75% phosphoric acid, once with
acetone, and counted by a scintillation counter.
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RESULTS |
Proliferation and apoptosis of EPO-independent HCD57-SREI cells.
Earlier studies in this laboratory5 showed that the
treatment of HCD57 cells with SCF in the absence of EPO increased
proliferation for 3 days but the effect was limited because the cells
underwent apoptosis. While SCF clearly acted as a mitogen, EPO was also required to prevent apoptosis. The proliferation of HCD57 cells in
either EPO or SCF is also shown in Fig 1A.
In contrast, other workers have shown that introduction of the EPOR
into nonerythroid cells will allow SCF to promote proliferation for
infinite generations, possibly through an interaction of c-kit with the
EPOR.24 We subcloned the HCD57 cells, hoping to select for
an exclusively SCF-dependent cell line which had enhanced signaling of
SCF through the EPOR. To accomplish this, the EPO-dependent HCD57 cells
were cultured in the absence of EPO but in the presence of 100 ng
SCF/mL.
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| Fig 1.
Proliferation and survival of HCD57 and HCD57-SREI cells.
(A) The cells were cultured in the indicated factor, 1 U EPO/mL, 100 ng
SCF/mL, a combination of 100 ng SCF and 1 U EPO/mL, or no added factors
in medium containing 25% fetal calf serum. At the indicated day after
culture, viable cells were counted in the presence of trypan blue.
Error bars indicate the standard deviation from triplicate
measurements. (B) HCD57-SREI cells have escaped apoptosis in the
absence of EPO. HCD57 cells and HCD57-SREI cells were cultured in the
presence of 1 U EPO/mL (+) absence of EPO () for 72 hours. The
genomic DNA was isolated and analyzed for fragmentation of DNA
characteristic of apoptosis as described previously.5
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After 5 days in medium without EPO but containing SCF, virtually all
the HCD57 cells had undergone apoptosis such that viable cells were
less than 5% of the cell population (Fig 1A, left panel). During the
next 2 weeks, the number of viable cells dropped below 1% of total
cells. However, after 2 weeks in culture, significant numbers of
healthy cells appeared in the culture. These cells were cultured for 10 weeks without EPO, at which point greater than 95% of the cells were
viable as determined by trypan blue exclusion. From these pooled cells,
two single-cell clones were isolated by limiting dilution. However, the
subcloned cell lines appeared to be identical to the three separate
isolations of pooled cells, suggesting a single population of cells was
selected by these conditions.
We have been able to select these EPO-independent cells only from
high-passage HCD57 cells and have tried in five different experiments
to select EPO-independent cells from low-passage HCD57 cells without
success. Therefore, it is probable that the selection in the EPO minus
culture (with or without SCF) repeatedly finds the same subpopulation
of EPO-independent cells that are present in the high-passage
population of HCD57 cells through a spontaneous mutation.
Whereas SCF had only a transitory effect of increased proliferation but
could not prevent apoptosis in the parental HCD57 cells, SCF stimulated
proliferation of these surviving cells (HCD57-SREI) over at least 5 days of culture (Fig 1A, right panel). EPO also stimulated
proliferation. Surprisingly, these cells required neither EPO nor SCF
for limited proliferation and total protection from apoptosis. As shown
in Fig 1B, these surviving cells did not undergo apoptosis in the
absence of EPO or SCF (Fig 1B, SREI-EPO) whereas the parental HCD57
cells degraded DNA to fragments that are characteristic of apoptosis
(Fig 1B, HCD57-EPO). Therefore, the cells were named HCD57-SREI (for
SCF-Responsive but EPO-Independent for protection from apoptosis).
HCD57-SREI cells have escaped apoptosis in the absence of EPO but
proliferation may be stimulated by either EPO or SCF.
HCD57-SREI do not produce EPO.
Some erythroleukemic cell lines may produce autocrine EPO. To
investigate whether the HCD57-SREI cells were secreting EPO into the
medium, we tested whether the parental HCD57 cells could survive in
medium conditioned by the HCD57-SREI cells. HCD57 cells did not
proliferate and underwent apoptosis in this conditioned medium even
when the HCD57-SREI medium was concentrated (data not shown). In
addition, the proliferation of HCD57-SREI cells was not dependent on a
high concentration of cells in culture and was not affected by the
addition of an anti-EPO antibody that blocked the proliferation of
HCD57 cells in EPO-containing medium (data not shown). To investigate
whether HCD57-SREI cells might produce EPO that is not secreted, we
probed a Northern blot of RNA prepared from HCD57-SREI cells with a
probe to EPO. No EPO mRNA was detected (data not shown). Thus, it
appeared that the EPO-independent phenotype of the HCD57-SREI cells did
not result from either autocrine EPO production or secretion of another
factor that maintains the viability of erythroid cells.
EPOR in HCD57-SREI is not activated through an auto-activating
mutation.
We tested whether the EPO-independent phenotype resulted from a
mutation of the EPOR gene. The arginine 129 to cysteine
mutation in the EPOR is an autoactivating mutation50 that
allows sulfhydryl bonding to form covalent dimers of EPOR in the
absence of EPO. Therefore, we isolated 10 cDNA clones of the EPOR from
these HCD57-SREI cells for sequencing. None of the 10 clones were found
to have the arginine 129 mutation. Full-length sequencing of two
HCD57-SREI cDNA clones showed four identical mutations compared with a
previously published sequence51 (amino acid 5, A to G
mutation in the second base of codon; amino acid 273, T to G mutation
of the first base of the codon; amino acid 334, mutation of A to G in
the third base of the codon; amino acid 350 mutation of T to G in the
third base of the codon). However, these mutations are conservative such that the amino acid sequence of the protein is not altered. Thus,
the EPOR peptide in HCD57-SREI cells is wild type and has not undergone
an autoactivating mutation.
Processing and expression of EPOR and c-Kit in HCD57-SREI cells.
In the parental HCD57 cells and other primary erythroid cells, most of
the cell-surface EPOR molecules are posttranslationally processed by
extensive N-linked glycosylation and phosphorylation of nontyrosine
residues into a 78-kD form.6,10 We examined the posttranslational processing and metabolism of the EPOR in HCD57-SREI cells to test whether overexpression of EPOR peptide, alternative posttranslational processing, or failure to downregulate in
the presence of EPO might contribute to EPO independence (or apparent
independence as a result of hypersensitivity to trace levels of EPO).
Figure 2A shows that the EPOR in HCD57-SREI
cells appears to be identically processed from the 62-kD peptide into the 78-kD EPOR as the parental HCD57 cells; however, the level of
expression of the EPOR was reduced in the HCD57-SREI cells. This
reduced level of EPOR expression correlated with the determination that
HCD57 cells (deprived of EPO overnight to upregulate receptors) had
3,800 binding sites for 125I-EPO whereas the HCD57-SREI
cells only expressed 500 binding sites for 125I-EPO. The
binding affinity for 125I-EPO was similar in both cell
lines (kd = 810 nmol/L and 780 nmol/L). In the presence
of EPO, the high-molecular-weight 68- to 78-kD forms of EPOR in both
HCD57 and HCD57-SREI cells were similarly downregulated within 40 minutes (Fig 2A, lanes D and H). Thus, there appeared to be no
alteration of the EPOR protein in HCD57-SREI cells that would explain
the EPO-independent phenotype.
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| Fig 2.
Processing and expression of c-Kit and EPOR in HCD57 and
HCD57-SREI cells. (A) HCD57 cells deprived of EPO overnight (lanes A
through D) or HCD57-SREI cells cultured without EPO (lanes D through G)
were left untreated (lanes A and E), or treated with 10 U EPO/mL for
the indicated times at 37°C. The cells were rapidly cooled, and the
forms of EPOR present were determined by immunoprecipitation of the
EPOR and detection of the receptor proteins using the same
affinity-purified anti-COOH terminal, anti-EPOR IgG. The form of EPOR
between 62 and 78 kD are marked (upper arrows). The IgG on the gel from
the heavy chain of anti-EPOR present in the immunoprecipitates is also
indicated (lower arrow). (B) HCD57-SREI cells exhibit upregulated
cell-surface c-Kit expression. HCD57 and HCD57-SREI cells were
incubated with an FITC-conjugated c-Kit antibody and detected using
flow cytometry. HCD57-SREI cells exhibit 81.4% more cell surface c-Kit
(peak C) than HCD57 (peak B). Peak A represents background staining
(CD4).
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We next examined cell-surface expression of c-Kit by incubation of
HCD57 and HCD57-SREI cells with a c-Kit antibody and detection of
surface-bound antibody by flow cytometry. Detection of surface c-Kit
expression showed an 81.4% increase in c-Kit surface expression in
HCD57-SREI cells (Fig 2B, peak C) as compared with HCD57 cells (Fig 2B,
peak B).
Expression of Bcl-XL and Bcl-2 in HCD57 and HCD57-SREI cells.
The above experiments indicated a nonreceptor explanation for
EPO-independence in HCD57-SREI cells. Overexpression of the Bcl family
of apoptosis modulating proteins can result in resistance to apoptosis.
Previous work had implicated Bcl-Xl but not Bcl-2 as a critical control
point in protecting from apoptosis in HCD57 cells.52
Therefore, we examined the expression of Bcl-Xl and Bcl-2 in the
HCD57-SREI cells. There was a similar level of expression of these two
apoptosis suppressing proteins in the HCD57-SREI cells (Fig
3, lanes D and H) as the parental HCD57
cells (Fig 3, lanes B and F). Thus, the protection from apoptosis in
the HCD57-SREI cells did not result from overexpression of the Bcl gene
products. However, we found that whereas the withdrawal of EPO
suppressed the expression of both Bcl-Xl and Bcl-2 proteins in HCD57
cells, Bcl-Xl levels were maintained in the presence or absence of EPO
in HCD57-SREI cells (Fig 3, lanes C and G). The EPO-independent
phenotype of HCD57-SREI cells, therefore, may be caused by an
alteration in signaling upstream of the regulation of Bcl-Xl gene that
maintains the expression of the Bcl-Xl protein even in the absence of
EPO.
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| Fig 3.
Bcl-Xl and Bcl-2 are maintained in HCD57-SREI cells when
EPO is withdrawn. HCD57 cells (lanes A, B, E, and F) and HCD57-SREI
cells (lanes C, D, G, and H) were cultured for 72 hours in either
medium containing 1 U EPO/mL (lanes B, D, F, H) or no EPO (lanes A, C,
E, G). Ten micrograms of cellular protein was analyzed by Western
blotting using anti-Bcl-Xl (lanes A through D) or anti-Bcl-2 (lanes E
through H) antisera. The proteins of interest were visualized with ECL
and are marked by an arrow.
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Tyrosine protein phosphorylation in HCD57 and HCD57-SREI cells.
Alterations in cellular signaling in HCD57-SREI cells might explain why
Bcl-Xl levels were maintained in the absence of EPO. Therefore, we
examined the phosphotyrosine-containing proteins in either untreated
cells or cells treated with SCF or EPO in HCD57-SREI and parental HCD57
cells. If an autoactivating mutation in the EPOR or a mutation
resulting in expression of EPO were present, we would expect to see
constitutive phosphorylation of the EPOR and JAK2 kinase as previously
reported.53 In addition, the constitutive secretion of SCF
would be expected to lead to constitutive tyrosine protein
phosphorylation of c-Kit. However, as shown in Fig
4A (top
panel), there was no apparent constitutive phosphorylation of bands
corresponding to EPOR, JAK2, c-Kit, or other major phosphotyrosine
[Tyr(P)] containing protein in HCD57-SREI cells, virtually ruling out
the possibility of an autoactivating mutation in either EPOR or JAK2 or
autocrine production of EPO or SCF.
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| Fig 4.
Effect of EPO and SCF on tyrosine protein phosphorylation
in HCD57 cell and HCD57-SREI cells. (A) HCD57 cells deprived of EPO
overnight (lanes A, B, C) or HCD57-SREI cells cultured without EPO
(lanes D, E, F) were left untreated (A, D) or treated with 10 U EPO/mL
for 10 minutes (B, E) or 100 ng SCF/mL for 10 minutes (C, F). The cells
were then rapidly chilled in an ice bath, a detergent lysate was
prepared, and phosphotyrosine containing proteins were
immunoprecipitated with a polyclonal anti-Tyr(P) antibody. The
immunoprecipitated proteins were then run on SDS-polyacrylamide gel
electrophoresis, Western blotted, and probed with a monoclonal
anti-Tyr(P) antibody (top panel) and then stripped of bound antibody
and reprobed with an anti-JAK2 antiserum (bottom panel). Bands were
visualized with ECL. (B) SHC protein tyrosine phosphorylation in
HCD57-SREI cells (lanes 1 through 3) and HCD57 cells (lanes 4 through
6). The cells were treated with nothing (lanes 1 and 4), SCF (lanes 2 and 5), and EPO (lanes 3 and 6) as described above and SHC proteins
were immunoprecipitated. The Western blot was probed with anti-Tyr(P)
(top panel) and then stripped of bound antibody and reprobed with
anti-SHC antiserum (bottom panel). (C) MAP kinase (ERK-1) activity in HCD57 and
HCD57-SREI cells. HCD57 cells were deprived of EPO overnight in IMDM
containing 25% serum. Aliquots of HCD57 cells that had been deprived
of EPO (lanes 1 through 4) and HCD57-SREI cell continually cultured
without EPO (lanes 5 through 8) were deprived of serum for 1 hour.
After this 1-hour incubation in serumless medium, either nothing (lanes
1 and 5), 1.0 U EPO/mL (lanes 2 and 6), 25% fetal calf serum (lanes 3 and 7), or a combination of 25% serum plus 1.0 U EPO/mL was added for
10 minutes at 37°C. After the 10-minute incubation, the cells were
rapidly chilled in cold medium, solubilized, and the ERK-1, MAP kinase
activity determined by immunoprecipitation and phosphorylation of
myelin basic protein as described in Materials and Methods. (D)
Constitutive STAT5 phosphorylation in HCD57-SREI cells. HCD57-SREI
cells (lanes A through C, G through I) and HCD57 cells (lanes E through
F, J through L) were treated with nothing (lanes A, D, G, J) or 10 U
EPO/mL for either 5 minutes (lanes B, E, H, K) or 10 minutes (lanes C,
F, I, L) at 37°C. Ten micrograms of total cellular extract (lanes
A through F) or STAT5 immunoprecipitated from 200 µg of cellular
extract (lanes G through L) were analyzed by Western blotting with
anti-Tyr(P) antiserum. Molecular-weight markers and migration of
proteins of interest are indicated.
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The Tyr(P)-containing proteins were very similar in the HCD57 and
HCD57-SREI cells after either EPO or SCF treatment with the exception
that the tyrosine-phosphorylated 78-kD band corresponding to EPOR was
more prominent in EPO-treated HCD57 cells, correlating with the
increased expression of the 78-kD EPOR. As shown by very careful
examination of Fig 4A, there was only a trace of Tyr(P) in the EPOR in
either HCD57 or HCD57-SREI cells treated with SCF. In data not shown,
we observed that there was twofold to threefold more tyrosine
phosphorylation of the 78-kD EPOR immunoprecipitated from HCD57-SREI
cells treated with SCF than the EPOR in comparably treated HCD57 cells.
This level of EPOR phosphorylation was only about 10% of the
phosphorylation achieved with EPO stimulation. A 150-kD band which
apparently corresponds to the SCF receptor was more strongly
phosphorylated in SCF-treated HCD57-SREI than HCD57 cells, correlating
with increased expression of c-Kit on HCD57-SREI cells. Another
approximately 30-kD protein of unknown identity was highly
phosphorylated in SCF-treated HCD57-SREI cells compared with HCD57
cells. Stripping this blot and reprobing with anti-JAK2 antiserum
showed that JAK2 was neither autophosphorylated in HCD57-SREI cells nor
tyrosine phosphorylated after SCF treatment (Fig 4A, bottom panel,
lane F).
We also studied the tyrosine phosphorylation of the SHC proteins in
both EPO-treated and SCF-treated HCD57 and HCD57-SREI cells (Fig 4B).
As suggested by the antiphosphotyrosine Western blots in Fig 4A,
immunoprecipitated SHC was not constitutively phosphorylated in either
cell type (Fig 4B, lanes 1 and 4). The lesser effect of EPO on SHC
phosphorylation in HCD57-SREI cells reflected the reduced expression
and phosphorylation of the EPOR.
In HCD57 cells, neither the EPO-dependent nor SCF-dependent SHC
tyrosine phosphorylation appeared to correlate with induced ERK-1
activity.5 However, recent information shows that ERK may
be activated by SHC-independent mechanisms.54 Therefore, we
tested MAP kinase (ERK-1) activity by immunoprecipitating ERK-1 proteins and measuring the activity that phosphorylated myelin basic
protein. In data not shown, a low level of constitutive activity of
ERK-1 was found in both HCD57 and HCD57-SREI cells in the absence of
growth factors but in the presence of 25% fetal calf serum. This
observation seems to eliminate the SHC/ERK pathway from a role in the
resistance to apoptosis upon EPO withdrawal seen in HCD57-SREI cells.
We then removed the serum for 1 hour and measured the basal levels of
ERK-1 in the absence of serum and the ERK activity after the addition
of 25% serum, 10 U EPO/mL, or a combination of serum and EPO. As shown
in Fig 4C, there was no detectable ERK-1 activity in either the HCD57
cells or HCD57-SREI cells in the absence of serum and EPO. It is
interesting to note that this experiment showed that MAP kinase
activity is poorly activated by either EPO alone or serum alone and
that there is a strong synergistic effect of serum and EPO in both
HCD57 and HCD57-SREI cells. However, the magnitude of the synergistic
effect of combining EPO and serum was much greater in the HCD57-SREI
cells than observed in the HCD57 cells. We found this surprising but
three separate experiments showed almost identical results as that
shown in Fig 4C.
STAT5A/B is constitutively phosphorylated, translocated to the
nucleus, and bound to DNA in the HCD57-SREI cells.
Although not evident in the above experiments, a band of about 100 kD constitutively tyrosine phosphorylated in the SREI
subclone but not the HCD57 cells, was seen in some experiments. To test whether this approximately 100-kD band might be an activated STAT protein, we immunoprecipitated STAT5A/B from HCD57 cells and HCD57-SREI cells and analyzed the tyrosine phosphorylation state of STAT5 in the
presence and absence of EPO (Fig 4D). This experiment confirmed that
STAT5 was constitutively phosphorylated on tyrosine residues in the
HCD57-SREI cells (Fig 4D, lane G). Additional experiments with STAT5A-
and STAT5B-specific antisera show that both forms of STAT5 were
phosphorylated, but we did not find other STATs to be constitutively
active. Interestingly, the major tyrosine phosphorylated band at 95 kD
seen in EPO-treated HCD57 and HCD57-SREI cells was not the STAT5
protein as many have assumed. STAT5 was a minor component of this 95-kD
band (STAT5 migrates at the very top of this broad band). Although
STAT1 is also activated by EPO in HCD57 cells, the level of
phosphorylation of STAT1 is an order of magnitude less than STAT5 and,
therefore, does not contribute significantly to this band, leaving the
identity of the band a mystery.
We did not see constitutively phosphorylated STAT5 in the Tyr(P)
immunoprecipitate of HCD57-SREI cells (Fig 4A); however, this was due
to the inability of the polyclonal anti-Tyr(P) antibody used to
immunoprecipitate the Tyr(P)-containing proteins (Zymed) to recognize
phosphorylated STAT5. Figure 4D shows that when the total cellular
extract was examined by blotting with anti-Tyr(P) [without
immunoprecipitation to concentrate Tyr(P) proteins] the STAT5A/B
constitutive tyrosine phosphorylation was not apparent. We were able to
detect constitutive phosphorylation of STAT5 only when
20-fold more HCD57-SREI cells were used to immunoprecipitate STAT5 A/B compared with the number of cells used in the total cellular
extract. However, parallel experiments with very concentrated cell
extracts did not show constitutively phosphorylated JAK kinases or EPOR.
We also tested whether there was constitutive activation of STAT
proteins in the nuclear fraction of SREI cells. As shown in Fig
5, HCD57-SREI cells showed constitutive
nuclear localization of STAT5 proteins (Fig 5A, lane 5), tyrosine
phosphorylation of a 95-kD band corresponding to STAT5 (Fig 5B, lane
5), and activation of DNA binding activity characteristic of STAT5 (Fig
5C, lane 5). The addition of EPO to HCD57-SREI cells led to additional activation of STAT5 phosphorylation, translocation, and DNA binding activity above the constitutive level (Fig 5C, lane 6). In
contrast, the HCD57 cells did not have any detectable constitutive
activation of STAT5 activity in the absence of EPO treatment (Fig 5C,
lane 1). SCF treatment of HCD57 cells did not activate STAT5 nor did SCF treatment of HCD57-SREI cells increase the activity of STAT5 above
the constitutive activation (Fig 5C, lane 7). To confirm that the DNA
binding activity to the PIE DNA element constitutively activated in
HCD57-SREI cells was STAT5 and to further discriminate whether that
activity was due to STAT5A or STAT5B, we used super-shift analysis with
various anti-STAT antisera. Antibodies to STAT1, STAT3, STAT5A, and
STAT5B were incubated with nuclear extracts to test the identity of the
proteins binding to the PIE DNA. As shown in Fig 5D, no super-shift was
detected when anti-STAT1 or anti-STAT3 antibodies were present,
indicating the likely absence or low abundance of these factors in the
DNA binding complex. However, both anti-STAT5A and anti-STAT5B
antibodies super-shifted the complex. This result suggests that
heterodimers of STAT5A and STAT5B are the predominant members of the
PIE DNA binding complex constitutively bound in HCD57-SREI cells in the
absence of factors and in EPO-treated HCD57 and HCD57-SREI cells.
Interestingly, two minor bands present in the complex indicated that
both STAT5A homodimers and STAT5B homodimers also bind to the DNA.
These homodimers could be discriminated from the heterodimers of STAT5A
and STAT5B by faster migration on the polyacrylamide gel, with
STAT5A:STAT5A homodimers migrating faster than the STAT5B:STAT5B
homodimers.
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| Fig 5.
Characterization of nuclear extracts made from EPO- and
SCF-treated HCD57 (lanes 1 through 4) and HCD57-SREI cells (lanes 5 through 7). Cells (2 × 107) were
treated with nothing (lanes 1 and 5), EPO (10 U/mL) (lanes 2 and 6),
interferon- (5 ng/mL) (lane 4), or SCF (100 ng/mL) (lanes 3 and 6)
for 10 minutes at 37°C. The immunoblot was probed with MoAb to STAT5
(A) and reprobed with MoAbs to PY (B). The 95K arrows mark the
molecular weight and position where the STAT5 proteins migrate on the
gel. (C) Mobility-shift assays of the PIE binding nuclear proteins from
EPO and SCF treatment. Nuclear protein preparation and gel shift
analyses with the PIE sequence were performed as previously
described.11 Nuclear protein (20 µg) from control (C)
lanes 1 and 5, and EPO-treated extracts (lanes 2 and 6) and SCF-treated
cells (lanes 3 and 7) were incubated with the radiolabeled
oligonucleotide, and shifted bands were visualized by autoradiography.
Only upon a very dark exposure can a minor band below the position of
the major band be seen that arises from STAT1 binding in either EPO- or
interferon-treated HCD57 cells. The HCD57 cells seem to be very
unresponsive to interferon- compared with primary erythroid cells.
(D) Super-shift analysis shows that STAT5A/B heterodimers are the
constitutively active DNA-binding proteins. The cells were treated as
before, or left untreated and the gel mobility shift assay was
performed as above except that either anti-STAT1, anti-STAT3,
anti-STAT5A, or anti-STAT5B were preincubated for 15 minutes on ice
with the nuclear extracts before the addition of the radiolabeled PIE
DNA. The bar indicates the supershift of DNA binding that occurred with
either anti-STAT5A or anti-STAT5B antisera. The deduced positions of
apparent shifts resulting from either STAT5A:STAT5B heterodimers
(STAT5A:B), STAT5A homodimers (STAT5A:A), or STAT5B homodimers
(STAT5B:B) are indicated by arrows.
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The finding of constitutive STAT5 activity without a corresponding
constitutive activity of JAK2 led us to further investigate if there
might be constitutive activation of other members of the Janus kinases.
Therefore, we immunoprecipitated JAK1, JAK2, JAK3, and TYK2 from
HCD57-SREI cells either untreated or treated with EPO. When the
immunoprecipitate was analyzed by Western blotting with anti-Tyr(P)
antiserum, only JAK2 from EPO-treated cells was observed to be
phosphorylated (data not shown).
Does a phosphatase defect explain the HCD57-SREI resistance to
apoptosis?
The observation showing that sodium vanadate
(Na3VO4), an inhibitor of tyrosine protein
phosphatase activity, can activate STAT protein independently of Janus
kinases suggested that constitutive activation of STAT5 might be due to
the loss of phosphatase activity in HCD57-SREI cells.55,56
The SHP-1 tyrosine protein phosphatase has been strongly implicated in
the modulation of both JAK2/EPOR and STAT
signaling.22,57-60 We and others have previously reported that HCD57 cells are hypersensitive to EPO.10,61 Therefore, we tested the hypothesis that the hypersensitivity of HCD57 cells to
EPO might be caused by a reduction of the SHP-1, and that the loss of
EPO-dependence in the HCD57-SREI cells could be the result of the
further reduction or complete loss of this phosphatase. We compared the
levels of SHP-1 in these two cell lines with a population of CFU-E and
proerythroblasts purified from normal human blood. Figure
6 shows that identical levels of SHP-1 were present in normal immature human erythroid cells, HCD57 cells, and the
apoptosis-resistant HCD57-SREI cells.
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| Fig 6.
The tyrosine protein phosphatase, SHP-1, is equally
expressed in HCD57, HCD57-SREI cells and primary human erythroid cells.
Frozen HCD57 and HCD57-SREI cells were sent to Dr Amittha Wickrema who
compared 10 µg of total cellular protein from these cell lines with
an equal amount of primary erythroid cells at the CFU-E stage of
development that develop in culture from more immature erythroid cells
purified from human blood. The Western blot was probed with anti-SHP-1
antiserum and visualized by ECL.
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Constitutive activation of PI3-kinase in HCD57 and HCD57-SREI cells.
PI3 kinase has been reported to be activated as part of the EPO
signaling pathway. Because PI3-kinase has been implicated in several
cell types as critical to the regulation of cell proliferation, differentiation, and survival,62-64 we measured PI3-kinase
activity and tested for interaction of PI3-kinase with proteins known
to associate with PI3-kinase. This study compared these parameters in
the EPO-dependent HCD57 cells and the EPO-independent HCD57-SREI cells.
Figure 7A shows that the PI3-kinase
activity measured by incorporation of 32P from
gamma-labeled ATP into phosphatidylinositol by the immunoprecipitated PI3-kinase (anti-p85 subunit of PI3-kinase) appeared constitutive in
both HCD57 and HCD57-SREI cells and was not additionally increased by
treatment of EPO. The bottom panel of Fig 7A shows the combined results
from five experiments that confirm the lack of EPO effect. To verify
that this unexpected constitutive activity which phosphorylated phosphatidylinositol was truly PI3-kinase activity, we added the PI3-kinase inhibitor, LY294002, to the reaction mixture and showed that
the activity was inhibited in a dose-responsive fashion at concentrations of inhibitor known to specifically block PI3-kinase activity (shown in Fig 7B). For a further control, we examined the
PI3-kinase activity in immunoprecipitated STAT5, JAK2, Vav, as well as
PI3-kinase. PI3-kinase activity was only detected in the
immunoprecipitated PI3-kinase (anti-p85 immunoprecipitate), as shown in
Fig 7C.
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| Fig 7.
PI 3-kinase was constitutively activated in HCD57 and
HCD57-SREI cells. (A) Analysis of PI 3-kinase activity in HCD57 and
HCD57-SREI cells. (Top panel, A) 3 × 106 HCD57 (lanes 1 and 2) and HCD57-SREI (lanes 3 and 4) cells were deprived of EPO
overnight and treated with 10 U/mL EPO (lanes 2 and 4) for 5 minutes or
left untreated (lanes 1 and 3). Cells were lysed and immunoprecipitated
by anti-p85 subunit of PI 3-kinase antibody, and the precipitates were
assayed for PI3-kinase activity as described in Materials and Methods.
The positions of the PI 3-phosphate product (PI3-P) and the origin are
indicated. (B) Inhibition of in vitro PI3-kinase activity by LY294002.
(Top panel, B) HCD57 cells were deprived of EPO overnight and treated
with EPO for 5 minutes. Immunoprecipitates were prepared from HCD-57
cells as described previously. PI 3-kinase assay was performed in the
presence of various concentrations of LY294002 (lanes 5 through 8) as
indicated. (Bottom panels, A and B) PI 3-kinase activities were
quantified and the results were expressed as fold activity compared to
the activity of HCD57 cells not treated with EPO. Data are means ± SD
from three independent experiments. (C) Analysis of PI 3-kinase
activity in different immunoprecipitates. Cell lysates were prepared
from HCD-57 cells cultured in normal medium and immunoprecipitated by
anti-p85 subunit of PI 3-kinase (lane 9), anti-JAK2 (lane 10),
anti-STAT5 (lane 11), or anti-Vav (lane 12) antibodies. The
precipitates were assayed for PI 3-kinase activity as described in
Materials and Methods. The positions of the PI3-phosphate product
(PI3-P) and the origin are indicated.
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As a further control that the EPO signaling pathway was not activated
in the EPO-independent HCD57-SREI cells, we also measured the
PI3-kinase activity that associated with the EPOR in either untreated
or EPO treated HCD57 and HCD57-SREI cells. As shown in Fig
8A, in either cell, there was no PI3-kinase
activity in untreated cells, but EPO treatment led to a large increase
in PI3-kinase activity bound to the EPOR.
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| Fig 8.
PI 3-kinase activity associates with the EPOR and IRS-2.
(A) HCD57 (lanes 1 and 2) and HCD57-SREI (lanes 3 and 4) cells were
deprived of EPO overnight and treated with 10 U/mL EPO (lanes 2 and 4)
for 5 minutes or left untreated (lanes 1 and 3). Cells were lysed and
immunoprecipitated by anti-EPOR antiserum, and the precipitates were
assayed for PI3-kinase activity as described in Materials and Methods.
The positions of the PI3-phosphate product (PI3-P) and the origin are
indicated. (B) HCD57 (lanes 5 and 6) and HCD57-SREI (lanes 7 and 8)
cells were deprived of EPO overnight and treated with 10 U/mL EPO
(lanes 6 and 8) for 5 minutes or left untreated (lanes 5 and 7). Cells
were lysed and immunoprecipitated by anti-IRS-2 antibody, and the
precipitates were assayed for PI3-kinase activity as described in
Materials and Methods. The positions of the PI3-phosphate product
(PI3-P) and the origin are indicated.
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During this study we noticed that there was a constitutively
tyrosine-phosphorylated protein of 185-kD that coprecipitated with
PI3-kinase in both HCD57 and HCD57-SREI cells. In unpublished work
(H.B. and S.T.S., 1998), we found that this protein was insulin receptor substrate-2 (IRS-2). To verify that the PI3-kinase that was
associated with the IRS-2 protein was active, we measured the
PI3-kinase activity in the IRS-2 immunoprecipitates. As shown in Fig
8B, PI3-kinase activity was constitutive and unaffected by EPO
treatment in either HCD57 or HCD57-SREI cells, strongly suggesting that
IRS-2 is responsible for the constitutive activity of PI3-kinase in the
in vitro assay. It is possible that the PI3-kinase is activated and
becomes associated with the tyrosine-phosphorylated IRS only after the
cells are disrupted and does not reflect the in vivo activity of
PI3-kinase.
To test the physiological role of either the constitutive or
factor-stimulated PI3-kinase in the EPO-dependent HCD57 cells and the
EPO-independent HCD57-SREI cells, the effect of inhibiting PI3-kinase
activity by the PI3-kinase selective inhibitor, LY294002, was tested by
measuring the number of viable cells and DNA fragmentation as
indicators of proliferation and apoptosis, respectively.
Figure 9A shows the effect of the indicated
concentrations of the LY294002 on viable cell number of either HCD57
cells in EPO or the EPO-independent HCD57-SREI cells in the
presence or absence of EPO. The HCD57-SREI cells in the absence of EPO
are more sensitive to the inhibitor compared with either HCD57 or
HCD57-SREI cells exposed to EPO. At 10 µmol/L LY294002, there are
only half as many viable HCD57-SREI cells in the absence of EPO
surviving compared with HCD57 or HCD57-SREI cells in the presence of
EPO. At 25 µmol/L LY294002, HCD57-SREI cells minus EPO have fallen to
20% of control while either the HCD57 or HCD57-SREI cells in EPO were
only moderately diminished (50% to 70% the number of cells that have
proliferated in the absence of inhibitor). To test whether the effect
of the PI3-kinase inhibitor could be determined to be an effect only on
proliferation versus an effect that might be the summation of
eliminating cells by inducing apoptosis or programmed cell death, we
isolated DNA from cells treated with various concentration of LY294002
for 24 hours and analyzed whether this DNA was intact or degraded into
the ladder pattern that distinguished apoptosis. As shown in Fig 9B, no
DNA fragmentation was observed in HCD57 until the highest concentration
of inhibitor was used. However, in the EPO-independent HCD57-SREI cells
(no EPO), DNA fragments characteristic of the intranucleosomal cleavage
induced in apoptosis were observed in the lowest concentration of
LY294002 used. Therefore, the reduction in viable HCD57-SREI cells that
results from inhibition of PI3-kinase certainly is in part caused by
apoptosis.
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| Fig 9.
Effect of inhibition of PI3-kinase activity on the
proliferation and apoptosis of HCD57 and HCD-SREI cells. (A) Cells
cultured in the presence of 1 U EPO/mL (HCD57,  ; HCD57-SREI,  ) or
absence of EPO (HCD57-SREI, x) were treated with LY294002 at the
indicated concentrations for 72 hours, and the viable cells were
counted in the presence of trypan blue. Values are expressed as a
percentage of the corresponding control (treated with vehicle
dimethylsulfoxide [DMSO]). Data are means ± SE of at least
triplicate measurements. (B) Cells were cultured in the presence of 1 U
EPO/mL (HCD57) or absence of EPO (HCD57-SREI). HCD-57 (lanes 2 through
6) and HCD57-SREI (lanes 7 through 11) cells were treated with vehicle
DMSO (lanes 3 and 8), LY294002 at indicated concentrations (lanes 4 through 6 and 9 through 11), or untreated (lanes 2 and 7) for 24 hours.
The genomic DNA was isolated and analyzed for fragmentation of
DNA characteristic of apoptosis as described in Materials and
Methods.
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Factor-dependent and constitutive activation of PKB/Akt.
The observation of both EPO-dependent and constitutive PI3-kinase
activity led us to test if the downstream effectors of PI3-kinase correlated with observed PI3-kinase activity. Activity of PKB/Akt was
assessed two ways, by activity (phosphorylation)-specific antibodies
and the ability of immunoprecipitated PKB/Akt to phosphorylate a
peptide substrate. In sharp contrast to the finding of constitutive activity of PI3-kinase in the anti-p85 PI3-kinase immunoprecipitate in
both EPO-dependent HCD57 cells and EPO-independent HCD57-SREI cells (in
Fig 7), there was constitutive activity of PKB/Akt activity only in the
EPO-independent HCD57-SREI. There was no detectable phosphorylation of
PKB/Akt in the HCD57 cells in the absence of added factors (Fig
10). The constitutive activation of
PKB/Akt in HCD57-SREI cells and EPO-dependent activation in HCD57 cells was confirmed by the in vitro assay of PKB/Akt. The in vitro assay of
PKB/Akt gave the following result when PKB/AKt activity was determined
(assayed as described in Materials and Methods and expressed as the
average of three assays to determine the cpm of 32P from
ATP incorporated into a substrate peptide ± standard deviation): HCD57 EPO = 132 ± 94 cpm; HCD57 + EPO = 6,725 ± 163 cpm; and HCD57-SREI cells EPO = 933 ± 328 cpm. Moreover, treatment of the
HCD57 cells with either EPO or SCF led to a rapid activation of PKB/Akt
(Fig 10A). Comparison of the EPO-dependent and SCF-dependent time
course of PKB/Akt phosphorylation showed that the EPO effect was
greater in magnitude than the SCF effect. PKB/Akt phosphorylation was
maximal after 5 minutes of treatment with either EPO or SCF, it then
declined by 1 hour and was virtually undetectable after 6 hours (Fig
10B).
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| Fig 10.
PKB/Akt activity is constitutive in HCD57-SREI cells but
activity is induced by EPO and SCF in HCD57 cells. (Top, A) HCD57 cells
deprived of EPO overnight (lanes 1, 2, 5, 6) or HCD57-SREI cells
cultured without EPO (lanes 3, 4, 7, 8) were left untreated (1, 3, 5, 7) or treated with 10 U EPO/mL for 5 minutes (lanes 6, 8) or 100 ng
SCF/mL for 5 minutes (lanes 2, 4). Cell lysates were prepared and
subjected to Western blot analysis as described in Materials and
Methods. The blot was probed with anti-phospho-PKB/Akt antibody (top
panel). After being stripped, the blot was reprobed with
a general anti-PKB/Akt antibody that recognizes all forms of the
protein (bottom panel). (B) Time course of EPO and SCF-induced PKB/Akt
activation. HCD57 cells deprived of EPO overnight were left untreated
(lanes 1, 5) or treated with 10 U EPO/mL (lanes 2 through 4) or 100 ng
SCF/mL (lanes 6 through 8) for indicated time periods. Cell lysates
were prepared and subjected to Western blot analysis as described in
Materials and Methods. The blot was probed with anti-phospho-PKB/Akt
antibody (top panel). After being stripped, the blot was reprobed with
the general anti-PKB/Akt antibody used above (bottom panel). (C)
PI3-kinase inhibitor blocked EPO-induced PKB/Akt phosphorylation in
HCD57-SREI cells. HCD57-SREI cells cultured without EPO were left
untreated (lanes 1, 7), pretreated with DMSO (lanes 2, 8), or
pretreated with LY294002 (lanes 3 through 6, 9 through 12) at the
indicated concentration for 4 hours. Then cells were left untreated
(lanes 1 through 6) or treated with 10 U EPO/mL for 5 minutes (lanes 7 through 12). Cell lysates were prepared and subjected to Western blot
analysis as described in Materials and Methods. The blot was probed
with anti-phospho-PKB/Akt antibody (top panel). The blot was stripped
of the phospho-specific antibody and reprobed with an antibody that
recognizes both phosphorylated and nonphosphophorylated PKB/Akt to
verify equal loading (bottom panel).
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Because of the possibility that the PKB/Akt might be activated by
upstream activators not in the PI3-kinase pathway (due to lack of
correlation of PKB/Akt activity with the constitutive PI3-kinase
activity in HCD57 cells), the activity of PKB/Akt detected with the
activation (phospho)-specific antibody was tested after inhibition of
PI3-kinase activity with LY294002. As shown in Fig 10C, treatment of
the HCD57-SREI cells in the presence or absence of EPO resulted in
inhibition of PKB/Akt, establishing that PI3-kinase was the upstream
activator. The effect of LY294002 on PKB/Akt activation was also tested
in HCD57 cells in the presence of EPO and PKB/Akt inhibition was very
similar to inhibition observed in HCD57-SREI cells in the presence of
EPO (data not shown); however, in the EPO-independent, HCD57-SREI cells
in the absence of EPO, PKB/Akt phosphorylation was more sensitive to
LY294002. PKB/Akt activity in HCD57-SREI cells without EPO was greatly
reduced by treatment with 5 µmol/L LY294002 whereas treatment with 25 µmol/L LY294002 was required for the same magnitude of inhibition of PKB/Akt phosphorylation in either HCD57 cells or HCD57-SREI cells in
EPO. This result established a correlation between the level of PKB/Akt
activity and cell proliferation and/or viability. HCD57-SREI cells in
the absence of EPO proliferated less (Fig 9A), had a higher percentage
of cells in apoptosis (Fig 9B), and had less PKB/Akt activity when
exposed to LY294002 (Fig 10C) than either the HCD57-SREI cells exposed
to EPO or HCD57 cells in EPO treated in parallel with LY294002.
| |
DISCUSSION |
Experiments reported here strongly suggest a major role of the
PI3-kinase/PKB(Akt) signaling pathway in maintaining the viability and/or proliferation of erythroid cells. Interestingly, whether PI3-kinase/PKB activity is stimulated by EPO treatment or is
constitutively activated through an unknown mechanism in
EPO-independent, apoptosis-resistant cells, blocking the activity of
this pathway results in apoptosis of either cell type. This strongly
implicates PI3-kinase/PKB activity as a necessary signal for survival.
The observation that SCF treatment also activates PI3-kinase/PKB in
erythroid cells but does not protect these cells from
apoptosis5 (and Fig 1A) indicates that PI3-kinase/PKB
signals may be necessary, but PI3-kinase/PKB activity are
not sufficient for the protection from apoptosis. Therefore, another
signaling pathway in addition to PI3-kinase/PKB pathway must also be
active to maintain the viability of erythroid cells. The
above-described study suggests that constitutive STAT5 activity in
combination with PI3-kinase/PKB signals might be sufficient to protect
the EPO-independent cells from apoptosis; however, signaling pathways
yet to be discovered or known signaling pathways not tested could also
be active and required for survival and proliferation.
We undertook the subcloning of the HCD57 cells in the presence of SCF
to test if SCF was capable of activating the complete EPO signaling
pathway through the interaction of the EPOR and c-Kit
molecules.5,24 Although we established the
HCD57-SREI cell line and found long-term responsiveness to SCF, no
subclones depended on SCF for survival. This strongly suggests that SCF (either through c-Kit or the EPOR) cannot generate the same
anti-apoptotic signal in the HCD57 erythroid cell that is triggered
through the EPOR by EPO. The current study is consistent with our past
work indicating a role of JAK2/STAT5 upstream of the EPO-dependent prevention of apoptosis.5 Whereas EPO activated the
JAK2/STAT5 pathway and prevented apoptosis, SCF failed to protect from
apoptosis and did not activate JAK2/STAT5. However, we cannot determine that STAT5 activity is required for protection from apoptosis in the
current study. The effect of antisense RNA or a dominant negative STAT5
mutant may shed some further light on the role of STAT5 in the
regulation of EPO-independent proliferation and survival.
The SCF effect on preventing apoptosis may be dependent on the cell
line selected for study. SCF can prevent apoptosis in the nonerythroid,
hematopoietic cell line FDC-2.65 Wu et al24 found that SCF could completely replace the requirement for other hematopoietic factors only through the cooperation of the c-Kit and
EPOR. The study by Joneja et al65 contradicts Wu et al and confirms that SCF does not activate JAK2, but the differences in these
published results may simply reflect that some cells are inherently
more resistant to apoptosis after growth factor withdrawal. We report
here that HCD57-SREI cells have escaped the apoptosis normally
triggered by EPO withdrawal. However, these cells will readily undergo
apoptosis when kinases are inhibited by a wide spectrum inhibitor,
staurosporine (not shown), or the inhibition of PI3-kinase activity by
LY294002 (Fig 9B), proving that the machinery for carrying out the
final stages of apoptosis is intact in HCD57-SREI cells.
HCD57-SREI cells have evolved a mechanism of resisting apoptosis that
includes the EPO-independent expression of the Bcl family of proteins.
We previously showed the induction of JunB and AP1 DNA binding activity
after EPO withdrawal in HCD57 cells and that a dominant negative form
of AP1 prevented both the apoptosis and the loss of expression of
Bcl-Xl after EPO withdrawal.66 In contrast to the
EPO-dependent cells, the apoptosis-resistant HCD57-SREI cells did not
show any changes in AP1 binding activity or express junB when
the cells were deprived of EPO.66 Because the
apoptosis-inducing activity of AP1 is suppressed in HCD57-SREI cells,
control of expression of Bcl-X1 through an AP1-dependent mechanism is
consistent with the hypothesis we have suggested
previously.66 The observation of a correlation of Bcl-Xl
expression with resistance to apoptosis extends earlier work of Silva
et al,52 who showed that unregulated expression of human
Bcl-Xl could prevent the murine HCD57 cells from undergoing apoptosis.
The LY294002 study showed that PI3-kinase activity was necessary for
erythroid cell survival and proliferation, even if other signaling
pathways were active. PKB/Akt activity correlated inversely with
susceptibility to apoptosis; PKB activity was inhibited by fivefold
lower concentrations of LY294002 and more apoptosis was induced in
HCD57-SREI cells in the absence of EPO than either cell line treated
with EPO. IRS-2 and PI3-kinase have both been reported to be activated
by EPO binding to the EPOR.20,21,47,67-69 However, the
constitutive activation of these signals in either the HCD57 cells
or the HCD57-SREI cells does not involve JAK2 or the EPOR. The
HCD57-SREI cells neither produced nor secreted EPO, did not appear to
secrete another factor that allowed HCD57 cells to survive in the
absence of EPO, and did not have a mutated EPOR activated in the
absence of EPO. The EPOR also did not become tyrosine phosphorylated
without the binding of EPO. In addition, there was no detectable
association of IRS-2 or PI3-kinase until EPO interacted with the EPOR.
We report here that IRS-2 and PI3-kinase were constitutively active in
both HCD57 and HCD57-SREI cells; however, only constitutive PKB/Akt
activity appeared to be a signal that protected the HCD57-SREI cells
from apoptosis in the absence of EPO. It is puzzling that the apparent
constitutive activity of PI3-kinase in the EPO-dependent HCD57 cells
did not result in the downstream constitutive activity of the PKB/Akt
activity. There are at least two explanations for this paradox: the
first is that the PI3-kinase detected in the in vitro activity is an
artifact of disrupting the cells and immunoprecipitating PI3-kinase or
IRS-2. The second possibility is that the PI3-kinase may truly be
activated by IRS-2 in HCD57 cells but that this activity is not
translocated to the plasma membrane until either EPO or SCF is added to
the cells.
There are reports that link constitutive activity of PI-3 kinase to
carcinogenesis43,44; however, the apparent constitutive activation of IRS-2/PI3-kinase in the HCD57 cells does not allow these
cells to proliferate or survive in the absence of EPO. This study
suggests that the activity of the downstream modulator of PI3-kinase,
PKB/Akt, is a better marker of transformed cells. BCR/ABL is known to
activate the PI3-kinase/PKB signaling pathway, implicating this signal
in the pathology of chronic myelogenous leukemia.47,70 PKB
was initially identified as the cellular homologue of the v-akt
oncogene and PKB/c-Akt is overexpressed in tumors.47
Preliminary evidence suggests that overexpressed PKB/Akt better
correlates with tumor aggressiveness rather than tumor induction. This
possibility is consistent with the finding here of an erythroleukemic
cell line progressing from a hormone-dependent cell line to a
hormone-independent cell line as a result of acquiring constitutively
active PKB/Akt.
Klingmüller et al71 have recently suggested that
PI3-kinase acts in the EPO signaling pathway as an intermediate in the activation of the MAP kinase cascade. The current study does not support this hypothesis. Constitutive activity of the PI3-kinase/PKB pathway (even in the absence of serum) compared with the lack of
detectable ERK-1 activity in unstimulated cells strongly suggests that
PI3-kinase activity alone is insufficient to trigger MAP kinase
activation. It is interesting to note that the synergistic effect of
serum and EPO on MAP kinase activation is magnified in the HCD57-SREI
cells compared with the parental HCD57 cells (Fig 4C). It is possible
that the constitutive PI3-kinase/PKB/Akt activity in the HCD57-SREI
cells may act upstream to magnify the synergistic activation of the MAP
kinase cascade by serum and EPO, even though the PI3-kinase/PKB pathway
does not activate either SHC or MAP kinase.
The HCD57-SREI cells expressed constitutively activated STAT5a and
STAT5b, yet this signaling pathway was completely dependent on EPO in
the parental, EPO-dependent HCD57 cells. Therefore, STAT5 may act
upstream of an intracellular signal that protects erythroid cells from
apoptosis (but this study shows that signals such as PKB/Akt are also
required). Recent studies showing that "knock out" of STAT5a/b
does not have significant effects on erythroid cell development do not
rule out the role of constitutive STAT5 activation acting to prevent
apoptosis.40-42 Indeed, even if STAT5 may not be required
for erythroid cell proliferation and protection from apoptosis,
constitutive activation of STAT5 may, nevertheless, have consequences
in erythroid cell signaling. Our experiments failed to show any
constitutive activation of JAK2 or other known member of the Janus
kinase family. Therefore, the mechanism by which STAT5 becomes
phosphorylated and activated to bind to DNA in HCD57-SREI cells is not apparent.
It has been reported that sodium vanadate activates STAT proteins
independently of JAK kinase.55,56 This work suggests a low
level of STAT activation by non-Janus kinase(s) that only becomes
apparent when tyrosine protein phosphatase activity is inhibited. This
observation and our own finding that sodium vanadate can activate STAT5
in FVA cells11 suggests that a possible reduction or loss
of tyrosine phosphatase activity in HCD57-SREI cells. The tyrosine
protein phosphatase SHP-1 has been implicated in regulating both the
activity of the EPOR/JAK222 and has been reported to
dephosphorylate STAT proteins in the nucleus.58 We tested
the hypothesis that SHP-1 might be reduced or absent in the
EPO-independent HCD57-SREI cells by measuring the expression of SHP-1
by Western blotting (Fig 6). We found that the levels of SHP-1 proteins
in both HCD57 and HCD57-SREI cell lines were identical to that of
primary human erythroid cells. Although this result suggests that the
defect in HCD57-SREI cells responsible for the escape from apoptosis in
the absence of EPO is not due to SHP-1, this experiment does not rule
out the loss of other phosphatase activity in these cells.
Constitutive activity of the JAK/STAT pathway is linked to
leukemogenesis and the transformation of other cancerous
cells.26-35 Recent reports have identified the constitutive
activation of STAT5 and STAT1 in peripheral cells from acute
lymphoblastic leukemia and constitutive STAT1, STAT3, and STAT5
activity in peripheral cells from patients with acute myeloid
leukemia.25 Constitutive activation of STAT5 is found in a
variety of primary lymphoid and myeloid leukemia cells and constitutive
STAT1 and STAT3 Epstein-Barr virus-related lymphoma
cells.26 Constitutive STAT3 has been associated with breast
cancer cells.27,28 The observation that oncogenes like
v-Src, v-Abl, and Bcr/abl may activate the JAK/STAT pathway
independently of growth factors30,33,35 further suggests that constitutive STAT activity may contribute to the transformation of
cells from factor-dependent to factor-independent proliferation. This
study suggests a role for constitutive STA5a/b activity in a supporting
role to constitutive PKB/Akt activity in the progression of an
erythroleukemic cell line to factor-independent viability and proliferation.
| |
ACKNOWLEDGMENT |
The authors thank Dr John Ryan (Virginia Commonwealth
University, Richmond, VA) for his assistance with the flow cytometry analysis.
| |
FOOTNOTES |
Submitted March 20, 1998; accepted February 1, 1999.
Supported by National Institutes of Health Grant No. RO1DK39781 to
S.T.S. and an American Heart Association Fellowship Grant to S.M.J.-H.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephen T. Sawyer, PhD, Department of
Pharmacology & Toxicology, Medical College of Virginia of Virginia
Commonwealth University, PO Box 980613, Richmond, VA 23298-0613. e-mail: ssawyer{at}hsc.vcu.edu.
| |
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INTRODUCTION