|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1568-1577
Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary
Cultured Human Erythroid Precursor Cells From Apoptosis
By
Yoshihito Haseyama,
Ken-ichi Sawada,
Atsushi Oda,
Kazuki Koizumi,
Hina Takano,
Takashi Tarumi,
Mitsufumi Nishio,
Makoto Handa,
Yasuo Ikeda, and
Takao Koike
From the Department of Internal Medicine II,
Hokkaido University School of Medicine, Sapporo; and the
Division of Hematology, Department of Internal
Medicine, and the Blood Center, Keio University,
Tokyo, Japan.
 |
ABSTRACT |
Little is known about the physiologic role of phosphatidylinositol
3-kinase (PI-3K) in the development of erythrocytes. Previous studies
have shown that the effects of the PI-3K inhibitor wortmannin on
erythropoietin (EPO)-dependent cell lines differed depending on the
cell type used. Wortmannin inhibited EPO-induced differentiation of
some cell lines without affecting their proliferation; however, the
EPO-induced proliferation of other cell lines was inhibited by
wortmannin. In neither case were signs of apoptosis observed. We have
previously reported that signaling in highly purified human colony
forming units-erythroid (CFU-E), generated in vitro from
CD34+ cells, differed from that in EPO-dependent cell
lines. In the current study, we examined the effects of a more specific
PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002
dose-dependently inhibits the proliferation of erythroid progenitor
cells with a half-maximal effect at 10 µmol/L LY294002. LY294002 at
similar concentrations also induces apoptosis of these cells, as
evidenced by the appearance of annexin V-binding cells and DNA
fragmentation. The steady-state phosphorylation of AKT at Ser-473 that
occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are
specific. Interestingly, the acceleration of apoptosis by LY294002 was
observed in the presence or absence of EPO. Further, deprivation of EPO
resulted in accelerated apoptosis irrespective of the presence of
LY294002. Our study confirms and extends the finding that signaling in
human primary cultured erythroid cells is significantly different from
that in EPO-dependent cell lines. These data suggest that PI-3K has an
antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from
apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive
and one that is EPO-dependent and at least partly insensitive to LY294002.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ERYTHROPOIETIN (EPO) is a glycoprotein
hormone essential for normal erythropoiesis.1-4 Signaling
through the EPO receptor (EPO-R) regulates the proliferation,
differentiation, and survival of erythroid progenitor cells.
Homodimerization of the receptor in response to EPO binding transiently
activates the receptor-associated protein tyrosine kinase
Jak2.5-8 It has been reported that activation of Jak2 is
accompanied by tyrosine phosphorylation of numerous proteins, including
Jak2 itself, STAT proteins, SHP-1, Shc, Vav, the EPO-R, and the classic
phosphatidylinositol 3-kinase (PI-3K), although tyrosine kinases other
than Jak2 may also phosphorylate these proteins.4,6-22 The
classic PI-3K is a heterodimeric enzyme composed of a regulatory p85
subunit and a catalytic p110 subunit that phosphorylates
phosphoinositides at the D3 position of the inositol
ring.23 Published data have implicated PI-3K and its downstream target, the serine/threonine kinase AKT, in a pathway that
conveys survival signals within various systems.24-26
Activation of PI-3K results in the generation of 2 lipid products
(PI-3,4-P2 and PI-3,4,5-P3), which serve as
second-messenger molecules and activate AKT. Activated AKT may
phosphorylate the proapoptotic factor BAD on a serine residue,
resulting in its dissociation from BCL-XL and its
association with 14-3-3.24 Released BCL-XL may
then suppress cell-death pathways that involve the activity of APO-1,
cytochrome c, and the caspase protease cascade.26 Such a
mechanism may function in human erythroid cells, as it has been
reported that these cells express BCL-XL and its expression is positively regulated during the final stage of
erythropoiesis.27,28 Another possible target of activated
AKT may be glycogen synthase kinase-3 (GSK-3), and it was reported that
phosphorylation of GSK-3 by activated AKT may promote the survival of
Rat-1 and PC12 cells.29
Little is known about the physiologic significance of PI-3K in the
survival, proliferation, and differentiation of erythroid progenitors.
Wortmannin, an inhibitor of PI-3K, was reported to inhibit the
proliferation of HCD-57 and DA-3 cells in response to
EPO.18,23 More recently, Sui et al30 reported
that wortmannin inhibited the proliferation of human erythroid
precursors expanded in vitro. The mechanism involved in the
antiproliferative effects was unknown. Moreover, in certain cells,
wortmannin had no antiproliferative effects. However, in some of the
studies cited, high concentrations of wortmannin were used that
probably had nonspecific effects. Lysophosphatidic acid (LPA) has been
shown to be a major survival factor for murine macrophages. While high
concentrations of wortmannin were necessary to inhibit the survival
effects of LPA,31 LY294002, a more specific inhibitor of
PI-3K,32 opposed the action of LPA at appropriate
concentrations. We postulate that the use of wortmannin and different
cell lines could explain the controversy regarding the role of PI-3K in erythropoiesis.
We previously used primary cultured human erythroid progenitor
cells33 to gain insight into the physiologic role of PI-3K activation to support proliferation and differentiation of erythroid progenitors. Using these cells, we found that EPO induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. In the present study, we
show that LY294002 induces apoptosis in human erythroid progenitors in
a time- and dose-dependent manner, and that it blocks phosphorylation of AKT at concentrations similar to those required for the induction of
apoptosis. Our results suggest that PI-3K may be essential for the
survival of human erythroid precursor cells by preventing apoptosis.
| |
MATERIALS AND METHODS |
Reagents.
HEPES, sodium dodecyl sulfate (SDS), 2-mercaptoethanol (2-ME), sodium
orthovanadate, bovine serum albumin (BSA), chicken egg albumin,
Iscove's modified Dulbecco's medium (IMDM), propidium iodide (PI),
protein A-Sepharose, Triton X-100, and Tris were purchased from Sigma
(St Louis, MO). Polyvinylidene difluoride (PVDF) membranes (pore size,
0.45 µm) were from Millipore Corp (Bedford, MA). SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) molecular standards and enhanced
chemiluminescence reagents including secondary antibodies were
purchased from Amersham (Arlington Heights, IL). Insulin (porcine
sodium; activity, 26.3 USP U/mg) was purchased from Calbiochem and
Behring Diagnostics (La Jolla, CA). Antibodies against AKT and
phosphorylated AKT were from Upstate Biotechnology Inc (Lake Placid,
NY) and New England Biolabs (Beverly, MA). Nitroblue tetrazolium
chloride and 5-bromo-4 chloro-3-indolyl phosphate p-toluidine
salt were from GIBCO-BRL (Gaithersburg, MD). Horse tendon type I
collagen was from Nycomed (Munich, Germany).
Recombinant human EPO (180,000 U/mg) was kindly donated by Chugai
Pharmaceutical Co (Tokyo, Japan). Recombinant human interleukin-3 ([IL-3] 108 chronic myelogenous leukemia U/mg) was from
Amgen Biologicals (Thousand Oaks, CA). Recombinant human stem cell
factor (SCF) was kindly donated by Kirin Brewery Co (Tokyo, Japan).
Vitamin B12 and folic acid were from Sankyo Pharmaceutical
Co (Tokyo, Japan) and Takeda Pharmaceutical Co (Osaka, Japan),
respectively. Fetal calf serum (FCS), penicillin, and streptomycin were
from Flow Laboratories Inc (McLean, VA). LY294002 was purchased from BIOMOL Research Laboratories Inc (Plymouth Meeting, PA).
Ex vivo generation of erythroid progenitor cells.
Human erythroid progenitor cells were generated ex vivo as previously
described.33 In brief, recombinant human granulocyte colony-stimulating factor ([G-CSF] Chugai Pharmaceutical Co and Kyowa
Hakko Pharmaceutical Co, Tokyo, Japan) was administered to healthy
subjects who previously signed consent forms approved by the Hokkaido
University School of Medicine and the Hokkaido Red Cross Blood Center
Committee for the Protection of Human Subjects, as described
previously.34 The mobilized peripheral blood (PB) CD34+ cells were isolated using immunomagnetic
beads.35,36 The cells were then cryopreserved and stored
until use in liquid nitrogen. The frozen PB CD34+ cells
were thawed, suspended in IMDM containing 30% FCS and 100 U/mL DNase,
and then centrifuged at 400g for 5 minutes at 4°C. The cells
were washed twice with IMDM containing 20% FCS and then resuspended in
IMDM containing 0.3% deionized BSA.37 The cells were next
cultured in liquid phase as described elsewhere.33,38 In
brief, cells at 0.5 × 104 to 2.0 × 104
cells/mL were suspended in a mixture containing 20% FCS, 10% heat-inactivated pooled human AB serum, 1% BSA, 10 µg/mL insulin, 10 µg/mL vitamin B12, 15 µg/mL folic acid, 100 U/mL IL-3,
100 ng/mL SCF, and 4 U/mL EPO in the presence of 5 × 10 5 mol/mL  -ME, 50 U/mL penicillin, 50 U/mL
streptomycin, and IMDM in a 50-mL polystyrene flask (Corning Coster
Corp, Cambridge, MA). After incubation for 8 days at 37°C in a 5%
CO2/95% O2 atmosphere, the cells were
collected, washed twice with IMDM containing 0.3% BSA (day 8 cells).
Semisolid culture of progenitors.
Day 8 cells were incubated in triplicate at a concentration of 500 cells/mL in flat-bottom 48-well tissue culture plates (Linbro; Flow
Laboratories) in 0.25 mL serum-containing or serum-free fibrin clots
with EPO at 2 U/mL.38-41 After 7 days of incubation at
37°C in a 5% CO2/5% O2 incubator, the clots
were fixed and stained with benzidine-hematoxylin.37 The
aggregates consisting of 8 to 49 hemoglobinized cells were defined as
colony-forming units-erythroid (CFU-E), while aggregates consisting of
2 to 7 hemoglobinized cells were defined as small erythroid. Erythroid
colony-forming cells (ECFCs) were defined as cells that yield colonies
of 2 to 49 hemoglobinized cells after 7 days of culture of day 8 cells.40
Liquid suspension culture of progenitors.
Day 8 cells were incubated at concentrations of 1 × 105
to 5 × 105 cells/mL in flat-bottom 24- to 96-well tissue
culture plates (Linbro; Flow Laboratories) in serum-free medium with
EPO at 2 U/mL.39 After incubation at 37°C in a 5%
CO2/5% O2 incubator for the indicated periods,
the cells were collected and washed twice with IMDM containing 0.3%
BSA before the subsequent colony assay of these cells.
Immunoprecipitation, gel electrophoresis, and Western blotting.
After starvation, day 8 cells were stimulated with EPO. Cells were
lysed by adding an equal amount of lysis buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L PMSF, 10 mmol/L EGTA, 1 mmol/L sodium
orthovanadate, 0.8 µg/mL leupeptin, and 2% Triton X-100 vol/wt, pH
7.4). After 20 minutes on ice, the lysates were centrifuged at
10,000× g (at 4°C) for 20 minutes. The supernatant was
removed and precleared with preimmune serum and protein A-Sepharose
(40 µL of 50% slurry) for 1 hour. Anti-AKT polyclonal antibody was then added and the preparation was incubated for 2 to 3 hours on ice.
Protein A-Sepharose (40 µL of 50% slurry) was added and followed by
1 hour of incubation. The immune complexes were washed 3 times with 1 mL cold washing buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L
PMSF, 10 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 0.8 µg/mL
leupeptin, and 1% Triton X-100 vol/wt, pH 7.4) and resuspended in
Laemmli sample buffer (10% glycerol, 1% SDS, 5% 2-ME, 50 mmol/L Tris
HCl (pH 6.8), and 0.002% bromophenol blue) with 10 mmol/L EGTA and 1 mmol/L sodium orthovanadate. After boiling at 95°C for 5 minutes,
1-dimensional electrophoresis was performed on SDS 10% or 7.5% to
15% polyacrylamide gels.42 Separated proteins were
electrophoretically transferred from the gel onto PVDF membranes or
nitrocellulose in a buffer containing Tris (25 mmol/L), glycine (192 mmol/L), and 20% methanol at 0.2 amps for 12 hours at room temperature. To block residual protein binding sites, membranes were
incubated in TBST (Tris-buffered saline [TBS], 10 mmol/L Tris, and
150 mmol/L NaCl, pH 7.6, with 0.1% Tween 20) with 10% chicken egg
albumin. The blots were washed with TBST and incubated overnight with
primary antibodies at a final concentration of 1.0 µg/mL in TBST. The
primary antibody was removed, and the blots were washed 4 times in TBST
and incubated with horseradish peroxidase-conjugated secondary
antibodies diluted 1:3,000 in TBST. The blots were then washed 4 times
in TBST. Antibody reactions were quantified by chemiluminescence
according to the manufacturer's instructions.
Flow cytometry.
Day 8 cells were cultured at a concentration of 5 × 105/mL in 1.0 mL serum-free medium supplemented with 2 U/mL
EPO with or without 50 µmol/L LY294002. After incubation for 24 hours, the cells were collected, washed twice with staining medium
(phosphate-buffered saline [PBS] containing 3% FCS and 0.005%
NaN3), counterstained with PI and annexin V conjugated with
FITC (Apoptosis Detection Kit; R&D Systems Inc, Minneapolis, MN), and
then analyzed by FACS Vantage (Becton Dickinson, Franklin Lakes, NJ).
DNA fragmentation.
DNA fragmentation was measured by quantitation of cytosolic
oligonucleosome-bound DNA using an enzyme-linked immunosorbent assay
(ELISA) kit (Cell Death Detection ELISA; Boehringer, Mannheim, Germany) according to the manufacturer's instructions.
Briefly, day 8 cells were incubated in 0.5 mL serum-free medium in the presence of 2 U/mL EPO with or without 50 mmol/L LY294002 at a concentration of 1 × 105 cells/mL. In the other set of
experiments, cells were incubated for 24 hours with various
concentrations of LY294002 (0, 1, 10, 20, 50, and 100 µmol/L). After
incubation for the indicated periods, the cells were collected and
washed twice with PBS and the cytosolic fraction (13,000g
supernatant) was extracted. The diluted cytosolic fraction equivalent
to 200 cultured cells was used as the Ag source in a sandwich ELISA
with a primary anti-histone Ab coated to the microtiter plate and a
secondary anti-DNA Ab coupled to peroxidase. From the absorbance
values, the percentage of fragmentation, in comparison to controls, was
calculated according to the following formula:
|
|
|
|
Platelet preparation.
Washed platelets were prepared from citrated whole blood as previously
described.43
| |
RESULTS |
Characteristics of expanded cells.
PB CD34+ cells cultured for 8 days in serum-containing
medium with SCF, IL-3, and EPO generated cells that predominantly
consist of erythroid cells (day 8 cells; Fig
1). The ECFCs in day 8 cells predominantly
consist of mature erythroid progenitor cells for which the maturation
level is equivalent to CFU-E as described elsewhere.33
View larger version (105K):
[in this window]
[in a new window]
| Fig 1.
PB CD34+ cells cultured for 8 days with
SCF, IL-3, and EPO (day 8 cells). May-Grünwald staining (original
magnification ×1,000).
|
|
Inhibition of erythroid colony growth by LY294002.
To gain insight into the role of PI-3K signaling in human erythroid
colony growth, day 8 cells were incubated in serum-free fibrin clots
with 2 U/mL EPO in the presence of various concentrations of LY294002,
a specific PI-3K inhibitor. The inhibition of erythroid colony growth
by LY294002 occurred in a dose-dependent manner, and half-maximal
inhibition occurred at 10 µmol/L LY294002 (P < .001).
Maximal inhibition occurred at about 50 µmol/L (Table 1).
Inhibition of proliferation and survival of erythroid progenitor
cells by LY294002.
To investigate the effect of LY294002 on the viability and
proliferation of erythroid progenitor cells, day 8 cells were incubated in liquid phase in serum-free medium with 2 U/mL EPO in the presence of
various concentrations of LY294002 (Table 2). After 7 days of culture,
the cells incubated with neither LY294002 nor 0.1% DMSO increased in
number by 27.8 ± 2.8-fold with 88.0% ± 5.3% viability, resulting
in a 24.5 ± 3.5-fold increase in the number of viable cells. The
presence of 0.1% DMSO, a vehicle of LY294002, did not affect the
proliferation or viability of erythroid progenitor cells. The
proliferation of day 8 cells was markedly inhibited by LY294002 in a
dose-dependent manner and was accompanied by a decrease in viability.
Inhibition of the proliferation and survival of day 8 cells was evident
at concentrations of 10 µmol/L (P < .001 for total cells
and P < .01 for viable cells) and 20 µmol/L LY294002
(P < .01), respectively, the maximum being about 50 µmol/L, which suggests that a blockage of PI-3K signaling by LY294002 inhibits the proliferation and differentiation of erythroid progenitor cells with an accompanying decrease in cell viability. These findings suggest that the inhibition of erythroid progenitor cells by LY294002 could be an early event during the course of erythroid proliferation and differentiation.
Time-course study of the effect of LY294002 on erythroid progenitor
cells.
To elucidate the early events initiated by LY294002, the time course of
the inhibitory effect of LY294002 on the proliferation and
differentiation of erythroid progenitor cells was investigated. Day 8 cells were suspended in liquid phase in serum-free medium with 2 U/mL
EPO in the presence of 50 µmol/L LY294002 and cultured for 0, 4, 8, 24, and 48 hours, and the number and viability of the cells were
assessed. To determine if the inhibitory effect of LY294002 is
reversible, the cells were collected and washed twice with IMDM
containing 0.3% BSA and then cultured in serum-containing fibrin clots
with 2 U/mL EPO for 7 days, the objective being to assess the
colony-forming ability after exposure to LY294002.
The viability of cells cultured with LY294002 decreased to 44% at 48 hours, while that of cells incubated without LY294002 remained at 75%
(Fig 2A). In addition, the viability of cells cultured without LY294002
was close to 90% for up to 7 days of culture (Table 2). Among the
viable cells, those incubated without LY294002 markedly increased from
4.85 × 105 to 8.8 × 105 during 48 hours of
incubation (Fig 2B); however, cells incubated with LY294002 did not
appear to increase until 24 hours and decreased to 2.20 × 105 during 48 hours (Fig 2B). When the reversibility of the
inhibitory effect of LY294002 on the proliferation and differentiation
of erythroid progenitor cells was assessed by erythroid colony
formation (Fig 2C), the decrease in erythroid colony growth caused by
LY294002 was evident in a time-dependent manner. The statistically
significant decrease of erythroid progenitor cells was noted after 24 hours' exposure (from 100% ± 6% to 18% ± 4%,
P < .001), while erythroid progenitor cells in cells
incubated without LY294002 continuously increased for up to 48 hours
(195% ± 17%). Therefore, inhibition of erythroid progenitor cells
by LY294002 occurs as an early event in the course of proliferation and
differentiation in a time-dependent manner, and the inhibition is
irreversible.
View larger version (55K):
[in this window]
[in a new window]
| Fig 2.
Time-course study of the effect of LY294002 on the
proliferation and survival of erythroid progenitor cells. Day 8 cells
that contained 69% ± 4% erythroid progenitor cells were
suspended at a concentration of 5 × 105/mL in 1.0 mL
serum-free medium supplemented with 2 U/mL EPO with ( ) or without
( ) 50 µmol/L LY294002. After incubation and at the indicated
periods, the cells were collected, washed twice in IMDM containing
0.3% BSA, and then plated into serum-containing fibrin clots with 2 U/mL EPO. After 7 days of incubation, the clots were fixed and stained
with benzidine-hematoxylin. (A) Viability; (B) absolute number of
viable cells; (C) percent expression for the number of erythroid
colonies in relation to the value of 0 hours as 100%. The mean ± SD
of triplicates is shown for C. +++P < .001, decrease v control (); ***P < .001, decrease
v 0 hours; #P < .05, ###P < .001, increase v 0 hours.
|
|
Titration of the effect of LY294002 on erythroid progenitor cells.
To titrate the inhibitory effect of LY294002 on erythroid progenitor
cells, day 8 cells were suspended in liquid phase in serum-free medium
with 2 U/mL EPO in the presence of various concentrations of LY294002
(0, 1, 10, 20, 50, and 100 µmol/L) and cultured for 4 and 24 hours,
and the number and viability were assessed. To investigate whether the
irreversibility of the inhibitory effect of LY294002 depends on the
dose of LY294002, the cells were collected and washed twice with IMDM
containing 0.3% BSA and then cultured in serum-containing fibrin clots
with 2 U/mL EPO for 7 days.
The viability of cells cultured for 4 hours was not affected by
LY294002 at the concentrations tested. However, when day 8 cells were
incubated for 24 hours with LY294002, viability decreased in a
dose-dependent manner (Fig
3A).
View larger version (87K):
[in this window]
[in a new window]
| Fig 3.
Titration of the effects of LY294002 on the proliferation
and survival of erythroid progenitor cells. Day 8 cells that contained
64% ± 6% erythroid progenitor cells were suspended at a
concentration of 5 × 105/mL in 1.0 mL serum-free medium
supplemented with 2 U/mL EPO with various concentrations of LY294002 as
indicated. After incubation for 4 or 24 hours, the cells were collected
and washed twice in IMDM containing 0.3% BSA and then plated into
serum-containing fibrin clots with 2 U/mL EPO. After 7 days of
incubation, the clots were fixed and stained with
benzidine-hematoxylin. (A) Viability; (B) absolute number of viable
cells; (C) percent expression for the number of erythroid colonies
relative to the value in 0.1% DMSO as 100%. The mean ± SD of
triplicates is shown for C. Cells incubated for 4 or 24 hours with
0.1% DMSO contained 69% ± 4% and 65% ± 9% erythroid
colony-forming cells, respectively. *P < .05, ***P < .001, decrease v control.
|
|
Among the viable cells, those incubated with LY294002 for 4 hours were
not affected by any of the concentrations tested (Fig 3B). After 24 hours of incubation of day 8 cells without LY294002, the number of
viable cells increased from 5.75 × 105 (after 4 hours) to
9.40 × 105. The presence of LY294002 for 24 hours
decreased the number of viable cells in a dose-dependent manner.
Figure 3C shows the significant decrease in erythroid colony growth of
cells exposed to LY294002 in a dose-dependent manner in both cultures
for 4 and 24 hours. The decrease of erythroid colony growth in cells
exposed to LY294002 for 24 hours was more prominent than that found in
cells exposed for 4 hours, which indicates that the inhibition of
erythroid proliferation and differentiation is augmented by prolonged
exposure to LY294002. It is of note that the significant inhibition of
erythroid progenitor growth occurred (Fig 3C) during periods in which
the viability of cells and the number of viable cells were not affected
(Fig 3A and B; incubation for 4 hours).
LY294002 induces apoptosis and DNA fragmentation in erythroid
progenitor cells.
A decrease in erythroid progenitor growth with intact viability after
exposure to LY294002 suggests that an early event in the induction of
an internal suicide program (apoptosis) could be responsible for the
mechanism. Because apoptosis is commonly associated biochemically with
the loss of phospholipid asymmetry on the cell surface and DNA
fragmentation, we examined these 2 issues. The loss of phospholipid
asymmetry on the cell surface was examined using FITC-conjugated
annexin V and FACS (Fig 4). When day 8 cells were incubated for 24 hours, 22.8% of cells incubated with LY294002 at 50 µmol/L were
detected as annexin V+/PI, in contrast to
9.8% found in the control.
View larger version (27K):
[in this window]
[in a new window]
| Fig 4.
Apoptosis of erythroid progenitor cells by LY294002. Day
8 cells that contained 71% ± 5% erythroid progenitor cells were
suspended at a concentration of 5 × 105/mL in 1.0 mL
serum-free medium supplemented with 2 U/mL EPO with (B) or without (A)
50 µmol/L LY294002. After incubation for 24 hours, cells were
collected, washed twice, counterstained with PI and annexin V
conjugated with FITC, and then analyzed using FACS Vantage.
|
|
Chromatin fragmentation into oligonucleosomes was determined using an
ELISA specific for cytosolic histone-bound DNA (Fig 5).
LY294002-induced DNA fragmentation was found after 4 hours of
incubation and continuously increased in a time-dependent manner (Fig
5A). For day 8 cells cultured for 24 hours, LY294002-induced DNA
fragmentation was found for a concentration of 20 µmol/L and increased in a dose-dependent manner (Fig
5B).
View larger version (65K):
[in this window]
[in a new window]
| Fig 5.
DNA fragmentation in erythroid progenitor cells
challenged with LY294002. Day 8 cells that contained 62% ± 13%
erythroid progenitor cells were suspended at a concentration of 5 × 105/mL in 0.5 mL serum-free medium supplemented with 2 U/mL
EPO in the presence of 50 µmol/L LY294002 (A) or with various
concentrations of LY294002 (B). After incubation for the indicated
periods (A) or for 24 hours (B), the cells were collected and washed
twice with PBS. DNA fragmentation was determined by quantifying the
amount of oligonucleosome-bound DNA in the 20,000× g
supernatant of cell lysates. Data represent the mean from 2 determinations.
|
|
Deprivation of EPO by itself causes apoptosis in erythroid precursor
cells.1-4 Therefore, the effects of LY294002 with or without EPO on the viability and survival of erythroid progenitor cells
and DNA fragmentation were examined (Fig 6). When day 8 cells were
incubated for 24 hours, LY294002 decreased the viability of the cells
cultured without EPO (Fig 6A; P < .05). Deprivation of EPO
decreased the number of erythroid progenitor cells, which was further
accelerated by the presence of LY294002 (Fig 6B). Deprivation of EPO
resulted in accelerated DNA fragmentation irrespective of the presence
of LY294002. LY294002 accelerated the DNA fragmentation of cells
cultured without EPO (Fig 6C; P < .001). These data suggest that the effects of LY294002 on apoptosis in erythroid precursor cells
cannot be fully explained by the blockade of EPO-induced signal
transduction.
View larger version (20K):
[in this window]
[in a new window]
| Fig 6.
Effects of LY294002 with or without EPO on the viability
and survival of erythroid progenitor cells and DNA fragmentation. Day 8 cells that contained 61% ± 3% erythroid progenitor cells were
suspended at a concentration of 1 × 106/mL in 1.0 mL
serum-free medium with or without 2 U/mL EPO or 50 µmol/L LY294002.
After incubation for 24 hours, cells were collected and washed twice in
IMDM containing 0.3% BSA and then plated into serum-containing fibrin
clots with 2 U/mL EPO. After 7 days' incubation, the clots were fixed
and stained with benzidine-hematoxylin. Data represent the mean ± SD
of quadruplicates. (A) Viability; (B) percent expression for the number
of erythroid colonies relative to the value of fresh day 8 cells as
100%; (C) DNA fragmentation using fresh day 8 cells as controls.
*P < .05, **P < .01, ***P < .001.
|
|
LY294002 inhibits phosphorylation of the PI-3K-dependent kinase AKT.
To determine if PI-3K activity in situ was indeed inhibited by LY294002
in primary cultured human erythroid progenitor cells, we next examined
the effect of LY294002 on the steady-state phosphorylation of AKT at
Ser-473, a recognized downstream event of PI-3K activation. AKT was
modestly and constitutively phosphorylated at Ser-473 in human
erythroid progenitor cells, and phosphorylation of AKT was blocked by
treatment of the cells with LY294002 at concentrations similar to those
which suppressed erythroid growth or induced apoptosis (Fig
7). We acknowledge that the degree of AKT
phosphorylation was small. However, it should be noted that we have
tried to measure steady-state levels of AKT phosphorylation instead of
induced levels. In preliminary experiments, we could not find any
steady-state phosphorylation over the background levels by simple
Western blotting on 3 different occasions. However, we confirmed that
the reagents work well, because of the results of the following
experiments. Human washed platelets were prepared and stimulated by
type 1 horse tendon collagen (50 µg/L) as previously
described.43 Whole-cell lysates (107
cells/lane) were subjected to SDS-PAGE followed by Western blotting to
detect AKT phosphorylation using the same system. As expected from the
previous report,44 collagen induced a clear increase in AKT
phosphorylation and the increase was abolished by LY294002 (50 µmol/L), suggesting that the detection system and LY294002 are
appropriate (Fig 8). Similar amounts of AKT were detected on each lane
(lower panel). In both panels, the bands appear broad, probably due to
the presence of different isoforms of
AKT.26
View larger version (29K):
[in this window]
[in a new window]
| Fig 7.
(A) Phosphorylation of AKT in human erythroid cells
stimulated by EPO (10 U/mL). Day 8 cells were washed once in IMDM and
lysed by adding an equal amount of a buffer containing 2% Triton X-100
after incubation for 1 hour in the presence of DMSO or various
concentrations of LY294002. AKT was immunoprecipitated with specific
AKT antisera. Immune complexes were resuspended in SDS-sample buffer
and divided into two. Phosphorylation of AKT was detected using an
anti-phospho-AKT antibody (A), and total AKT was detected using an
anti-AKT polyclonal antibody (B). Bands were visualized by
chemiluminescence.
|
|
View larger version (39K):
[in this window]
[in a new window]
| Fig 8.
Collagen-induced phosphorylation of AKT in human
platelets. Washed platelets were pretreated with DMSO or LY294002 (50 µmol/L) as indicated for 15 minutes. Platelets were then treated with
horse tendon type I collagen (50 µg/mL) or buffer (control) for 5 minutes. Whole-cell extracts (107 cells/lane) were
subjected to SDS-PAGE and Western blot analysis with an
anti-phospho-AKT (A) or AKT antibody (B).
|
|
| |
DISCUSSION |
We examined the role of PI-3K in the proliferation and viability of
human erythroid progenitors expanded in vitro. Because these cells,
unlike genetically engineered murine cell lines expressing the EPO-R or
murine and human leukemic cell lines, regularly undergo full terminal
differentiation and become reticulocytes, these studies are more likely
to reflect actual physiologic situations. However, we acknowledge the
possible presence of artifacts arising from in vitro
culture.30,32
We found that LY294002, a specific PI-3K inhibitor, dose- and
time-dependently inhibits the proliferation and survival of human
erythroid progenitor cells. The inhibition of erythroid progenitor
growth by LY294002 was irreversible, and half-maximal inhibition was
observed at 10 µmol/L LY294002 and was maximal at about 50 µmol/L.
One of the mechanisms involved in this inhibitory effect is the
induction of apoptosis in the erythroid progenitor cells as observed at
20 µmol/L. The effect was dependent on the dose and the exposure
time, as evidenced by the appearance of annexin V-binding cells and
DNA fragmentation. Phosphorylation of AKT at Ser-473, a recognized
downstream event of PI-3K activation, was inhibited by LY294002 at
similar concentrations for suppressing proliferation and inducing
apoptosis, suggesting that the effects of LY294002 may be specific.
These results suggest that PI-3K has antiapoptotic effects on erythroid
progenitor cells and such effects may be mediated through downstream
molecules such as AKT. One of the possible pathways that are inhibited
by LY294002 includes the ras/MAP kinase pathway. Sui et
al30 have reported that wortmannin inhibited EPO- or
SCF-induced activation of MAP kinase and that PD98059, an MEK
inhibitor, inhibited the proliferation of primary erythroid cells. In
preliminary experiments, we also observed that PD98059 inhibited the
proliferation of erythroid cells, consistent with the report by Sui et
al.30
Although antagonistic effects of wortmannin on EPO-induced cell
proliferation have been reported,18,23,30 Kubota et
al16 noted that wortmannin prevents EPO-induced
differentiation but not proliferation of these cells. Miura et
al15 demonstrated that the C-terminus-deleted EPO-R, which
lacks the ability to associate with PI-3K and to activate it, is still
mitogenically active in 32D cells, an IL-3-dependent cell line, and
hence PI-3K is not required for growth signal transduction from EPO-R.
Taken together, these data suggest that in cells that proliferate or differentiate in response to EPO, the results of PI-3K activation may
vary depending on the cell lines used in each study. Furthermore, in
these reports, wortmannin did not induce apoptosis in erythroid-like cells. Thus, to our knowledge, our data suggest for the first time that
PI-3K may have a role in protecting human erythroid cells from
apoptosis. In view of a recent report on murine
macrophages,31 the use of LY294002 instead of wortmannin
may be one reason that our study found induction of apoptosis in
erythroid cells by inhibition of PI-3K while the other studies did not.
This view is strengthened by the report by Sui et al,30 as
they did not observe induction of apoptosis in human erythroid
precursors by wortmannin. Indeed, in preliminary experiments, we also
found that greater than 10 µmol/L wortmannin is required to induce
apoptosis in human erythroid cells. Such a high dose of wortmannin
probably has nonspecific effects. Interestingly, insulin (or
insulin-like growth factor [IGF]), EPO and c-kit ligand all activate
PI-3K in various cells.15-29 In our system, we regularly
add insulin; our previous studies have shown that albumin (fraction V)
contains IGF-1, which may induce PI-3K activation.26,37
These factors also have proliferative and antiapoptotic effects on
human erythroid precursors. The steady-state phosphorylation of AKT at
Ser-473 observed in our current studies may reflect, in part, the
steady-state activation of PI-3K,26 which may in turn be
induced by a combination of these factors. It is possible that EPO
supports the viability of cells by stimulating activation of PI-3K, but
that even without EPO some other factor maintains partial activation of
PI-3K and thus prolongs cell viability without EPO. Although more
studies are necessary to elucidate the factors responsible for
activation of PI-3K in primary cells, it seems likely that EPO protects
erythroid cells from apoptosis in a pathway that is not directly
related to PI-3K. Although such conclusions seem heretical in view of
previous studies using cell lines, it was recently reported that SCF,
but not EPO, induces activation of AKT in primary erythroid
cells.45 On the other hand, deprivation of
EPO facilitated apoptosis in the presence and absence of LY294002 (Fig
6).
A potential major limitation to the interpretation of our study is the
use of the PI-3K inhibitor LY294002, which may or may not be absolutely
specific for PI-3K. However, irrespective of the specificity of
LY294002, our studies revealed the presence of at least 2 different
pathways in the protection from apoptosis in primary erythroid cells.
One is LY294002-sensitive, and this pathway may not be directly related
to the presence of EPO. The other pathway is apparently EPO-dependent
and at least partly insensitive to LY294002, and the molecular basis of
this pathway remains to be determined. Very recently, abnormalities of
PTEN, which dephosphorylates D3-polyphosphoinositides, were noted in hematopoietic cell lines and some primary specimens.46 The
reduced levels of PTEN expression were associated with enhanced AKT
phosphorylation.46 The levels of D3-polyphosphoinositides
may partially regulate the survival of normal erythroid cells (and
possibly cells of different lineage), and their deregulation may lead
to malignant transformation of hematopoietic cells.
| |
ACKNOWLEDGMENT |
We thank Dr C.I. Civin for the generous gift of the monoclonal
antibodies, without which this study could not have been performed. We
also thank Chugai Pharmaceutical Co, Kirin Brewery Co, Sankyo Pharmaceutical Co, and Takeda Pharmaceutical Co for the generous gift
of recombinant human growth factors and reagents. Finally, we thank S. Okazaki for manuscript preparation, M. Ohara for helpful comments, and
M. Kitayama and I. Sato for technical assistance.
| |
FOOTNOTES |
Submitted November 6, 1998; accepted May 3, 1999.
Supported in part by grants-in-aid from the Ministry of Education,
Science, Sports and Culture of Japan (K.S., A.O., and Y.I.), a research
grant from the Idiopathic Disorders of Hematopoietic Organs Research
Committee of the Ministry of Health and Welfare of Japan (K.S.), and
the Ryoichi Naito Foundation for Medical Research (A.O.).
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 Ken-ichi Sawada, MD, Department of Medicine
II, Hokkaido University School of Medicine, N-15, W-7, Sapporo,
Hokkaido 060-8638, Japan.
| |
REFERENCES |
1.
Krantz SB:
Erythropoietin.
Blood
77:419, 1991[Free Full Text]
2.
Koury MJ, Bondurant MC:
The molecular mechanism of erythropoietin action.
Eur J Biochem
210:649, 1992[Medline]
[Order article via Infotrieve]
3.
Youssoufian H, Longmore G, Neuman D, Yoshimura A, Lodish HF:
Structure, function, and activation of the erythropoietin receptor.
Blood
81:2223, 1993[Free Full Text]
4.
Sayer ST, Penta K:
Erythropoietin cell biology.
Hematol Oncol Clin North Am
8:895, 1994[Medline]
[Order article via Infotrieve]
5.
Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN:
Jak2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
Cell
74:227, 1993[Medline]
[Order article via Infotrieve]
6.
Miura O, Nakamura N, Quelle FW, Witthuman BA, Ihle JN, Aoki N:
Erythropoietin induces association of the Jak2 protein tyrosine kinase with the erythropoietin receptor in vivo.
Blood
84:1501, 1994[Abstract/Free Full Text]
7.
Wakao H, Harada N, Kitamura T, Mui AL, Miyajima A:
Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.
EMBO J
14:2527, 1995[Medline]
[Order article via Infotrieve]
8.
Ohashi H, Maruyama K, Liu YC, Yoshimura A:
Ligand-induced activation of chimeric receptors between the erythropoietin receptor and receptor tyrosine kinases.
Proc Natl Acad Sci USA
1:158, 1994
9.
Sawyer ST, Penta K:
Association of Jak2 and STAT5 with erythropoietin receptors.
J Biol Chem
271:32430, 1996[Abstract/Free Full Text]
10.
Quell FW, Wang D, Nosaka T, Thierfelder WE, Stravopodis D, Weinstein Y, Ihle JN:
Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for mitogenic response.
Mol Cell Biol
16:1622, 1996[Abstract]
11.
Ohashi T, Masuda M, Ruscetti SK:
Induction of sequence-specific DNA-binding factors by erythropoietin and the spleen focus-forming virus.
Blood
85:1454, 1995[Abstract/Free Full Text]
12.
Klingmuller U, Bergelson S, Hsio JG, Lodish HF:
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Proc Natl Acad Sci USA
93:8324, 1996[Abstract/Free Full Text]
13.
Penta K, Sawyer ST:
Erythropoietin induces the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1 and STAT5 in erythroid cells.
J Biol Chem
270:31282, 1995[Abstract/Free Full Text]
14.
Mellitzer G, Wesseley O, Decker T, Meinke A, Hayman MJ, Beug H:
Activation of STAT5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.
Proc Natl Acad Sci USA
93:9600, 1996[Abstract/Free Full Text]
15.
Miura O, Nakamura N, Ihle JN, Aoki N:
Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.
J Biol Chem
269:614, 1994[Abstract/Free Full Text]
16.
Kubota Y, Tanaka T, Yamaoka G, Yamaguchi M, Ohnishi H, Kawanishi K, Takahara J, Irino S:
Wortmannin: A specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells.
Leukemia
10:720, 1996[Medline]
[Order article via Infotrieve]
17.
He TC, Zhuang H, Jiang N, Waterfield MD, Wojchowski DM:
Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.
Blood
82:3530, 1993[Abstract/Free Full Text]
18.
Jaster R, Bittorf T, Brock J:
Involvement of phosphatidylinositol 3-kinase in the mediation of erythropoietin-induced activation of p70S6k.
Cell Signal
9:175, 1997[Medline]
[Order article via Infotrieve]
19.
Joneja B, Wojchowski DM:
Mitogenic signaling and inhibition of apoptosis via the erythropoietin receptor Box-1 domain.
J Biol Chem
272:11176, 1997[Abstract/Free Full Text]
20.
Verdier F, Chretien S, Billat C, Gisselbrecht S, Lacombe C, Mayeux P:
Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.
J Biol Chem
272:26173, 1997[Abstract/Free Full Text]
21.
Shigematsu H, Iwasaki H, Otsuka T, Ohno Y, Arima F, Niho Y:
Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity.
J Biol Chem
272:14334, 1997[Abstract/Free Full Text]
22.
Gobert S, Porteu F, Pallu S, Muller O, Sabbah M, Dusanter-Fourt I, Courtois G, Lacombe C, Gisselbrecht S, Mayeux P:
Tyrosine phosphorylation of the erythropoietin receptor: Role for differentiation and mitogenic signal transduction.
Blood
86:598, 1995[Abstract/Free Full Text]
23.
Damen JE, Cutler RL, Jiao H, Yi T, Krystal G:
Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.
J Biol Chem
270:23402, 1995[Abstract/Free Full Text]
24.
Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M:
3-Phosphoinositide-dependent protein kinase-1 (PDK1): Structural and functional homology with the Drosophila DSTPK61 kinase.
Curr Biol
7:776, 1997[Medline]
[Order article via Infotrieve]
25.
Blume-Jensen P, Janknecht R, Hunter T:
The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136.
Curr Biol
18:779, 1998
26.
Downward J:
Mechanisms and consequences of activation of protein kinase B/Akt.
Curr Opin Cell Biol
10:262, 1998[Medline]
[Order article via Infotrieve]
27.
Chao DT, Korsmeyer SJ:
BCL-2 family: Regulators of cell death.
Annu Rev Immunol
16:395, 1998[Medline]
[Order article via Infotrieve]
28.
Gregoli PA, Bondurant MC:
The roles of Bcl-X(L) and apopain in the control of erythropoiesis by erythropoietin.
Blood
90:630, 1997[Abstract/Free Full Text]
29.
Pap M, Cooper GM:
Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway.
J Biol Chem
273:19929, 1998[Abstract/Free Full Text]
30.
Sui X, Krantz SB, You M, Zhao Z:
Synergistic activation of MAP kinase (ERK1/2) by erythropoietin and stem cell factor is essential for expanded erythropoiesis.
Blood
92:1142, 1998[Abstract/Free Full Text]
31.
Koh JS, Lieberthal W, Heydrick S, Levine JS:
Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway.
J Clin Invest
102:716, 1998[Medline]
[Order article via Infotrieve]
32.
Vlahos CJ, Matter WF, Hui KY, Brown RF:
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:5241, 1994[Abstract/Free Full Text]
33.
Oda A, Sawada K, Druker BJ, Ozaki K, Takano H, Koizumi K, Fukada Y, Handa M, Koike T, Ikeda Y:
Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.
Blood
92:443, 1998[Abstract/Free Full Text]
34.
Sato N, Sawada K, Takahashi TA, Mogi Y, Asano S, Koike T, Sekiguchi S:
A time course study for optimal harvest of peripheral blood progenitor cells by granulocyte colony stimulating factor in healthy volunteers.
Exp Hematol
22:973, 1994[Medline]
[Order article via Infotrieve]
35.
Sawada K, Krantz SB, Dai CH, Koury ST, Horn ST, Glick AD, Civin CI:
Purification of human blood burst forming units erythroid and demonstration of the evolution of erythropoietin receptors.
J Cell Physiol
142:219, 1993
36.
Yamaguchi M, Sawada K, Sato N, Koizumi K, Sekiguchi S, Koike T:
A rapid nylon fiber syringe system to deplete CD14+ cells for positive selection of human blood CD34+ cells. Use of immunomagnetic microspheres.
Bone Marrow Transplant
19:373, 1997[Medline]
[Order article via Infotrieve]
37.
McLeod DL, Shreeve MM, Axelrad AA:
Improved plasma culture system for production of erythrocytic colonies in vitro. Quantitative assay method for CFU-E.
Blood
44:517, 1974[Abstract/Free Full Text]
38.
Sawada K, Krantz SB, Dai CH, Sato N, Ieko M, Sakurama S, Yasukouchi T, Nakagawa S:
Transitional change of colony stimulating factor requirements for erythroid progenitors.
J Cell Physiol
149:1, 1991[Medline]
[Order article via Infotrieve]
39.
Sawada K, Krantz SB, Dessypris EN, Koury ST, Sawyer ST:
Human colony forming units erythroid do not require accessory cells, but do require direct interaction with insulin like growth factor I and/or insulin for erythroid development.
J Clin Invest
83:1701, 1989
40.
Sawada K, Krantz SB, Kans JS, Dessypris EN, Sawyer S, Glick AD, Civin CI:
Purification of human erythroid colony forming units and demonstration of specific binding of erythropoietin.
J Clin Invest
80:357, 1987
41.
Sawada K, Sato N, Notoya A, Tarumi T, Hirayama S, Takano H, Koizumi K, Yasukouchi T, Yamaguchi M, Koike T:
Proliferation and differentiation of myelodysplastic CD34+ cells: Phenotypic subpopulations of marrow CD34+ cells.
Blood
85:194, 1995[Abstract/Free Full Text]
42.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
43.
Oda A, Ochs HD, Druker BJ, Ozaki K, Watanabe C, Handa M, Miyakawa Y, Ikeda Y:
Collagen induces tyrosine phosphorylation of Wiskott-Aldrich syndrome protein in human platelets.
Blood
92:1852, 1998[Abstract/Free Full Text]
44.
Franke TF, Kaplan DR, Cantley LC, Toker A:
Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.
Science
275:665, 1997[Abstract/Free Full Text]
45.
Sui X, Krantz SB, Zhao Z:
Distinct roles of phosphoinositide-3 (PI-3) kinase in erythropoietin and stem cell factor signaling in primary human erythroid progenitor cells.
Blood
92:66a, 1998 (suppl 1, abstr)
46.
Dahia PLM, Aguiar RCT, Alberta J, Kum JB, Caron S, Sill H, Marsh DJ, Ritz J, Freedman A, Stiles C, Eng C:
PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies.
Hum Mol Genet
8:185, 1999[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
G. Grech, M. Blazquez-Domingo, A. Kolbus, W. J. Bakker, E. W. Mullner, H. Beug, and M. von Lindern
Igbp1 is part of a positive feedback loop in stem cell factor-dependent, selective mRNA translation initiation inhibiting erythroid differentiation
Blood,
October 1, 2008;
112(7):
2750 - 2760.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Jana, A. Jana, X. Liu, S. Ghosh, and K. Pahan
Involvement of Phosphatidylinositol 3-Kinase-Mediated Up-Regulation of I{kappa}B{alpha} in Anti-Inflammatory Effect of Gemfibrozil in Microglia
J. Immunol.,
September 15, 2007;
179(6):
4142 - 4152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bugarski, A. Krstic, S. Mojsilovic, M. Vlaski, M. Petakov, G. Jovcic, N. Stojanovic, and P. Milenkovic
Signaling Pathways Implicated in Hematopoietic Progenitor Cell Proliferation and Differentiation
Experimental Biology and Medicine,
January 1, 2007;
232(1):
156 - 163.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Tada, Y. Kagaya, M. Takeda, J. Ohta, Y. Asaumi, K. Satoh, K. Ito, A. Karibe, K. Shirato, N. Minegishi, et al.
Endogenous erythropoietin system in non-hematopoietic lineage cells plays a protective role in myocardial ischemia/reperfusion
Cardiovasc Res,
August 1, 2006;
71(3):
466 - 477.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Liu, R. Pop, C. Sadegh, C. Brugnara, V. H. Haase, and M. Socolovsky
Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo
Blood,
July 1, 2006;
108(1):
123 - 133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Schade, J. J. Powers, M. W. Wlodarski, and J. P. Maciejewski
Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis
Blood,
June 15, 2006;
107(12):
4834 - 4840.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
W. Zhao, C. Kitidis, M. D. Fleming, H. F. Lodish, and S. Ghaffari
Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway
Blood,
February 1, 2006;
107(3):
907 - 915.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Montoye, I. Lemmens, D. Catteeuw, S. Eyckerman, and J. Tavernier
A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach
Blood,
June 1, 2005;
105(11):
4264 - 4271.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. D. Sinor and L. Lillien
Akt-1 Expression Level Regulates CNS Precursors
J. Neurosci.,
September 29, 2004;
24(39):
8531 - 8541.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chu, M. Holtz, M. Gupta, and R. Bhatia
BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells
Blood,
April 15, 2004;
103(8):
3167 - 3174.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. J. Bakker, M. Blazquez-Domingo, A. Kolbus, J. Besooyen, P. Steinlein, H. Beug, P. J. Coffer, B. Lowenberg, M. von Lindern, and T. B. van Dijk
FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1
J. Cell Biol.,
January 19, 2004;
164(2):
175 - 184.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Myssina, S. M. Huber, C. Birka, P. A. Lang, K. S. Lang, B. Friedrich, T. Risler, T. Wieder, and F. Lang
Inhibition of Erythrocyte Cation Channels by Erythropoietin
J. Am. Soc. Nephrol.,
November 1, 2003;
14(11):
2750 - 2757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Huddleston, B. Tan, F.-C. Yang, H. White, M. J. Wenning, A. Orazi, M. C. Yoder, R. Kapur, and D. A. Ingram
Functional p85{alpha} gene is required for normal murine fetal erythropoiesis
Blood,
July 1, 2003;
102(1):
142 - 145.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bouscary, F. Pene, Y.-E. Claessens, O. Muller, S. Chretien, M. Fontenay-Roupie, S. Gisselbrecht, P. Mayeux, and C. Lacombe
Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation
Blood,
May 1, 2003;
101(9):
3436 - 3443.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A.-K. Boer, A. L. Drayer, H. Rui, and E. Vellenga
Prostaglandin-E2 enhances EPO-mediated STAT5 transcriptional activity by serine phosphorylation of CREB
Blood,
June 28, 2002;
100(2):
467 - 473.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles
Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism
Blood,
February 15, 2002;
99(4):
1117 - 1129.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. P. Miller, D. W. Heilman, and D. M. Wojchowski
Erythropoietin receptor-dependent erythroid colony-forming unit development: capacities of Y343 and phosphotyrosine-null receptor forms
Blood,
February 1, 2002;
99(3):
898 - 904.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ghaffari, C. Kitidis, M. D. Fleming, H. Neubauer, K. Pfeffer, and H. F. Lodish
Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2-/- hematopoietic progenitors
Blood,
November 15, 2001;
98(10):
2948 - 2957.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. C. P. Somervaille, D. C. Linch, and A. Khwaja
Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax
Blood,
September 1, 2001;
98(5):
1374 - 1381.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Kashii, M. Uchida, K. Kirito, M. Tanaka, K. Nishijima, M. Toshima, T. Ando, K. Koizumi, T. Endoh, K.-i. Sawada, et al.
A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction
Blood,
August 1, 2000;
96(3):
941 - 949.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Choi, K. Muta, A. Wickrema, S. B. Krantz, J. Nishimura, and H. Nawata
Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin
Blood,
June 15, 2000;
95(12):
3742 - 3749.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nishigaki, C. Hanson, T. Ohashi, D. Thompson, K. Muszynski, and S. Ruscetti
Erythroid Cells Rendered Erythropoietin Independent by Infection with Friend Spleen Focus-Forming Virus Show Constitutive Activation of Phosphatidylinositol 3-Kinase and Akt Kinase: Involvement of Insulin Receptor Substrate-Related Adapter Proteins
J. Virol.,
April 1, 2000;
74(7):
3037 - 3045.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Miyakawa, P. Rojnuckarin, T. Habib, and K. Kaushansky
Thrombopoietin Induces Phosphoinositol 3-Kinase Activation through SHP2, Gab, and Insulin Receptor Substrate Proteins in BAF3 Cells and Primary Murine Megakaryocytes
J. Biol. Chem.,
January 19, 2001;
276(4):
2494 - 2502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Geddis, N. E. Fox, and K. Kaushansky
Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors
J. Biol. Chem.,
September 7, 2001;
276(37):
34473 - 34479.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION