(Received for publication, August 20, 1996, and in revised form, October 18, 1996)
From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the § Department of Physiology, University of Michigan, School of Medicine and the Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105
The role of insulin-like growth factor 1 (IGF-1)
in preventing apoptosis was examined in differentiated PC12 cells.
Induction of differentiation was achieved using nerve growth factor,
and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 109
M and as early as 4 h. The phosphatidylinositol 3
(PI3
)-kinase inhibitors wortmannin (at concentrations of
10
8 M) and LY294002 (10
6
M) blocked the effect of IGF-1. The pp70 S6 kinase
(pp70S6K) inhibitor rapamycin (10
8
M) was, however, less effective in blocking IGF-1 action.
Moreover, stable transfection of a dominant-negative p85 (subunit of
PI3
-kinase) construct in PC12 cells enhanced apoptosis provoked by
serum deprivation. Interestingly, in the cells overexpressing the
dominant-negative p85 protein, IGF-1 was still capable of inhibiting
apoptosis, suggesting the existence of a second pathway involved in the
IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein
kinase/extracellular-response kinase kinase inhibitor PD098059
(10
5 M) inhibited the IGF-1 effect. When
wortmannin and PD098059 were given together, the effect was
synergistic. The results presented here suggest that IGF-1 is capable
of preventing apoptosis by activation of multiple signal transduction
pathways.
Apoptosis or programmed cell death plays an important role in embryonic development, involution of organs, and tumorigenesis. During development of the nervous system, a large proportion of neurons die by this process; about 50% of embryonic postmitotic neurons ultimately die during the period when synapses are formed between neurons and their targets (1, 2). The survival of neurons is dependent on neurotrophins secreted by the target cells (3). In PC12 cells (rat pheochromocytoma cells) differentiated in the presence of nerve growth factor (NGF),1 NGF itself, as well as other growth factors (including platelet-derived and epidermal growth factors, and insulin at high doses), protect cells maintained in serum-free media from apoptosis (4).
Insulin-like growth factor 1 (IGF-1) prevents apoptosis in a number of cell types. For example, IGF-1 at physiological concentrations was effective in inhibiting apoptosis in fibroblasts overexpressing c-myc (5, 6), in cells treated with the topoisomerase II inhibitor etoposide (7), in neuroblastoma cells under hyperosmotic stress (8), and potassium-deprived cerebellar granule cells (9, 10). Most cell types require IGF-1 for growth in culture (11), and a decrease in IGF-1 receptor number induces apoptosis in tumor cells (12). IGF-1 can also act as a survival factor in the absence of other factors. Specifically, IGF-1 inhibits apoptosis of several interleukin-3-dependent cell lines when IL-3 is removed (13).
Insulin and the IGFs exert their biological effects by binding to their
respective transmembrane receptors. Insulin and IGF-1 receptors are
similar, heterotetrameric proteins with intrinsic tyrosine kinase
activity (14, 15). Both receptors are capable of binding insulin and
IGF-1, but each receptor binds its own ligand with a 100-1000-fold
higher affinity than that of the heterologous peptide. In addition,
IGF-1 activity is also regulated by binding to specific IGF-binding
proteins that do not bind insulin (16). IGF-1, the IGF-1 receptor, and
the binding proteins are expressed in many tissues, creating an
autocrine-paracrine hormonal system. One of the earliest steps in
signal transduction by both insulin and IGF-1 is the extensive
phosphorylation of IRS-1, a 185-kDa protein. Tyrosyl-phosphorylated
IRS-1 then interacts with numerous SH2 domain-containing proteins,
including PI3-kinase and the guanine-nucleotide exchange factor
Grb2/SOS. PI3
-kinase then initiates phospholipid turnover, and
Grb2/SOS activation results in initiation of the MAP kinase signal
transduction cascade by sequential phosphorylation and activation of
proto-oncogenes Ras and Raf and the MAPK/ERK kinases (MEK1 and MEK2).
While the effectiveness of IGF-1 on inhibition of apoptosis is well
established, the signaling pathways leading to apoptosis and the
mechanisms of action by which IGF-1 and other agents prevent apoptosis
are largely unknown. The PI3-kinase inhibitor wortmannin is known to
block the protective action of NGF and platelet-derived growth factor
in serum-deprived PC12 cells (4); this finding suggests an important
role of the PI3
-kinase pathway in apoptosis prevention by growth
factors. PI3
-kinase is an important component of intracellular signal
transduction processes linked directly or indirectly to diverse
receptor types. PI3
-kinase is an heterodimer composed of an 85-kDa
regulatory subunit and a 110-kDa catalytic subunit (17). The p85
subunit, through its SH2 domains, mediates the association of
p110 with activated protein tyrosine kinase receptors or
phosphotyrosine-containing peptides such as IRS-1 (18).
In the present work, the involvement of PI3-kinase in mediating IGF-1
prevention of apoptosis in PC12 cells was further explored using the
PI3
-kinase inhibitors wortmannin and LY294002, and PC12 cells were
stably transfected with either a wild-type p85 (Wp85) construct or a
dominant-negative p85 construct (
p85) (19). The
p85 protein lacks
the inter-SH2 region required for binding to p110, thereby blocking
activation of p110 (20). Rapamycin, which inhibits pp70 S6 kinase
(pp70S6K) activation, was also used (21). In addition, the
involvement of the MAP kinase pathway in the prevention of apoptosis by
IGF-1 was tested using PD098059, a specific inhibitor of MEK and the MAP kinase cascade (22, 23).
Recombinant IGF-1 and mouse 2.5S NGF were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY), wortmannin
was from Sigma, and LY294002 was from BioMol Research
Labs Inc. (Plymouth Meeting, PA). Rapamycin was purchased from LC
Laboratories (Woburn, MA). The specific MEK inhibitor PD098059 has been
previously described (22, 23). Cell culture media and reagents were
purchased from Biofluids, Inc. (Rockville, MD). TACS Apoptotic DNA
laddering kit was purchased from Trevigen (Gaithersburg, MD).
Radionuclides, [-32P]dCTP (6000 Ci/mmol) and [
-
32P]ATP (6000 Ci/mmol), were from DuPont NEN.
PC12 cells (a gift
from Dr. G. Guroff, NICHD, NIH, Bethesda, MD) were typically maintained
in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10%
horse serum and 5% fetal bovine serum, in a humidified atmosphere of
95% air and 5% CO2 at 37 °C. For each experiment,
cells were grown in 100-mm culture dishes in DMEM until they reached
60-70% confluence. Cell differentiation was then allowed to proceed
as described previously (24) by changing cells to low serum (2% horse
serum and 1% fetal bovine serum) medium supplemented with 100 ng/ml
2.5S NGF. After 6-7 days, the cells were washed twice with
phosphate-buffered saline (PBS), and the medium was changed overnight
to serum containing DMEM without added NGF. The next day, the cells
were incubated in the absence or presence of IGF-1 (concentrations
ranging from 1010 to 10
7 M) in
serum-free DMEM. At various intervals as stated in the figure legends,
the cells remaining attached to the plate were harvested and combined
with those cells suspended in the medium, which were collected by
centrifugation.
In some experiments, the PI3-kinase inhibitors wortmannin
(10
9-10
7 M) or LY294002
(10
6-10
5 M) were added 6 h prior to harvesting the cells to the serum-free DMEM, either in the
absence or presence of IGF-1 (10
8 M). For
cells growing in either absence or presence of IGF-1, rapamycin
(10
8-10
7 M) was added 6 or
24 h prior to collecting the cells. In other experiments, the MEK
inhibitor PD098059 (10
6-10
4 M)
was added to cells in serum-free medium as described (22, 23). Cells
were preincubated for 30 min with PD098059 prior to the addition of
IGF-1.
Insulin and IGF-1 receptor number in PC12 cells were determined by the Scatchard method as described previously (25) using tracer amounts of Tyr-A14 monoiodinated insulin (2000 Ci/mmol specific activity, Amersham Life Science, Inc., Arlington Heights, IL) or des(1-3)IGF-1 (44 µCi/µg, GroPep, Adelaide, Australia).
DNA LadderingGenomic DNA was prepared from PC12 cells
using the TACS Apoptotic DNA laddering kit. The concentration and
purity of DNA were determined by measuring UV absorbance at 260 and 280 nm. Equal amounts of DNA from each sample (1 µg) were 3-end-labeled
for 10 min at room temperature using the Klenow fragment of DNA Pol I
and the radiolabeled nucleotide (0.5 µCi [
-32P]dCTP,
6000 Ci/mmol). The reaction was stopped by adding 6 × DNA loading
buffer, and samples were then electrophoresed through a 1.5% TreviGel
500 matrix (26) in 1 × Tris acetate/EDTA buffer. Following
electrophoresis, the gel was fixed in 10% acetic acid for several
hours, dried on Whatman 3 MM paper (Whatman Int., Maidstone, United
Kingdom) in a gel dryer at 70 °C, and analyzed by autoradiography
using Kodak X-Omat AR film and Cronex Lightning Plus enhancing
screens.
PC12 cells were grown in
60-mm plates until 60-70% confluent. Cells were co-transfected with
15 µg of plasmid DNA (SR-Wp85 or SR
-
p85, the kind gift of
Professor Masato Kasuga, Kobe University, Japan) (19) and 1 µg
pMC1-neo (Clontech, Palo Alto, CA), using Lipofectin reagent (Life
Technologies, Inc.). Transfections were performed for 12 h, and
the medium was then changed to DMEM for an additional 24 h. After
24 h of recovery, the cell mixtures were serially diluted and
plated onto 150-mm dishes. Stably transfected cells were selected using
500 µg/ml geneticin (Life Technologies, Inc.). Independent colonies
were picked using cloning cylinders (Specialty Media Inc., Lavallette,
NJ). Individual clones overexpressing bovine Wp85 or
p85 were
selected by Western blotting using a polyclonal anti-p85 antibody
(Transduction Laboratories, Lexington, KY).
PI3K
activity was measured as described previously (25) with some minor
modifications. Cells were stimulated with 3 × 108
M IGF-1 for 2 min, and lysates were immunoprecipitated with
a polyclonal anti-p85 antibody (Transduction Laboratories).
Phosphatidylinositol-4-monophosphate (Sigma) was used
as substrate in the kinase assay.
MAPK/ERK activity was
assayed as described (27) with some minor modifications. Briefly, cells
maintained overnight in serum-free medium were stimulated with IGF-1
(107 M) for 12 min at 37 °C. The cells
were then rapidly washed in ice-cold 1 × PBS and solubilized in
ice-cold lysis buffer (50 mM
-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM
dithiothreitol, 100 µM sodium orthovanadate, 10 µg/ml
aprotinin, 1 mM benzamidine, and 2 µg/ml pepstatin A).
The lysates were cleared by centrifugation, and equal amounts of
protein from each sample (5 µg) were incubated for 15 min at 30 °C
in the presence of 400 µg/ml specific MAP kinase substrate peptide
(myelin basic protein 95-98, Upstate Biotechnology Inc.) and 100 µM [
32P]ATP. The reaction was stopped by
spotting samples onto Whatman P81 paper squares followed by extensive
washing in 150 mM phosphoric acid. The papers were air
dried and counted in a
-counter with scintillation liquid.
A modification of the MTT assay
that measures mitochondrial function was used (28). Differentiated PC12
cells were plated on 24-well plates (50,000-80,000 cells/well) and
maintained overnight in complete medium. Cells were then changed to
serum-free medium in the absence or presence of IGF-1
(108 M) and the different inhibitors. At the
indicated periods of time, the medium was aspirated from the wells, and
200 µl of MTT reagent (1 mg/ml) were added to each well. The cells
were then incubated for 1 h at 37 °C and lysed by addition of
200 µl of isoamyl alcohol and shaking for 20 min. A 200-µl aliquot
of each sample was then translated to 96-well plates and read in an
enzyme-linked immunosorbent assay reader at 570-690 nm.
In parallel, direct counting of the cells remaining attached to the plate was performed. After treatment, medium was aspirated, and plates were washed twice with 1 × PBS. Cells still attached to the plate were trypsinized and counted in a Neuebauer chamber.
Differentiation
of PC12 cells was induced by incubating the cells with NGF-containing
medium for 6-7 days. Apoptosis in these differentiated cells was
induced by removal of NGF and incubation of the cells in serum-free
DMEM (SFM). Within the first 4-6 h, apoptosis was detected by
increased DNA laddering (Fig. 1A). The ability of IGF-1 to inhibit the process of apoptosis following the
withdrawal of NGF was studied by incubating the cells in SFM with
varying concentrations of IGF-1. IGF-1 was extremely effective in
inhibiting apoptosis in both a time- and a dose-dependent
fashion (Fig. 1, A and B). The effect of IGF-1
was detected as early as 4 h and at concentrations as low as
109 M, with maximal effects seen at
10
8 M (Fig. 1B). At the same time,
6 h of serum deprivation provoked a 29 ± 10% decrease in
cell number (Fig. 2A) as well as a decrease in MTT activity (Fig. 2B). IGF-1, on the other hand, was
capable of inhibiting both effects. The effect of insulin on inhibition of apoptosis was detected only at higher concentrations of the hormone
(10
7 M) (Fig. 1C). Since the PC12
cells used in these experiments expressed ~39,000 IGF-1
receptors/cell and ~7,600 insulin receptors/cell, we suggest that
insulin may affect apoptosis in these cells by its interaction with the
IGF-1 receptor.
The Effect of Wortmannin, LY294002, and Rapamycin on Apoptosis
To determine whether the effect of IGF-1 on inhibition
of apoptosis involved the PI3-kinase pathway, we used the specific PI3
-kinase inhibitors wortmannin, a fungal protein, and LY294002 (29),
a synthetic inhibitor of PI3
-kinase. The cells were incubated with
IGF-1 (10
8 M) and varying concentrations of
wortmannin (10
9-10
7 M).
Wortmannin at 10
8 M inhibited the action of
IGF-1. This inhibition was maximal at 10
7 M
wortmannin (Fig. 3). The presence of DNA laddering as a
consequence of the incubation with wortmannin was detected at both
6 h (Fig. 3) and 24 h (data not shown), being maximal at
6 h. This is not unexpected, due to the instability of the
inhibitor at the physiological temperature of the medium, as previously
reported (4, 30). To confirm that the PI3
-kinase pathway was involved,
we used LY294002, a reversible PI3
-kinase inhibitor. Similarly to
wortmannin, concentrations of 10
6 and 10
5
M LY294002 blocked the effect of IGF-1 on inhibition of
apoptosis as measured by increased DNA laddering (data not shown),
decrease in cell number (Fig. 2A), and mitochondrial
activity (Fig. 2B).
Interestingly, both wortmannin and LY294002 in the absence of IGF-1
enhanced the degree of apoptosis detected (Fig. 3, lanes 6-8), possibly resulting from the inhibition of basal PI3-kinase activity. However, the level of apoptosis seen with the inhibitor alone
is greater than when the inhibitor and IGF-1 are combined (Fig. 3,
lane 3 versus 7 and lane 4 versus 8). These
results support the conclusion that the PI3
-kinase pathway plays a
role in mediating apoptosis. Furthermore, the IGF-1 effect on
inhibition of apoptosis is, at least in part, via PI3
-kinase
activation.
pp70S6K is known to play a role in the signaling cascade
initiated by PI3-kinase (21). Stimulation of pp70S6K can
also be blocked by wortmannin (31, 32) or LY294002 (21). The inhibitor
rapamycin is known to block the activation of pp70S6K at
some point downstream of PI3
-kinase although its specific target
remains unknown (31, 32). In our system, 10
7
M rapamycin completely blocked IGF-1-stimulated
phosphorylation of pp70S6K as detected by a shift of the
band in a 15% SDS-polyacrylamide electrophoresis gel blotting with
anti-pp70S6K antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) (data not shown). However, incubation of PC12 cells with
10
8 M or 10
7 M
rapamycin resulted only in partial inhibition of the IGF-1 effect on
protecting the cells from apoptosis. This effect was detected as a
faint laddering (data not shown) concomitant with a loss of cells. The
effect of 10
8 M rapamycin was significant
only after a 24-h incubation (Fig. 2A). Incubation with
10
7 M rapamycin, on the other hand, resulted
in a significant, although moderate, decrease in cell number after
6 h (87 ± 4% of cells survived), which was more prominent
after 24 h (73 ± 9% of cells survived). Rapamycin alone at
10
8 or 10
7 M did not
significantly increase the degree of apoptosis in cells maintained in
SFM without IGF-1.
To further establish that the
PI3-kinase pathway is involved in apoptosis, we stably transfected
PC12 cells with vectors containing either a dominant-negative p85
construct (
p85) or a wild-type p85 construct (Wp85) as a control.
Multiple clones of each cell type were obtained, and Western blot
analyses established enhanced expression of the proteins (Fig.
4A). Overexpression of the wild-type p85
construct (Wp85) did not significantly affect the IGF-1 effect on
apoptosis (data not shown). Conversely, expression of
p85 resulted
in enhanced apoptosis (Fig. 4, B and C),
concomitant with a partial decrease in IGF-1-stimulated PI3
-kinase
activity (Fig. 4D). The addition of LY294002 yielded an even
greater effect on induction of apoptosis (Fig. 4B,
lane 6 versus 3). Similar results were obtained using
wortmannin (data not shown). After 16 h of incubation in SFM, the
difference in the degree of apoptosis between the parental cells and
the different
p85 clones studied was maintained or increased (data
not shown). Interestingly, in the cells overexpressing the dominant
negative p85 protein, IGF-1 was still capable of inhibiting apoptosis
although its sensitivity was decreased compared with its effect on
parental cells (Fig. 4C). The IC50 of the IGF-1
effect was 10
8 M in two
p85 clones studied
as compared with 10
9 M in the parental cells,
suggesting that inhibition of PI3
-kinase activity resulted in a
decreased effectiveness of IGF-1 on inhibition of apoptosis. On the
other hand, the IGF-1 effect was not completely eliminated by
inhibiting the PI3
-kinase pathway. This could be explained in part by
the fact that the level of expression of the dominant-negative p85 is
insufficient to totally block the PI3
-kinase pathway (Fig.
4D). Alternatively, other signaling pathways may be involved
in the IGF-1 effect.
The Role of the MAP Kinase Pathways
Since the MAP kinase
pathway mediates many of the known effects of IGF-1, we next blocked
this pathway to determine its role in IGF-1 prevention of apoptosis.
For this purpose, we used a specific MEK inhibitor, PD098059 (22, 23),
that inhibits MAPK activity in a dose-dependent fashion
(Fig. 5A). When incubated with IGF-1,
PD098059 at concentrations of 105 and 10
4
M inhibited the IGF-1 effect on apoptosis prevention (Fig.
5B, lanes 3-4 versus lane 1). This effect was
detectable at 10 h but was maximal at 25 or 30 h (Fig.
5C). PD098059 alone (10
5-10
4
M) also increased apoptosis (Fig. 5B,
lanes 6-7 versus lane 5). These results suggest that the
MAPK pathway is also involved in apoptosis and mediates the IGF-1
prevention of apoptosis. In agreement with the DNA laddering
experiments, a decrease in cell number was found at 24 h but not
at 6 h (Fig. 2A) although a decrease in MTT activity
was already detected at 6 h, being significant at 12 h (Fig.
2B).
Fig. 5.
Since both wortmannin and PD098059 were effective in inhibiting the
IGF-1 action, we next determined whether their effects were additive or
synergistic. As can be seen in Fig. 6, the effect of
addition of both inhibitors is synergistic. For example, the degree of
apoptosis in lane 8 is greater than the degree of apoptosis predicted by the addition of lanes 2 and 4.
Moreover, the extensive DNA laddering obtained by incubation of the
cells with both inhibitors was easily visible in electrophoretic gels
after staining the DNA with ethidium bromide. In contrast, the
laddering provoked after the incubation with either one or the other
inhibitor could not be visualized that way, needing radioactive
labeling (data not shown). Macroscopically, incubation with both
inhibitors simultaneously resulted in 70-80% of the cells detaching
from the plate, in contrast to about 30% when either of the inhibitors
was used separately (data not shown).
Wortmannin and PD098059 have synergistic
effects on apoptosis induction in PC12 cells. Differentiated PC12
cells were maintained for 18 h in 108 M
IGF-1 in the absence (
) or presence (+) of different concentrations of PD098059. Wortmannin was then added at various final concentrations, and incubation was allowed to proceed for 6 h. DNA was then
extracted and analyzed. The results presented are representative of two separate experiments.FIG. 5. PD098059 blocks MAPK activity and induces apoptosis in PC12 cells. A, PD098059 blocks MAP kinase activity in PC12 cells. PC12 cells were maintained in SFM for
12 h and preincubated with PD98059 (PD) for 18 h
or with wortmannin (WT) for 6 h at the indicated
concentrations. The cells were then stimulated with 10
7
M IGF-1 for 12 min and lysed. Lysates were cleared by
centrifugation and analyzed for MAPK activity as described under
"Experimental Procedures." Results are presented as radioactivity
incorporated into the substrate as a percentage of the basal values
(those in absence of IGF-1 stimulation). Results shown are means ± S.E. of three experiments done in triplicate. *, p < 0.05. B, specific MEK inhibitor PD098059 induces
apoptosis in PC12 cells. Differentiated PC12 cells were maintained in
SFM without (
) or with (+) IGF-1 10
8 M in
the presence of the indicated concentrations of PD098059 for 24 h
prior to collection and labeling of the DNA. The results presented are
representative of two separate experiments. C, time-course of apoptosis initiation by PD098059. PC12 cells were maintained for the
indicated periods of time in SFM in the absence (
) or presence (+) of
10
8 M IGF-1, with or without
10
4 M PD098059. Results are representative of
three separate experiments.
During embryonic development, rapid cellular proliferation is necessary for organ growth. However, appropriate function of each organ also depends on an orderly removal of certain cells. This is achieved by the process of apoptosis, also known as programmed cell death. Apoptosis is detected initially as internucleosomal fragmentation of genomic DNA, followed by chromatin condensation, nuclear disintegration, and cellular fragmentation (33, 34). In addition, apoptosis may play an important role in regulating tumorigenesis. For example, the tumor suppressor gene product, p53, may regulate tumorigenesis by inducing apoptosis in the presence of oncogenic factors (35). Mutations in the p53 gene are found in patients with a variety of malignancies, and a poor clinical prognosis is often correlated with the existence of these mutations. Therefore, understanding the mechanisms controlling apoptosis should enhance our ability to treat many diseases.
Insulin and the IGFs have been shown in a number of systems to inhibit apoptosis. In cerebellar granule neurons cultured in low potassium or PC12 cells following NGF withdrawal, these growth factors inhibit apoptosis (4, 9, 10). IGF-1 is a survival factor for neuroglial cells following ischemia (36). Similarly, c-myc-induced apoptosis can be prevented by IGF-1 (5, 6). Induction of apoptosis in a human colorectal carcinoma cell line is inhibited by IGF-1 (37). Conversely, a reduction in the expression of the IGF-1 receptors in C6 rat glioblastoma cells using antisense strategies enhanced apoptosis and delayed tumor growth (12).
While the importance of the IGF system in regulating cellular proliferation and apoptosis is well established, the cellular mechanisms involved in these processes are as yet undefined. We have begun to investigate these pathways using differentiated PC12 cells that undergo apoptosis following withdrawal of NGF from the culture medium. In these cells, we demonstrate that IGF-1 inhibits apoptosis at physiological concentrations, whereas much higher concentrations of insulin are required for comparable effects. Similar results using insulin to inhibit apoptosis have been previously described (4, 38). This finding suggests that insulin exerts its effect via the IGF-1 receptor. However, since the number of IGF-1 receptors in PC12 cells is almost 5-fold greater than that of the insulin receptor, we cannot rule out the possibility of the insulin receptor being effective.
Wortmannin, a fungal metabolite, demonstrates a substantial degree of
specificity for PI3-kinase compared with a number of other lipid
kinases, especially when wortmannin is used at low concentrations
(10
9 or 10
8 M) (39).
PI3
-kinase comprises an 85-kDa regulatory subunit and a 110-kDa
catalytic subunit that phosphorylates phosphatidylinositol at the
D-3-hydroxyl of the inositol ring. Wortmannin binds
irreversibly to the catalytic subunit (p110), thereby inhibiting the
signaling pathway following PI3
-kinase activation. Wortmannin has
previously been shown to completely inhibit PI3
-kinase activity in
PC12 cells at 10
7 M, with a half-maximal dose
(IC50) of approximately 3 × 10
9
M (4, 30). Due to the instability of the inhibitor in the medium, the peak inhibitory effect of wortmannin takes place after 3-4
h of incubation in the cells (30). In the present study, wortmannin at
low concentrations enhanced apoptosis and inhibited the effect of IGF-1
when added to PC12 cells, suggesting that PI3
-kinase is involved in
the regulation of apoptosis. Support for this hypothesis comes from the
use of the synthetic PI3
-kinase inhibitor LY294002 (29), which gave
essentially the same results as wortmannin. In both cases, detection of
characteristic DNA laddering coincided with a 20-30% decrease in the
number of cells attached to the plate and with an even more marked
decrease in mitochondrial activity as measured by MTT assay. The
acceleration of apoptosis by addition of wortmannin to cells in the
absence of growth factor has been observed previously (40) and can be attributed to the inhibition of the basal PI3
-kinase activity.
Since wortmannin (and LY294002) may not be absolutely specific for
PI3-kinase, we chose to further modulate the PI3
-kinase system by
stably transfecting PC12 cells with a dominant-negative p85 or a
wild-type p85 construct. Overexpression of these proteins has enabled
us to confirm the role of PI3
-kinase in apoptosis. Overexpression of
wild-type p85 did not significantly affect IGF-1 effects on apoptosis
following NGF withdrawal. The dominant-negative p85 subunit, on the
other hand, can bind phosphorylated IRS-1 but, because it lacks the
inter-SH2 domain, cannot bind the p110 subunit. Therefore, PI3
-kinase
activation by IGF-1 is effectively inhibited by blocking access of
functional p85 to phosphorylated IRS-1 (19). Overexpression of
p85
thus resulted in a partial decrease of PI3
-kinase activity and a
consequent increase in the degree of apoptosis observed in serum-free
medium and an increased sensitivity to the presence of wortmannin or
LY294002. Thus, our results support the findings of Yao and Cooper (4)
who similarly demonstrated the importance of PI3
-kinase in mediating
the NGF modulation of apoptosis in PC12 cells. The involvement of the PI3
-kinase pathway on prevention of apoptosis by IGF-1, but not by
IL-3, was also recently observed in hemopoietic progenitor cells (41).
The effect of rapamycin, an inhibitor of pp70S6K (21),
further supports the role of the PI3
-kinase pathway in mediating the
IGF-1 effect on apoptosis.
However, we have noted that overexpression of p85 did not result in
a complete inhibition of the IGF-1 action, suggesting the possibility
that IGF-1 may inhibit apoptosis via other signal transduction
pathways. We chose, therefore, to study the role of the MAP kinase
pathway using a synthetic inhibitor of MEK. The MAP kinase pathway is
responsible for mediating numerous effects of both insulin and IGF-1.
Both insulin and IGF-1 receptors activate the MAP kinase cascade
through a p21ras-dependent signal transduction
pathway. Activated Ras interacts with the serine-threonine kinase Raf
and localizes it to the membrane, thereby initiating Raf activation.
Activated Raf then initiates the kinase cascade by phosphorylating and
activating MEK, which in turn phosphorylates and activates the
MAPK/ERK.
PD098059 is a small molecular weight inhibitor of MEK activity, as
measured by MAP kinase activity (22, 23). PD098059 is a specific
noncompetitive inhibitor of MEK, with respect to ATP binding, and does
not inhibit several other kinases tested (22). PD098059 inhibits MAP
kinase activity in PC12 cells with an IC50 of approximately
106 M (22, 23). In our study, incubation of
PC12 cells with concentrations of PD098059 capable of significantly
inhibiting MAP kinase activity resulted in increased apoptosis when
given alone. PD098059 could also block the protective effect of IGF-1.
After 6 h of incubation with PD098059, neither a decrease in cell
number nor DNA laddering could be found although a decrease in the
metabolic activity in the cells treated with the inhibitor could be
already detected by this time. This decrease in mitochondrial activity
precedes by some hours the initiation of apoptosis as detected by loss of cell number and the appearance of DNA laddering. This loss of
metabolic activity preceding apoptosis triggering has been previously
found in sympathetic neurons deprived of growth factor (42). Thus, our
results demonstrate that the MAP kinase pathway is also involved in
IGF-1 prevention of apoptosis. A different conclusion was reached by
Yao and Cooper (4) using PC12 cells. However, their studies involved
the use of the expression of the dominant inhibitory mutant Ras N17,
which interferes with normal Ras function and Raf activation. Since Raf
and MEK activation may be achieved by pathways other than Ras (31, 32,
43), we suggest that inhibition of the pathway downstream of Raf, as in
the present study, is important to establish the role of the MAP kinase
pathway. Moreover, in PC12 cells, it has been described that the
expression of an activated Raf mutant did not activate the MAP kinases
although it resulted in gene expression similar to that induced by NGF
(44). This result suggests that the MAP kinases may be activated by
other pathways in addition to the Ras/Raf pathway in this cell type.
Further confirmation for our conclusion that the MAP kinase pathway is
involved in IGF-1 inhibition of apoptosis comes from a recent study
(38) that demonstrates a role for the MAP kinases in apoptosis
prevention by NGF in PC12 cells.
Since the PI3-kinase and MAP kinase pathways converge at some point
before MEK activation (31, 32), it is possible that the wortmannin
inhibition of PI3
-kinase in our cells affects MAP kinase activation.
Furthermore, recent studies have also shown that following certain
stimuli, MEK may be activated by a PI3
-kinase-dependent pathway (32). Consistent with this are our findings that wortmannin is
also capable of inhibiting IGF-1-stimulated MAP kinase activity to a
certain extent. However, while the possibility that the wortmannin effect is entirely due to the inhibition of the MAP kinase pathway is
consistent with our data, it is less likely because the wortmannin effect on IGF-1 inhibition of apoptosis occurs much earlier than the
effect of PD098059 on IGF-1 action. Furthermore, rapamycin alone was
also capable of inhibiting the effect of IGF-1 in preventing apoptosis.
We therefore postulate that both pathways are involved separately in
this process and that the synergistic effect we obtained by using the
MEK inhibitor together with wortmannin suggests some convergence of the
two pathways either at the level of MEK or at a more distal point. The
convergence of the two pathways is supported by our findings that the
effectiveness of wortmannin was greater than the effect of
rapamycin.
While we have described in this study two separate IGF-1-stimulated pathways that inhibit apoptosis, it is also likely that IGF-1 inhibition of apoptosis involves other as yet unidentified pathways. The elucidation of these pathways should ultimately lead to a better understanding of cellular growth and apoptosis and enable investigators to design new and effective therapeutic agents.
We thank Professor Masato Kasuga (Kobe University, Japan) for the gifts of the wild-type and dominant-negative p85 constructs and Drs. V. Blakesley and E. Wertheimer (National Institutes of Health, Bethesda, MD) for critical reading of the manuscript.