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HEMATOPOIESIS
From the Department of Hematology, Royal Free and
University College Medical School, London, United Kingdom.
The prevention of apoptosis is a key function of growth
factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human
erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 ( Erythropoietin (Epo) and stem cell factor (SCF) are
essential for normal erythropoiesis. Through binding and
activating their respective receptors, EpoR and c-kit, an intracellular
cascade of biochemical events is triggered, which, in addition to
permitting proliferation and differentiation, results in the
transduction of a survival signal to developing erythroid
progenitors.1-6 In many cell types, the
phosphoinositide-3-kinase (PI3K)/Akt (or protein kinase B) pathway is
central to survival signaling in response to a variety of
factors7 and this may also be the case in erythroid
cells.8-10 However, other pathways, in particular the
mitogen-activated protein (MAP) kinase pathway, have also been
implicated in survival signal transduction.11-14
Targets of the PI3K/Akt pathway include the Forkhead transcription
factors, the proapoptotic Bcl2 family member Bad, caspase 9, I In erythropoiesis, progenitors are critically dependent on growth
factors for survival and proliferation until the intermediate normoblast stage of development, and controlling the proportion of
cells that undergo apoptosis during development is one major way in which erythrocyte production is regulated.1
Recently, erythroid progenitors have been shown to be dependent on PI3K for survival,8 but it is unclear in this context which of
its downstream targets is critical in survival signal transduction.
We have examined the role of the PI3K/Akt target GSK3 in the regulation
of apoptosis of primary human erythroid progenitors and find that both
Epo and SCF suppress GSK3 activity. Furthermore, inhibition of GSK3 by
lithium chloride (LiCl) prevents apoptosis induced by growth factor
deprivation, maintains clonogenicity, and reduces the appearance
of the conformational form of the proapoptotic Bcl2 family member Bax
associated with apoptosis induction. These results implicate high GSK3
activity in apoptosis regulation in human erythroblasts and also
suggest Akt is involved in its suppression.
Materials
Erythroid primary cell culture
Time-course experiments and Western blotting To remove growth factors from cultured erythroblasts, cells on days 5 to 7 of culture were pelleted by centrifugation at 350g for 5 minutes and then washed and pelleted 3 times in phosphate-buffered saline (PBS; Life Technologies). Cells were then returned to IMDM and 10% FCS. For stimulation time courses, cells were kept without growth factors for 2 hours before stimulation with either 20 U/mL Epo or 100 ng/mL SCF, or both together. Where indicated, cells were incubated for 15 minutes with LY294002 (25 µM), U0126 (10 µM), or PD98059 (50 µM) before growth factor stimulation. At the time points shown, cells were pelleted rapidly (2000g for 30 seconds) and then resuspended in ice-cold lysis buffer. Lysates were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels and transferred onto nitrocellulose membranes as described.23 Membranes were incubated with primary antibodies (diluted 1:1000 in PBS and 3% bovine serum albumin [BSA; Sigma]) overnight at 4°C, washed 4 times with PBS and 0.1% Tween, and then incubated for a further 60 minutes at room temperature with the appropriate secondary horseradish peroxidase-conjugated antimouse or antirabbit antibody (Amersham, Arlington Heights, IL) at a dilution of 1:10 000 in PBS and 3% BSA. After membranes were washed a further 4 times, protein bands were photographically visualized with enhanced chemiluminescence (ECL or ECL Plus; Amersham).GSK3 kinase assay The method used was modified from that of Ryves and coworkers.24 Erythroid cells were washed and incubated for 1 hour in IMDM and 1% FCS (2 experiments) or 10% FCS (3 experiments) before stimulation with growth factors. At the indicated time points, cells were pelleted and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 10 mM DTT, 0.5 mM Na3VO4, 5 µM Microcystin, and 1 mM Pefabloc) at 250 000 cells/12.5 µL lysis buffer. Lysate (12.5 µL) was mixed with 6.25 µL substrate mix (containing 5 mg/mL synthetic phospho-CREB substrate peptide (KRREILSRRP[pS]YR; Research Genetics, Huntsville, AL) with or without 200 mM LiCl) and the assay, performed in duplicate, was commenced with 6.25 µL ATP mix (200 mM HEPES, pH 7.5, 50 mM MgCl2, 8 mM DTT, 400 µM ATP, 4.6 kBq/µL [ -32P]ATP). After 8 minutes at room temperature
reactions were terminated by spotting on phosphocellulose disks (Life
Technologies). Dry disks were washed 3 times in 0.75% phosphoric acid
and once in acetone before bound radioactivity was counted with a
 -scintillation counter. Specific activity due to GSK3 was determined
by calculating the difference in bound quantified radioactivity between
paired lithium-treated and untreated samples.
Apoptosis assay To determine the proportion of apoptotic cells after 8 hours' incubation in various conditions, cells were pelleted at 350g for 5 minutes and then washed once in annexin V binding buffer (140 mM NaCl, 10 mM HEPES, pH 7.4, and 5 mM CaCl2) before incubation for 10 minutes on ice with 1 µL annexin V-FLUOS (Roche) in 50 µL annexin V binding buffer per sample. Specific fluorescence was then determined using an EPICS Elite flow cytometer (Beckman-Coulter, High Wycombe, United Kingdom).Caspase 3 activity assay Caspase 3 activity in lysates from growth factor-deprived erythroblasts was determined using a colorimetric caspase 3 assay kit (ApoAlert) according to the manufacturer's instructions (Clontech, Palo Alto, CA).Clonogenic assays Erythroid cells on days 4 to 6 of culture were washed and equal numbers were treated for 8 hours in IMDM, 20% FCS, and one of the following: Epo (2 U/mL) and SCF (20 ng/mL), LiCl (20 mM), zVAD-FMK (100 µM), or nil. Cells were then returned to IMDM with 20% FCS, Epo 2 U/mL, and SCF 20 ng/mL and plated out in 96-well plates at a predicted density of 0.66 cells/well. After 72 hours, wells containing 4 cells or more were scored as containing a cell that survived the 8-hour conditioning period with intact clonogenicity.Fluorescence microscopy Cytospin preparations were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 20% normal goat serum (Dako, Cambridge, United Kingdom). Anti-Bax (6A7) was applied at a dilution of 1:50 in PBS and 20% normal goat serum for 1 hour at room temperature. Slides were then washed 4 times in PBS before incubation for 30 minutes with an anti-mouse fluorescein-conjugated secondary antibody (Vector Laboratories, Burlingame, CA) at a dilution of 1:250 in PBS and 20% normal goat serum. Slides were then washed 4 further times with PBS prior to application of Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Cells were visualized with an Olympus IX70 inverted fluorescence microscope (Olympus, Hamburg, Germany). A minimum of 200 cells were scored for Bax* positivity on each cytospin.
GSK3 phosphorylation in primary human erythroblasts In insulin signaling, Akt phosphorylation of GSK3 at S21 ( isoform) and S9 ( isoform) leads to reduced GSK3
activity.15 Akt requires PI3K-dependent phosphorylation at
S473 for optimal activity.25 The p44 MAP kinase (or
extracellular signal-regulated kinase-1 [Erk1]) requires
phosphorylation by Mek1 (MAP kinase or Erk kinase) on T202/Y204 for its
activity.26 The pattern of phosphorylation of these 3 kinases at their key regulatory sites was examined in primary human
erythroid progenitor cells. Erythroblasts lysed directly from
steady-state culture contained high levels of phospho-GSK3 and
GSK3, phospho-Akt, and phospho-p42/p44 MAP kinase. On withdrawal of
growth factor, phosphorylation of all 3 was rapidly and substantially
reduced within half an hour and remained low until cells were
re-exposed to growth factors, when it was rapidly reinduced (Figure
1). Treatment of cells with Epo and SCF
together did not have a noticeable additive effect over the degree of
phosphorylation observed with either cytokine alone (data not
shown).
To confirm that GSK3 phosphorylation was PI3K dependent, growth
factor-deprived cells were preincubated with the PI3K-specific inhibitor LY294002 (25 µM) for 15 minutes or not, before stimulation with either Epo or SCF (Figure 2A). The
Mek1 inhibitor U0126 (10 µM) had no effect on GSK3 phosphorylation
(data not shown). The pathway specificity of LY294002 and U0126 was
confirmed by exposing cells preincubated with either one inhibitor or
the other to a maximal stimulation of Epo 20 U/mL plus SCF 200 ng/mL.
Figure 2B shows that LY294002 blocked Akt phosphorylation without
altering MAP kinase phosphorylation and similarly U0126 blocked MAP
kinase phosphorylation without altering Akt phosphorylation. Similar patterns of blockade were observed when cells were stimulated with Epo
alone or SCF alone (data not shown).
Changes in GSK3 phosphorylation correlate with changes in kinase activity To determine whether changes in GSK3 phosphorylation correlated with alterations in kinase activity, we used a total cell extract kinase assay method,24 which utilized a synthetic phospho-CREB peptide as a GSK3 substrate. In growth factor-deprived primary human erythroblasts, addition of Epo significantly reduced GSK3 activity by 22% ± 8% (mean ± SEM) at 5 to 10 minutes (n = 5, P = .05) and by 24% ± 7% at 20 minutes (n = 4, P = .003; Figure 2C). This reduction in activity was blocked by LY294002. SCF significantly reduced GSK3 activity by 27% ± 9% at 5 to 10 minutes (n = 5, P = .03) and by 33% ± 1% at 20 minutes (n = 4, P < .001), in a similar PI3K-dependent manner.Use of pharmacological inhibitors implicates PI3K and not MAP kinase in survival signaling in human erythroblasts Withdrawal of growth factors from primary cultured human erythroblasts on days 5 to 7 of culture rapidly induced apoptosis. Eight hours after growth factor withdrawal, 49% ± 3% of cells were apoptotic compared with only 11% ± 1% of those supplemented with Epo and SCF (n = 13; P < .001). As described elsewhere,27 we found erythroid survival over this time course was optimal where cells were treated with both Epo and SCF. Either Epo alone or SCF alone, although reducing apoptosis significantly, was not as effective as the combination of the 2 together (data not shown). During the culture and expansion of CD34+ precursors to erythroblasts, a small proportion of cells was always found to be apoptotic, typically in the range 5% to 15%.LY294002 was used to block PI3K and Akt activity in growth
factor-replete erythroid cells and this induced apoptosis to a degree
similar to that seen with growth factor withdrawal. At 8 hours,
39% ± 5% of LY294002-treated cells were apoptotic, significantly higher than the proportion seen in normal growth factor conditions (14% ± 2%; P < .001) and not substantially different
from cells in growth factor-deprived conditions (47% ± %;
P = .08; n = 6; Figure
3A). Blockade of p42/p44 MAP kinase
activation using either U0126 (Figure 3B) or PD98059 (data not shown)
did not affect the proportion of apoptotic cells. Blockade of the
p42/p44 MAP kinase pathway for the duration of the experiment was
confirmed by finding absent p42/p44 MAP kinase phosphorylation in
lysates of cells incubated with Epo, SCF, and U0126 for 8 hours (data
not shown).
Blockade of either GSK3 activity or caspase activity prevents apoptosis of growth factor-deprived primary human erythroblasts Lithium chloride, which potently inhibits GSK3 activity,28 was added at 20 mM to growth factor-deprived erythroblasts and reduced apoptosis significantly from 45% ± 4% to 20% ± 3% (n = 8, P < .001), with 10% ± 1% apoptotic cells in control growth factor-replete conditions. In 5 further experiments, LiCl was added to cells 15 minutes before washing and additionally during the PBS wash steps and this reduced the proportion of apoptotic cells further to 13% ± 1%, representing a mean reduction in apoptosis consequent on growth factor withdrawal of 88% ± 2% (Figure 4A). The addition of either 20 mM KCl or an extra 20 mM NaCl to growth factor-deprived cells had no effect on apoptosis (data not shown). In 4 further experiments, LiCl was found to inhibit apoptosis in a dose-dependent manner at concentrations near to the 50% inhibitory concentration (IC50) for GSK3 under intracellular ionic conditions (2 mM)28 (Figure 4B). Rottlerin, a compound from Mallotus philippinensis,29 also inhibits GSK3,28 and we found that 20 µM rottlerin also reduced erythroblast apoptosis, consequent on growth factor withdrawal, by 78% ± 7% (n = 3).
Caspase activation is instrumental in apoptosis and we found
caspase 3 activity increased after growth factor withdrawal within 2 hours (Figure 5A), as shown by
others.30 Blockade of caspase activity using the
pan-caspase inhibitor zVAD- FMK prevented apoptosis. Only
11% ± 2% of growth factor-deprived cells treated with zVAD-FMK were apoptotic at 8 hours compared with 48% ± 6% of untreated cells (n = 6, P < .001, Figure 5B). Furthermore,
examination of nuclear morphology on cytospin preparations using DAPI
fluorescent nuclear staining showed that where cells had been treated
with zVAD-FMK, despite 8 hours with no growth factors, nuclear
morphology remained normal, unlike control cells (NIL), many of which
showed nuclear changes characteristic of apoptosis (A) (Figure
6B).
Clonogenicity of human erythroblasts is maintained by GSK3 inhibition but not by caspase inhibition To determine whether the clonogenic potential, and therefore actual survival, of erythroblasts was maintained by either LiCl or zVAD-FMK, single-cell cultures were performed. Cells were washed and equal numbers then treated for 8 hours in medium with either Epo and SCF, no growth factors, no growth factors and zVAD-FMK, or no growth factors and LiCl. Cells were then transferred to 96-well plates in culture medium containing both Epo and SCF at a predicted density of 0.66 cells/well. Results showed that 45% ± 10% of growth factor-treated cells yielded a colony of 4 cells or more at 72 hours, compared with 20% ± 4% of cells with no growth factors, 42% ± 4% of cells with LiCl, and 25% ± 7% of cells with zVAD-FMK (n = 4, Figure 7). GSK3 inhibition with LiCl therefore maintained clonogenicity when compared with untreated cells (P = .006), whereas caspase inhibition did not.
GSK3 inhibition, but not caspase inhibition, reduces the appearance of the conformational form of Bax associated with apoptosis During apoptosis, the proapoptotic Bcl2 family member Bax undergoes a conformational change (Bax*), which is required for its translocation from cytosol to the mitochondrial outer membrane, where it may form pores and permit cytochrome c release.31-34 The conformational change is associated with the appearance of an NH2-terminal epitope that may be recognized by specific antibodies.35 Cytospins of human erythroblasts cultured for 8 hours with either Epo and SCF, no growth factors, no growth factors and LiCl, or no growth factors and zVAD-FMK were probed with a conformation sensitive anti-Bax antibody. Figure 6A shows that 20% ± 2% of cells treated with Epo and SCF were Bax* positive, compared with 61% ± 3% with no growth factors, 27% ± 2% with LiCl, and 51% ± 4% with zVAD-FMK (n = 3). Therefore, whereas LiCl treatment of growth factor-deprived cells significantly reduced Bax* positivity at 8 hours compared with untreated cells (P = .02), blockade of caspase activity did not. Figure 6B shows a mixture of apoptotic Bax*-positive cells with condensed nuclear material (A) and intact Bax*-negative cells (N) in the no growth factor condition (NIL). In the zVAD condition, nuclear morphology is intact in all cells although some cells are seen to be Bax* positive (B).
This work suggests that a pathway involving
phosphoinositide-3-kinase, Akt, GSK3, and Bax contributes to the
control of apoptosis induction in human erythroid progenitors. In cells
mainly at the late burst-forming unit and colony-forming unit stages of
development, GSK3 Both the PI3K/Akt pathway7 and the MAP kinase pathway11-14 have been implicated in survival signaling in primary cells. Although we found that Epo and SCF induced phosphorylation of both Akt and Erk 1/2 at their key regulatory sites in human erythroblasts, the PI3K/Akt pathway and not the MAP kinase pathway was implicated in survival signal transduction in erythroblasts because pharmacological blockade of the PI3K/Akt pathway induced apoptosis, whereas blockade of the MAP kinase pathway did not. Of the downstream targets of Akt, we examined GSK3 for a possible role
in survival signaling in erythroid cells. This is because GSK3 has been
implicated in the regulation of apoptosis in Rat-1 fibroblasts, the
PC12 rat pheochromocytoma cell line,36
sympathetic18 and cortical neurons,17 in
addition to its other well-defined biological roles. GSK3 is regulated
by Akt in insulin signal transduction, reducing kinase activity by 40%
in the rat L6 skeletal muscle line.15,19 Similar
reductions in GSK3 activity are induced by epidermal growth factor
(EGF) in A431 cells (50%)37 and nerve growth factor (NGF)
in PC12 cells (30%).36 Human erythroblasts in
steady-state culture show high levels of GSK3 phosphorylation at
S21/S9, consistent with low GSK3 activity. This disappears with the
withdrawal of growth factors and is reinduced by either Epo or SCF. As
in insulin signal transduction, this effect is PI3K and not MAP kinase
dependent. This suggests that the upstream kinase here is likely to be
Akt, although a role for serum and glucocorticoid inducible kinase 1 (SGK1) cannot be ruled out, because this is also PI3K dependent for
activity and may phosphorylate GSK3.38 The degree of
inhibition of GSK3 activity induced by Epo and SCF was in the range of
25% to 30%, broadly comparable to the inhibition of GSK3 obtained by
others with insulin, NGF, and EGF. It is, however, unclear how
relatively small changes in GSK3 kinase activity can have important
cellular effects. One possible explanation is that although whole cell
GSK3 activity is assayed in a kinase assay, only a fraction of cellular
GSK3 may be accessible to Akt for regulation, with the rest sequestered elsewhere in a different cellular context, with potentially different function and substrate availability.39 GSK3 requires
tyrosine phosphorylation on Y279 ( To determine whether GSK3 might have a role in apoptosis induction, we used LiCl to block GSK3 activity. LiCl is a potent inhibitor of GSK328 and has been shown to mimic inhibition of GSK3 in Wnt signaling in Xenopus and Dictyostelium.42-44 This effect was not found with inhibitors of inositol monophosphatase,44 which is also inhibited by lithium.45 We found that 20 mM LiCl reduced the proportion of erythroblasts undergoing apoptosis consequent on growth factor withdrawal for an 8-hour period by 88%. This protective effect was found to be dose dependent over a range of lithium concentrations around and above the 2 mM IC50 reported for GSK3 with lithium under ionic conditions comparable to those found intracellularly.28 Furthermore, a similar degree of protection was achieved using rottlerin, which is also a potent inhibitor of GSK3 activity.28 Together, this evidence strongly suggests that the reduction in apoptosis seen in these experiments is due to inhibition of GSK3. Recently, a GSK3 To determine a possible mechanism by which GSK3 might influence the cellular apoptotic machinery in human erythroblasts, we examined the proapoptotic Bcl2 family member Bax. Bax is normally located in the cytosol in peripheral association with the mitochondria but following a death signal translocates to the mitochondrial outer membrane31,32 consequent on a conformational change.33,49 Bax oligomers then assemble,50 which have the ability to form pores and release cytochrome c.34 Concomitant with this process, an NH2-terminus epitope lying between amino acids 13 to 19 is exposed.35 This conformational change is induced by binding of Bax to the BH3-domain-only proapoptotic protein Bid, a process that may be caspase dependent51 or independent.52 Other reported inducers of conformational change in Bax include increased p38 MAP kinase activity in cortical neurons53 and blockade of pp125FAK (focal adhesion kinase) activity in mammary epithelial cells.54 We found that apoptotic human erythroblasts contained conformationally altered Bax and that its appearance was reduced by inhibiting GSK3 activity but not caspase activity. This suggests the existence of a caspase-independent, GSK3-regulated pathway that controls commitment to cell death in human erythroblasts. Although our data suggest an important role for GSK3 in the regulation
of apoptosis in erythroid cells, it is probable that other mechanisms
also contribute to survival signal transduction. For example, the
Forkhead transcription factor FKHRL1, a target of Akt, is
phosphorylated at T32 and S253 in response to Epo and SCF in primary
human erythroid cells.55 Another Akt target, the
proapoptotic Bcl2 family member Bad is phosphorylated by Akt at S136,
inducing dissociation from Bcl-X and binding to cytoplasmic 14-3-3 protein to promote cell survival.7 However, in this case,
we did not find evidence for its regulation in response to Epo or SCF
in primary human erythroid cells either by using phosphospecific
antibodies or by looking for a shift in the position of Bad in
SDS-PAGE-resolved lysates obtained from erythroid cells before and
after growth factor stimulation (data not shown). Bcl-XL, an antiapoptotic member of the Bcl2 family of proteins, is essential for the survival through to terminal differentiation and enucleation of
erythroid progenitors.56,57 Its expression increases
substantially during human erythroid differentiation30 and
may be induced by Epo through a Stat5 binding element in the Bcl-X
promoter.58,59 However, apoptosis of in
vitro-differentiated Bcl-X Caspase activity has also been implicated in the regulation of erythropoiesis. Late erythroblasts express Fas ligand, which may interact with Fas expressed by earlier erythroid progenitors.61,62 This interaction activates caspases 3, 7, and 8, which are specifically targeted to the nucleus to cleave GATA-1, delaying erythroid differentiation without inducing apoptosis. This effect was reported to be dose dependently prevented by Epo acting downstream of caspase activation and reversed with inhibitors of caspase activity.62 At odds with this, limited caspase activation is suggested to be absolutely required for terminal erythroid differentiation, which may be blocked by inhibitors of caspase activity.63 We found evidence of increased caspase 3 activity as little as 2 hours after growth factor withdrawal in human erythroblasts, which correlated with rapid apoptotic cell death. Although caspase inhibition blocked apoptosis, it did not maintain clonogenicity, a finding also reported elsewhere,64 suggesting that caspase activation in growth factor-deprived erythroblasts occurs subsequent to commitment to cell death and may not be amenable to regulation by growth factors. In summary, we find that a key role of the PI3K/Akt prosurvival pathway in human erythroid progenitors is to suppress GSK3 activity. Growth factor deprivation results in increased GSK3 activity, which is associated with a conformational change in Bax and induction of cell death, irrespective of caspase blockade. Exactly how GSK3 effects these cellular changes will require further work.
Submitted February 21, 2001; accepted April 23, 2001.
Supported by the Medical Research Council (United Kingdom) with personal fellowships to T.S. and A.K. and a cooperative group grant to A.K. and D.L.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Tim C. P. Somervaille, Department of Hematology, Royal Free and University College Medical School, 98, Chenies Mews, London, United Kingdom, WC1E 6HX; e-mail: t.somervaille{at}ucl.ac.uk.
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Top
Abstract
Introduction
B
kinase,
/
plays a central role in neutrophil apoptosis.
Blood.
1999;94:291-301