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*
Laboratory of Immunophysiology, Department of Animal Sciences, and
College of Medicine, Department of Pathology, University of Illinois, Urbana, IL 61801; and
Institut National de la Recherche Agronomique (INRA)-Institut National de la Santé et de la Recherche Médicale (INSERM) U394, Institut François Magendie, Bordeaux, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of cytokines and growth factors can activate PI 3-kinase via diverse mechanisms (15). Intrinsic tyrosine kinase receptors, such as platelet derived growth factor (16), nonreceptor tyrosine protein kinases like JAK-1 (17, 18), the src family kinases involved in TCR signaling (19), and the high m.w. docking proteins IRS-1 and IRS-2 (7, 20), are all involved in the activation of PI 3-kinase. IL-4 activates PI 3-kinase in several types of hemopoietic cells, including T lymphocytes (18) and myeloid progenitors (21). This activity appears to be dependent upon JAK-1 tyrosine phosphorylation of IRS-1/IRS-2. Phosphorylated tyrosine residues in YXXM motifs of IRS-1 (22) and IRS-2 (23) are bound by the src homology domain 2 (SH2) domains of the p85 regulatory subunit of PI 3-kinase. Tyrosine phosphorylation of IRS-1 and the subsequent association with the p85 subunit of PI 3-kinase have now been shown to be critical for IL-4-mediated inhibition of apoptosis (7, 8). Similarly, activation of IGF-IR in human T cells leads to the phosphorylation of IRS-1 and the subsequent association with the p85 subunit of PI 3-kinase (24). Two recent studies have suggested that a member of the antiapoptotic Bcl-2 family, Bcl-xL, is a downstream mediator of IGF-I-stimulated PI 3-kinase and the subsequent survival of neuronal cells (5, 25).
Activation of PI 3-kinase is not always required for cell survival. For example, we have shown that IL-3 increases the lipid kinase activity of PI 3-kinase, but this activity is not necessary for the survival of myeloid progenitor cells (6). This finding has been confirmed (7), clearly establishing that there are PI 3-kinase-dependent and -independent cell survival pathways. Here we show that IL-4 and IGF-I, but not IL-3, activate both S6-kinase and PI 3-kinase, which suggested that S6-kinase might play a role in cell survival via a pathway that is linked to PI 3-kinase. However, inhibition of S6-kinase activity did not affect the capability of either IGF-I or IL-4 to promote cell survival. Instead, IL-3, IGF-I, and IL-4 enhanced expression of Bcl-2. PI 3-kinase activity was required for IGF-I and IL-4, but not IL-3, to increase expression of Bcl-2 and to promote cell survival. Therefore, these data establish that, although Bcl-2 is a common target for cytokines that promote survival of promyeloid cells, only IL-4 and IGF-I increase expression of this protein via a pathway that is dependent upon PI 3-kinase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 (Media Tech, Herndon, VA) or DMEM (Life
Technologies, Gaithersburg, MD) was prepared with 2 g/L of sodium
bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma;
St. Louis, MO). Cultures were supplemented with 5% heat inactivated
horse serum (Sigma; <25 pg endotoxin/ml, as assessed by
Limulus amebocyte assay, Associates of Cape Cod, Woods Hole,
MA) and 0.25 U/ml of recombinant murine IL-3 (Biosource, Camarillo,
CA). FDCP-1 cells (FDCP; a gift from Dr. Lawrence
Rohrschneider, Fred Hutchinson Cancer Center, Seattle, WA) were
maintained at 37°C at 95% humidity and 7% CO2. In
experiments that measured apoptotic populations in FDCP cells treated
with growth factors or cytokines, the cells were washed three times in
RPMI (400 x g) and incubated in serum-free DMEM for
4 h with inhibitors of PI 3-kinase or S6-kinase, at the indicated
concentrations, before the addition of growth factors. For the in vitro
kinase assays, the cells were washed three times in RPMI and then
treated for the indicated times with growth factors in serum-free
medium. IL-3 and IL-4 were purchased from Biosource, IFN-
was from
Life Technologies, and IGF-I was obtained from Intergen (Purchase, NY).
The ELISA kit used to measure IL-3 was from Biosource.
Double staining of FDCP cells with Hoechst 33342 and propidium iodide to measure the apoptotic population
To determine the apoptotic population of FDCP cells, flow
cytometric analysis was used (EPICS V; Coulter Instruments, Miami, FL).
Cells (5 x 105/ml) were incubated 24 h with
IGF-I (100 ng/ml), IL-3 (25 U/ml), IL-4 (25 ng/ml), growth
hormone (250 ng/ml), prolactin (250 ng/ml), or IFN-
(250 U/ml) in the presence or absence of 1 nM rapamycin, 100 nM
wortmannin, or 10 µM LY294002 in serum-free medium. Hoechst 33342 (7
ng/ml; Sigma) was added to the cells for 7 min at 37°C, and the cells
were placed on ice. Immediately before analysis, 2 µg/ml of propidium
iodide (PI; Sigma) was added to the cells. At least 104
cells were analyzed for each sample by a double exclusion staining
protocol using PI and Hoechst 33342, which excluded PI-positive cells
and evaluated Hoechst 33342 staining vs forward angle light scatter
(FALS) to determine the apoptotic populations, as previously described
(6).
Detection of S6-kinase enzymatic activity
Specific activity of S6-kinase was determined by 32P
incorporation into S6 peptide (Upstate Biotechnology, Lake Placid, NY)
as previously described (26). Cells (5 x 106) were
washed in RPMI 1640 and treated with indicated concentrations of IGF-I,
IL-3, or IL-4 for 20 min in the presence or absence of 1 nM rapamycin,
100 nM wortmannin, or 10 µM LY294002 before lysis with 1 ml of lysis
buffer (10 mM potassium phosphate, 1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM ß-glycerophosphate, 1 mM
Na3VO4, 2 mM DTT, 40 µg/ml PMSF, and 0.1%
Nonidet P-40, pH 7.4). Clarified lysates (14,000 x g)
were added to a mixture containing a rabbit anti-p70 S6-kinase IgG
specific for residues 511–525 of rat/human S6 kinase (2
µg/sample; Upstate Biotechnology) complexed to 50 µl of protein
G Sepharose (Pharmacia, Piscataway, NJ) and incubated for 4 h at 4
C. The beads were washed once with lysis buffer and once with kinase
buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.4 mM DTT).
Following the final wash, the immune complexes were suspended in 50
µl of kinase buffer containing 100 µM unlabeled ATP, 200 µCi/nmol
[-32P]ATP (Amersham, Arlington Heights, IL), and 125
µM S6 peptide. The reaction was allowed to proceed for 30 min at
30°C and was terminated by adding 20 µl of 250 mM EDTA and boiling
for 5 min. Following a brief centrifugation, duplicate samples of the
supernatant (25 µl) were applied to phosphocellulose paper (Upstate
Biotechnology), and radioactivity was determined using a Beckman liquid
scintillation counter, LS 6000IC (Irvine, CA). S6-kinase was not
detected in immunocomplexes precipitated with a control rabbit Ig (data
not shown).
Measurement of PI 3-kinase enzymatic activity
FDCP cells were washed three times in RPMI 1640 (400 x
g) and were incubated for 4 h in serum-free medium with
or without 1 nM rapamycin, 100 nM wortmannin, or 10 µM LY294002.
Inducible phosphotyrosine-associated activity was determined by
incubating 5 x 107 cells with IGF-I (100 ng/ml), IL-4
(25 ng/ml), or medium alone for 30 s. Cells were then homogenized
in 1 ml containing 1% Nonidet P-40, 50 mM Tris-HCl, 100 mM
NaCl, 50 mM NaF, 10 mM tetrasodium pyrophosphate, 2 mM
Na3VO4, 2.5 mM benzamidine, 1 mM PMSF, and 1
µM DTT, pH 7.4. Clarified lysates (20 min, 14,000 x
g) were then added to 50 µl of protein G Sepharose
(Pharmacia) conjugated with 2 µg of anti-phosphotyrosine Ab 4G10
(Upstate Biotechnology) that had been diluted with 100 µl of fast
flow Sepharose CL4 beads (Sigma). The immune complexes were then washed
twice with wash buffer A (1% Nonidet P-40, 1 mM DTT, and PBS, pH 7.4),
buffer B (0.5 M LiCl, 1 mM DTT, and 100 mM Tris-HCl, pH 7.4), and
buffer C (10 mM NaCl, 1 mM DTT, and 100 mM Tris-HCl, pH 7.4). PI
3-kinase activity was not detected in immunocomplexes precipitated with
a control isotype-matched mouse IgG2b
Ab (data not shown).
The lipid kinase assay was initiated by adding 60 µl of sonicated
L-
-phosphatidylinositol (0.33 mg/ml) in kinase buffer
containing 20 mM HEPES, 0.04 mM EGTA, 0.4 mM NaPO4, 48 µM
[-32P]ATP (2.1 µCi/nmol; Amersham), and 10
mM MgCl2 at pH 7.1. The reactions were performed at room
temperature for 15 min and terminated by the addition of 15 µl of 4 N
HCl. An equal volume mixture of chloroform/methanol (200 µl) was used
to extract the lipids following vortexing and centrifugation
(14,000 x g) for 10 min. The chloroform-containing
lipid phase was reextracted with 150 µl of an equal volume mixture of
0.15 N HCl/methanol, followed by vortexing and centrifugation at
14,000 x g for 10 min. Ten microliters were then
resolved by thin layer chromatography using a
chloroform/methanol/ammonium hydroxide (75:58:17 (v/v)) running buffer,
as previously described (6). Detection of phosphorylated
phosphoinositol was performed by autoradiography on XAR film (Eastman
Kodak, Rochester, NY), and measurement of band intensity was by
Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA).
Western blotting to detect Bcl-2 and Bax
Cells were treated with medium alone, IGF-I, IL-3, or IL-4 in the presence or absence of 100 nM wortmannin or 10 µM LY294002 in serum-free medium for 24 h, and blots were performed with 50 µg of protein from lysates of 106 cells in homogenization buffer (1% Nonidet P-40, 50 mM Tris-HCl, 100 mM NaCl, 1 mM PMSF). Protein determination was performed on the clarified lysates (20 min; 14, 000 x g) using the MicroBCA protein assay (Pierce, Rockford, IL). Protein from the lysates was then boiled in SDS PAGE loading buffer (10% SDS, 10 mM NaCl, 1 mM EDTA, 1% bromophenol blue, and 200 mM DTT) for 5 min and separated on 12% polyacrylamide gels. The proteins were then transferred to Trans-blot PVDF membrane (Bio-Rad, Hercules, CA), blocked for 1 h in 5% nonfat dry milk at room temperature, and incubated with hamster anti-murine Bcl-2 Ab (2.0 µg/ml) for 2 h at room temperature in PBS-0.1% Tween 20. The blot was then incubated with murine anti-hamster IgG (1 µg/ml) and subsequently with horseradish peroxidase-labeled sheep anti-mouse IgG (1:3000). The blots were then developed with enhanced chemiluminescence substrate (Amersham) and subsequently exposed to autoradiographic XAR film (Eastman Kodak).
In additional experiments, 50 µg of protein from cells treated with medium, IGF-I, IL-3, or IL-4 in the presence or absence of wortmannin (100 nM) or LY294002 (10 µM) was separated as above and transferred to PVDF for detection of Bax. The membranes were probed with 1:3000 dilution of rabbit anti-mouse Bax Ab (PharMingen) in PBS-0.1% Tween 20 for 2 h at room temperature. The membranes were subsequently incubated with horseradish peroxidase-labeled murine anti-rabbit IgG (Amersham) at room temperature for 4 h. Proteins were identified using enhanced chemiluminescence substrate and subsequent exposure to autoradiographic film (Eastman Kodak).
Statistical Analysis
All experiments were repeated a minimum of three times. Results were analyzed using a general linear model with the Statistical Analysis System (SAS Institute, Cary, NC) (27), and differences between treatments were detected with Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overexpression of IRS-1 in myeloid progenitors permits both IL-4
and insulin to activate S6-kinase and stimulate the proliferation of
these cells (12), but it is unknown whether PI 3-kinase is associated
with this activity. To determine the induction of S6-kinase by specific
ligands in FDCP cells, optimal concentrations of IGF-I (6), IL-3 (6),
and IL-4 (data not shown) that activate PI 3-kinase were added to
106 cells in serum-free medium for 20 min, and cell lysates
were immunoprecipitated with an Ab specific for S6-kinase. As shown in
Fig. 1
A, both IGF-I (100
ng/ml) and IL-4 (25 ng/ml) increased the ability of these
immunoprecipitates to phosphorylate a specific peptide substrate of
S6-kinase by 10-fold ± 1 and 11-fold ± 1
(p < 0.01; n = 3).
Interestingly, although we have previously established that IL-3 (25
U/ml) increases PI 3-kinase activity in FDCP cells (6), treatment with
IL-3 did not significantly stimulate S6-kinase activity.
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B; p < 0.01; n = 3). More
significantly, wortmannin (100 nM; 28) also potently reduced both
the IGF-I- and IL-4-mediated increase in S6-kinase activity by 82% ±
7 and 81% ± 8, respectively (Fig. 1B; p <
0.01; n = 3). When the chemical inhibitor of PI
3-kinase activity was used, LY294002 (10 µM; 29), the inhibition
of S6-kinase activity induced by IGF-I was 87% ± 9, whereas that for
IL-4 was 84% ± 11 (Fig. 1B; p < 0.01;
n = 3). When cells were cultured in either medium alone
or IL-3, none of the inhibitors affected S6-kinase activity (data not
shown). Collectively, these data demonstrate that IGF-I and IL-4, but
not IL-3, potently increase the activity of S6-kinase, an effect that
may be dependent upon the activation of PI 3-kinase. Survival of FDCP cells does not depend upon activation of S6-kinase
To determine the role of ligand-activated S6-kinase in inhibiting
apoptosis of myeloid progenitor cells, FDCP cells (5 x
105 cells/ml) were treated with IGF-I (100 ng/ml), IL-4 (25
ng/ml), or IL-3 (25 U/ml) for 24 h. The early apoptotic population
was characterized as those cells negative for PI that expressed low
forward angle light scatter and high Hoechst 33342 staining, as we
previously described (6, 31). All three ligands significantly reduced
the apoptotic population from 43% ± 6 in medium-treated cells to 17%
± 4, 6% ± 2, and 21% ± 5 in IGF-I-, IL-3-, and IL-4-treated cells,
respectively (Fig. 2; p
< 0.01; n = 3). Addition of a blocking dose of
rapamycin (1 nM; Fig. 1) did not prevent either IGF-I or IL-4 from
promoting the survival of these cells (Fig. 2). Similarly, rapamycin
had no effect on the survival of cells treated with IL-3. These data
demonstrate that, although IGF-I and IL-4 activate S6-kinase, this
enzyme is not essential for IGF-I and IL-4 to enhance the survival of
myeloid progenitor cells.
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We tested the possibility that IL-4 and IGF-I act indirectly to inhibit apoptosis by inducing the synthesis and release of IL-3. We measured the amount of IL-3 in supernatants from FDCP cells cultured with medium alone, IL-4 (25 ng/ml), or IGF-I (100 ng/ml) for 4, 12, or 24 h. In all cases, the level of IL-3 was below the sensitivity of the ELISA assay (<3 pg/ml). It is therefore unlikely that IL-4 and IGF-I inhibit apoptosis in FDCP cells by inducing the secretion of IL-3.
We (6) and others (7, 25) have established that there are at
least two survival pathways in myeloid progenitor cells based upon
their requirement for PI 3-kinase. To determine whether PI 3-kinase is
required for the inhibition of apoptosis in IL-4-treated FDCP cells, we
investigated whether IL-4 would induce PI 3-kinase and whether
inhibition of this IL-4-induced enzymatic activity would prevent
IL-4-mediated cell survival. We treated 5 x 107 FDCP
cells with IGF-I (100 ng/ml), a well-characterized inducer of PI
3-kinase activity in these cells (6), or an optimal concentration of
IL-4 (25 ng/ml) that inhibits apoptosis (Fig. 2). Induction of PI
3-kinase activity was measured following immunoprecipitation of cell
lysates with a phosphotyrosine Ab, followed by an in vitro lipid kinase
assay that measures the phosphorylation of phosphatidylinositol. A
representative example is shown in Fig. 3, A and B, and the
summary of three independent experiments is given below. The positive
control treatment, IGF-I, potently stimulated PI 3-kinase activity by
11-fold ± 2, and a similar 13-fold ± 2 increase in PI
3-kinase activity was observed following treatment with IL-4
(p < 0.01; n = 3).
Pretreatment of FDCP cells with wortmannin (100 nM) for
4 h potently inhibited ligand-induced PI 3-kinase activity,
reducing lipid phosphorylation by 90% ± 5 in the positive control
treatment, IGF-I, and by 86% ± 8 in cells stimulated with IL-4
(p < 0.01; n = 3). A similar
reduction in PI 3-kinase activity was obtained in IGF-I- and
IL-4-treated cells (87% ± 6 and 84% ± 9, respectively) following a
4-h pretreatment with the chemical inhibitor of PI 3-kinase LY294002
(p < 0.01; n = 3). As
expected, incubation of FDCP cells with rapamycin, at a concentration
that inhibited S6-kinase activity (Fig. 1) but did not affect cell
survival (Fig. 2), did not reduce the capability of either IGF-I
(inhibition of 4% ± 1) or IL-4 (reduction of 6% ± 2) to stimulate
the activity of PI 3-kinase. These data demonstrate that IL-4
stimulates anti-tyrosine-precipitable PI 3-kinase activity in FDCP
cells and confirm that this activity is blocked by both wortmannin and
LY294002, but not rapamycin.
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A) and measured
the apoptotic population. As expected, both IGF-I and IL-3
significantly reduced the apoptotic population from 43% ± 4 to 17%
± 2 and 6% ± 2, respectively (Fig. 4;
p < 0.01; n = 3). The effect of IGF-I
was blocked by both PI 3-kinase inhibitors, as demonstrated by the
findings that wortmannin and LY294002 increased the apoptotic
population in IGF-I-treated cells from 17% ± 2 to 39% ± 4 and 42%
± 4, respectively (p < 0.01;
n = 3). Treatment of cells with IL-4 reduced the
apoptotic population from 43% ± 4 to 23% ± 2 (Fig. 4;
p < 0.01; n = 3), and this protection
was totally blocked in the presence of either wortmannin (39% ± 4) or
LY294002 (38% ± 4). Although we have previously demonstrated that
IL-3 activates PI 3-kinase activity (6), neither wortmannin nor
LY294002 increased the apoptotic population in IL-3-treated FDCP cells
(5% ± 2 and 7% ± 2, respectively) compared with cells treated with
IL-3 alone (6% ± 2; p > 0.10; n =
3). These data reinforce the original concept that there are both PI
3-kinase-dependent (IGF-I) and -independent (IL-3) pathways involved in
inhibition of apoptosis and confirm the results of Zamorano et al. (7)
by establishing that IL-4, like IGF-I, requires PI 3-kinase to enhance
the survival of myeloid progenitor cells.
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Overexpression of Bcl-2 permits FDCP cells to survive in the
absence of IL-3 (32, 33) and protects monocytic cells from apoptosis
following treatment with inhibitors of PI 3-kinase (25). Similarly,
recent evidence in neuroblastoma cells suggests that PI 3-kinase may be
involved in the regulation of the Bcl-family members (34). To determine
the role of PI 3-kinase in the regulation of the antiapoptotic protein
Bcl-2 in hemopoietic cells, we compared the amount of Bcl-2 protein in
IL-3-deprived FDCP cells that were subsequently incubated with IL-3 (25
U/ml), IL-4 (25 ng/ml), or IGF-I (100 ng/ml) in serum-free medium for
24 h. The amount of Bcl-2 in 50 µg of lysates was measured by
Western analysis using a hamster anti-mouse Ab. A representative
Western blot is shown in Fig. 5A, and the densitometric
results of three independent experiments are summarized in Fig. 5, B and C. Cells cultured in medium alone expressed
little, but detectable, amounts of the 26-kDa Bcl-2 protein. IGF-I
induced a 4.1-fold ± 0.5 increase (p <
0.01) in expression of Bcl-2, and similar results were observed with
IL-4 (4.3-fold ± 0.3; p < 0.01) and IL-3
(5.1-fold ± 0.3; p < 0.01) (Fig. 5B).
All three cytokines also caused a similar increase in the expression of
Bcl-2 in the experiments shown in Fig. 5C.
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, A and B) and
LY294002 (Fig. 5C) were used to inhibit the activity of this
enzyme. Wortmannin significantly reduced the induction of Bcl-2 in
IGF-I-treated cells from 4.1-fold ± 0.5 to 0.9-fold ± 0.3
(Fig. 5B), amounting to an inhibition of 90% ± 9
(p < 0.01; n = 3). Similarly,
inhibition of PI 3-kinase activity by wortmannin reduced the capability
of IL-4 to induce Bcl-2 expression from 4.3-fold ± 0.3 to
0.9-fold ± 0.3 (Fig. 5B), or a 98% ± 12 decline
(p < 0.01; n = 3). However,
this inhibitor of PI 3-kinase activity did not affect the expression of
Bcl-2 in cells treated with IL-3 (5.1-fold ± 0.3 vs 4.7-fold
± 0.3; n = 3). Similarly, wortmannin did not affect
expression of Bcl-2 in cells incubated in medium alone (1-fold ±
0.4 vs 0.8-fold ± 0.3). When the LY294002 inhibitor of PI
3-kinase activity was used, nearly identical results were obtained for
IGF-I (92% ± 11 inhibition; p < 0.01;
n = 3) and IL-4 (94% ± 11 inhibition;
p < 0.01; n = 3), with little affect
on Bcl-2 expression in FDCP cells cultured in either medium or IL-3
(Fig. 5C). Collectively, these data establish that, even
though all three proteins increase expression of Bcl-2, only IGF-I and
IL-4 do so by a pathway that requires PI 3-kinase. Since neither
inhibitor affected the ability of IL-3 to induce Bcl-2, these data
further suggest that there exists an alternative pathway for
cytokine-mediated maintenance of this antiapoptotic protein. Bax expression is independent of PI 3-kinase activity
The 21-kDa protein Bax has recently been shown to act as a
tumor suppressor by stimulating apoptosis in vivo (35). To determine
whether PI 3-kinase also might regulate expression of this
apoptosis-inducing protein, FDCP cells (106) were cultured
in medium, IGF-I, IL-3, or IL-4 for 24 h, and Bax was measured in
50 µg of cell lysates using a rabbit anti-mouse Bax Ab (Fig. 6A). When averaged over three
independent experiments, treatment with IGF-I, IL-3, or IL-4 did not
significantly alter Bax protein expression (1.1-fold ± 0.5,
1.0-fold ± 0.4, or 1.1-fold ± 0.3 increase, respectively,
above medium-treated cells; Fig. 6B), suggesting that this
apoptotic-inducing protein is independent of growth factor or cytokine
stimulation. To determine whether PI 3-kinase might regulate Bax
expression, we pretreated cells with either wortmannin or LY294002. At
concentrations that effectively inhibit PI 3-kinase activity, neither
wortmannin (100 nM; Fig. 6B) nor LY294002 (10 µM; Fig. 6C) significantly affected Bax levels in cells cultured in
medium or with IGF-I, IL-4, or IL-3. These data establish that Bax is
not subject to the same regulatory mechanisms as those for Bcl-2,
supporting the idea that IL-3, IL-4, and IGF-I increase expression of
antiapoptotic proteins rather than reducing the amount of proapoptotic
proteins.
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enhances the survival of IL-3-deprived FDCP cells
All members of the hemopoietic receptor superfamily activate the
nonreceptor JAK protein tyrosine kinases (36), so we tested the
ability of other members of this family to promote the survival of FDCP
cells. IFN-, growth hormone, and prolactin are members of this
receptor superfamily, and both IFN- and growth hormone have also
been reported to tyrosine phosphorylate IRS proteins and recruit PI
3-kinase (37). Using flow cytometry, we measured the low forward angle
light scatter, high Hoechst 33342 early apoptotic population of growth
factor-deprived FDCP cells (5 x 105 cell/ml) treated
with IFN- (250 U/ml), IL-3 (25 U/ml), growth hormone (500 ng/ml), or
prolactin (500 ng/ml) for 24 h. IFN- was nearly as effective as
IL-3 in enhancing the survival of FDCP cells, reducing the apoptotic
population from 43% ± 4 to 9% ± 2 (p <
0.01; n = 3). Neither growth hormone nor prolactin
inhibited apoptosis in these cells (data not shown). These data
establish that not all members of the hemopoietic receptor superfamily
are capable of protecting promyeloid cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although these data
suggest that S6-kinase is downstream of IL-4- and
IGF-I-stimulated PI 3-kinase, the activation of S6-kinase is not
critical for biological responses of FDCP cells to IL-4 or IGF-I, as
assessed by an increase in cell survival (Fig. 2). Both IL-4 and IGF-I
stimulate PI 3-kinase activity (Fig. 3), and addition of wortmannin or
LY294002 abrogates IL-4- and IGF-I-mediated inhibition of apoptosis
(Fig. 4) and potently reduces IL-4- and IGF-I-stimulated expression of
Bcl-2 in these cells (Fig. 5). Addition of either inhibitor does not
prevent IL-3 from promoting the survival of FDCP cells or the
expression of Bcl-2 protein, suggesting that IL-3 increases the
expression of Bcl-2 and the subsequent survival of myeloid progenitor
cells via a PI 3-kinase-independent pathway. Not all ligands that bind
to members of the hemopoietic receptor superfamily are able to protect
myeloid progenitors from cell death (Fig. 7). Finally, none of the cytokines or
inhibitors that we tested reduced expression of the apoptotic inducer
Bax (Fig. 6), suggesting that Bax is not regulated via a PI
3-kinase-dependent pathway. Collectively, these data indicate that
increased expression of Bcl-2 is critical for IL-4, IGF-I, and IL-3 to
inhibit apoptosis in FDCP cells. However, only IGF-I and IL-4 require
PI 3-kinase to enhance expression of Bcl-2 and promote the survival of
myeloid progenitors.
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The serine/threonine S6-kinase may be a critical substrate of
ligand-activated PI 3-kinase (41). However, inhibition of S6-kinase
activity with rapamycin does not affect survival of FDCP cells cultured
with either IGF-I or IL-4. Similarly, survival of neurons (4), as well
as skeletal muscle cell differentiation (42), requires the activity of
PI 3-kinase but not S6-kinase. It therefore appears that S6-kinase is
not required for IL-3, IGF-I, or IL-4 to protect cells from apoptosis.
The recent identification of novel high-molecular mass serine/threonine
kinases has revealed a new family of proteins that contain a
carboxyl-terminal catalytic domain that is closely related to PI
3-kinase (43). One of these PI 3-kinase homology of mammalian origin is
mTOR (mammalian targets of rapamycin), and wortmannin and LY294002 have
been shown to directly inhibit the activation of mTOR (44). Our
results in vivo are in accord with this observation (Fig. 1). More
importantly, however, the mTOR PI 3-kinase family member does not
appear to be involved in the survival-promoting activity of IGF-I or
IL-4 because rapamycin, which also totally blocked the activation of
S6-kinase but not that of PI 3-kinase, had no effect upon the ability
of IGF-I or IL-4 to promote cell survival. These data are consistent
with the idea that closely related PI 3-kinase family members can have
separate and distinct biological functions, at least in myeloid
progenitor cells. We have previously established that IGF-I can
increase cell survival in the presence of actinomycin D (6), so it is
possible that IGF-I and IL-4, acting via PI 3-kinase, maintain
expression of Bcl-2 by reducing the degradation of Bcl-2 mRNA or
protein. We are now exploring the idea that PI 3-kinase maintains Bcl-2
and cell survival via an alternative mechanism that does not utilize
S6-kinase, such as activation of Akt/protein kinase B that has
been reported for the IGF-I-promoted survival of fibroblasts (45).
Indeed, preliminary results are consistent with the possibility because
IGF-I increases the activity of Akt-1 more effectively than IL-3 and
because this IGF-I-induced Akt-1 serine kinase activity is inhibited by
both wortmannin and LY294002, but not rapamycin.
PI 3-kinase may potentially down-regulate the expression of proapoptotic members of the Bcl-family and subsequently promote cell survival (40, 46). Bax has recently been shown to act as a tumor suppressor, inhibiting tumor growth (35). Here we measured the expression of Bax in FDCP cells and showed that addition of IL-3, IL-4, or IGF-I does not affect the expression of Bax protein. A similar finding has been reported by Akbar et al. (47), who demonstrated that Bax expression remains unchanged in IL-2-deprived T lymphocytes, again suggesting that Bax is not under cytokine control. Since here we show that survival factors act to increase expression of Bcl-2, our findings are not inconsistent with earlier experiments that established that induction of apoptosis following cytokine deprivation is related to a reduction in the amount of Bcl-2 relative to Bax (48).
In this report we significantly extend our previous findings that
there are two mechanisms that mediate the survival of myeloid
progenitor cells (6). Enhanced expression of Bcl-2 in IGF-I- and
IL-4-treated cells requires PI 3-kinase activity, while IL-3 maintains
Bcl-2 expression in FDCP cells via a PI 3-kinase-independent pathway.
IGF-I stimulates the intrinsic tyrosine kinase activity of its receptor
and induces the direct phosphorylation of IRS-2 in myeloid cells (49)
whereas IL-4 is dependent upon JAK-1 to mediate tyrosine
phosphorylation of IRS-2 (20, 50, 51). These data suggest that other
ligands that stimulate JAK activity, such as those in the hemopoietic
receptor superfamily that have been shown to affect a number of immune
events (52, 53), may inhibit apoptosis in FDCP cells. Here, we
demonstrate that IFN-, but not growth hormone or prolactin, enhances
the survival of myeloid progenitor cells. IFN- may act through
either the PI 3-kinase-dependent or -independent pathway.
In summary, we have established that IL-4 shares with IGF-I the requirement for PI 3-kinase to promote the survival of myeloid progenitor cells and that this process is independent of S6-kinase. Expression of Bcl-2 is maintained by IL-3 as well as IGF-I and IL-4, but neither cell survival nor expression of Bcl-2 is regulated by PI 3-kinase in IL-3-treated cells. In contrast, both IGF-I and IL-4 depend upon PI 3-kinase activity to promote cell survival and to increase expression of Bcl-2. This PI 3-kinase regulation is specific for Bcl-2 because the antiapoptotic protein Bax is unaffected by either of the cytokines or their downstream inhibitors. Collectively, these data demonstrate that there are at least two pathways that regulate Bcl-2 protein expression and the subsequent inhibition of apoptosis in myeloid progenitor cells and that neither pathway requires the activation of S6-kinase.
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Footnotes |
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2 Current address: Department of Biology, 206 Felmley Hall, Illinois State University, Normal, IL 61790.
3 Address correspondence and reprint requests to Dr. Keith W. Kelley, Laboratory of Immunophysiology, University of Illinois, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. E-mail address:
4 Abbreviations used in this paper: PI 3-kinase, phosphatidylinositol 3'-kinase; FDCP, factor-dependent cell progenitor; Bcl-2, B cell lymphoma-2; S6-kinase, p70 S6-kinase; IGF, insulin-like growth factor; IRS, insulin receptor substrate; JAK, Janus kinase; PI, propidium iodide; PVDF, polyvinylidene difluoride.
Received for publication September 23, 1998. Accepted for publication February 2, 1999.
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: Y497 and Y713, but not the STAT-6-docking tyrosines, signal protection from apoptosis. J. Immunol. 161:859.-110 phosphoinositide 3-kinase complex in vivo. J. Biol. Chem. 271:21614. and leukemia inhibitory factor utilized insulin receptor substrate in intracellular signaling. J. Biol. Chem. 271:29415.-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur. J. Immunol. 26:294.[Medline]
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