From INSERM U.402, Institut Fédératif de Recherche 65, Laboratoire de Biologie Cellulaire, Faculté de Médecine
Saint-Antoine, 27 rue Chaligny, 75571 Paris, Cedex 12, France, the
INSERM U.482, Institut Fédératif de Recherche
65, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine,
75571 Paris, Cedex 12 France, and the ¶ Centre de Biochimie, CNRS,
Unité Mixte de Recherche 134, Faculté des Sciences, Parc
Valrose, 06108 Nice, Cedex 02, France
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ABSTRACT |
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We previously reported that insulin activates
nuclear factor B (NF-
B) in Chinese hamster ovary (CHO)-R cells
overexpressing wild-type insulin receptors (IRs) through a pathway
requiring IR tyrosine kinase and Raf-1 kinase activities. We now
investigated whether the activation of NF-
B by insulin could serve
an antiapoptotic function. Insulin
(10
9-10
7 M) inhibited
apoptosis induced by serum withdrawal in CHO-R cells in a
concentration-dependent manner. Insulin antiapoptotic
signaling: (i) was dependent on IR number and IR tyrosine kinase
activity since it was reduced in parental CHO cells and was abolished
in CHO-Y2 cells overexpressing IRs mutated at Tyr1162/1163;
(ii) was, like insulin activation of NF-
B, dependent on Raf-1 but
not on mitogen-activated protein kinase activity since both processes
were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas
they persisted in mitogen-activated protein kinase-depleted cells; and
(iii) required NF-
B activation since it was decreased by proteasome
inhibitors and the dominant-negative I
B-
(A32/36) mutant and was
mimicked by overexpression of the NF-
B c-Rel subunit. We also show
that insulin antiapoptotic signaling but not insulin activation of
NF-
B involved phosphatidylinositol 3-kinase (PI 3-kinase), as
supported by the inhibition of the former but not of the latter process
by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-
B and
PI 3-kinase totally abolished insulin antiapoptotic signaling. Thus
insulin exerts a specific antiapoptotic function which is dependent on
IR tyrosine kinase activity and is mediated by both a
Raf-1-dependent pathway that leads to NF-
B activation and a PI 3-kinase-dependent pathway.
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INTRODUCTION |
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Insulin has long been known as an anabolic hormone that stimulates
a number of transporters in the plasma membrane and regulates, at the
mitochondrial and cytoplasmic levels, a variety of rate-limiting enzymes involved in the intermediary metabolism. In the past decade, evidence has been provided that insulin also regulates gene expression through the control of various nuclear factors (1). We (2) and others
(3) recently reported that insulin activated nuclear factor B
(NF-
B) in mammalian cells through a Raf-1-dependent pathway. The NF-
B/Rel family members identified so far include NF-
B1 (p50 and its precursor, p105), NF-
B2 (p52 and its
precursor, p100), p65 (Rel A), c-Rel, and Rel B. Prototypical NF-
B
is a p50/p65 heterodimer which is usually retained in the cytoplasm of
unstimulated cells in an inactive form by I
B-
, the best
characterized member of the I
B inhibitory protein family (4-6).
Upon cell stimulation, I
B-
is rapidly phosphorylated at
Ser32 and Ser36 near its amino terminus (7, 8),
which targets this subunit for proteolytic degradation via the
ubiquitin-proteasome pathway (9, 10). The released NF-
B p50/p65
heterodimer can then translocate to the nucleus where it directly binds
to its cognate DNA sequence to regulate gene transcription (9, 10).
NF-
B is involved in a number of different cellular processes
including the control of apoptosis, a physiological mechanism of
programmed cell death that is characterized by nuclear condensation and
fragmentation and degradation of DNA into oligonucleosome fragments
(11-13).
The role that NF-B plays in apoptosis appears to be complex since it
has been found to depend on the cell type. Some studies have implicated
NF-
B in promoting apoptosis in certain cells such as neurons (14),
Schwan cells (15), prostate carcinoma cells (16), and embryonic kidney
cells (17). Conversely, several recent reports provided convincing
evidence that NF-
B was involved in apoptosis inhibition. Cells from
transgenic mice deficient in p65/RelA are highly susceptible to
TNF-
1-induced apoptosis
and this susceptibility is reversed by transfection of the cells with
the wild-type Rel A gene (18). Inhibition of NF-
B by protease
inhibitors induces apoptosis in murine B cells, a cell type expressing
constitutively active NF-
B (19). Furthermore, inhibition of NF-
B
nuclear translocation by expression of a dominant-negative form of
I
B-
that cannot be phosphorylated (I
B-
A32/36) increased
cell death induced by apoptotic stimuli known to activate NF-
B
such as TNF-
(20-22), ionizing radiation and daunorubicin
(21) but not by the apoptotic inducer staurosporine, a compound
which does not activate NF-
B (21).
Like insulin-like growth factor I (IGF-I), insulin was shown to exert
an antiapoptotic function. However, in most (23-29) but one (30)
studies, this function was observed at high concentrations of insulin
which are known to activate IGF-I receptors. Since the inhibition of
apoptosis by IGF-I was shown to require the activation of signaling
molecules such as IRS1 (31), phosphatidylinositol 3-kinase (PI
3-kinase) (29, 32), Akt (28), and less frequently mitogen-activated
protein kinase (MAPK) (29), all of which are also specifically
activated by insulin, we decided to investigate a specific
antiapoptotic function of insulin and to examine the role of NF-B in
this function. To this end, we used parental Chinese hamster ovary
(CHO) cells and CHO cells overexpressing either wild-type human insulin
receptors (IRs) or kinase-defective IRs mutated at Tyr1162
and Tyr1163, two autophosphorylation sites playing a
crucial role in receptor activation (33-36). Our study provides
evidence for the ability of insulin to trigger antiapoptotic signaling
in mammalian cells through the activation of its own receptors. Our
results moreover indicate that the antiapoptotic function of insulin
requires the integrity of the IR tyrosine kinase and is mediated by
both a Raf-1-dependent pathway that leads to NF-
B
activation and a PI 3-kinase-dependent pathway.
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EXPERIMENTAL PROCEDURES |
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Reagents--
[-32P]ATP (10 Ci/mmol), ECL
detection kit, Hybond N+ membranes, and hyperfilm MP were
from Amersham Corp. [acetyl-3H]Acetyl coenzyme
A (200 mCi/mmol), CAT assay grade, was obtained from NEN Life
Technologies Inc. Insulin was purchased from Novo Laboratories. IGF-I
and the proteasome inhibitor I
(Z-Ile-Glu-(OtBu)-Ala-leucinal (Z-AL) were from Calbiochem.
The calpain I inhibitor
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL), myelin basic protein, and protein A-Sepharose were obtained from Sigma. The MAPK R2 antibody was obtained from Upstate
Biotechnology, Inc. The ERK1 (C-16) and the rabbit anti-c-Rel
polyclonal antibodies were purchased from Santa Cruz Biotechnology,
Inc.
Cell Lines-- The three different CHO cell lines (generous gift from Professor E. Clauser, INSERM U.36, Paris) used in this study have been previously described (2, 34, 35). These include the parental cell line (CHO), the CHO cell line transfected with a plasmid coding for the native form of human IRs (CHO-R), and the CHO cell line expressing human IRs in which tyrosines at positions 1162 and 1163 have been replaced with phenylalanine residues by site-directed mutagenesis (CHO-Y2). Cells were grown in Ham's F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS).
Plasmids and Transfections--
The green fluorescent protein
plasmid pEFGP and the pCMV5/lacZ and pRSV-CAT plasmids were
previously described (2, 37). The reporter plasmid (Ig)3-conaluc and
its control counterpart conaluc, the mutated plasmid construct
I
B-
(A32/36) (generous gifts from Dr A. Israël, Institut
Pasteur, Paris, France), and the plasmid RSV-C4
that expresses the
Raf-1 dominant-negative mutant Raf-C4 (generous gift from Dr. U. R. Rapp, Institute of Medical Radiobiology and Cell Biology, Wurzburg,
Germany) have been described (8, 38, 39). The full-length
p44MAPK antisense construct that inhibits the expression of
ERK1 (p44MAPK) and ERK2 (p42MAPK) and the
selection vector pEAP (NHE 1 cDNA) have been described (40). The
expression plasmid coding for the c-Rel subunit of NF-
B was a
generous gift from Dr. N. R. Rice (Laboratory of Molecular Virology and Carcinogenesis, Frederick, MD). Transfections were performed by the calcium phosphate precipitation method. The amounts of
the different reporter or expression plasmids used in each transfection
assay are indicated in the legends to Table I and Figs. 5 and 6.
DNA Fragmentation Assay--
For analysis of DNA laddering,
3-5 × 106 cells were used according to the procedure
of Herrmann et al. (41). Briefly, control or treated cells
were harvested, collected by centrifugation, and washed twice in cold
phosphate-buffered saline. Pellets were then suspended in 50 µl of
lysis buffer containing 1% Nonidet P-40, 20 mM EDTA, 50 mM Tris-HCl (pH 7.5). After centrifugation for 5 min at
1,600 × g, the supernatant was collected and the extraction was repeated once. The supernatants were incubated with 5 mg/ml RNase A for 2 h at 56 °C in the presence of 1% SDS (w/v). Then 2.5 mg/ml proteinase K were added and the incubation continued for at least 2 h at 37 °C. DNA fragments were
precipitated with 2.5 volumes of ethanol in the presence of 0.5 volume
of 10 M ammonium acetate at 20 °C overnight. After
centrifugation, samples were washed with 70% ethanol and resuspended
in loading buffer. Electrophoresis was performed in 1 × TBE
buffer on 1% agarose gels containing ethidium bromide.
Apoptosis Assays--
CHO-R cells and CHO-R cells expressing
c-Rel (clone Rel-3) were maintained for 24 h in serum-free medium
(SFM) with or without 107 M insulin. After
24 h, cells were fixed and stained with the Hoechst 33258 DNA dye.
In some experiments, CHO-R cells were cotransfected with
pCMV5/lacZ or pEFGP and different expression plasmids. At the indicated times after transfection, cells were maintained for
24 h in SFM in the presence or absence of 10
7
M insulin and then fixed and revealed with
5-bromo-4-chloro-3-indoyl
-D-galactoside or Hoechst
33258. Cells were observed microscopically and quantitation of
apoptosis was performed as indicated in the legends to Figs. 3, 5, and
6.
Luciferase and CAT Assays-- The lysates from transfected cells were prepared and assayed for luciferase and CAT activities, as described previously (2). Results were normalized as indicated in the legend to Table I.
Immunocomplex MAP Kinase Assay--
CHO-R cells seeded in 6-well
plates were serum-deprived for 24 h and treated for 12 min with or
without 107 M insulin or 20% FCS. Then cells
were lysed for 30 min at 4 °C in lysis buffer containing 1% (w/v)
Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM
NaCl, 50 mM sodium fluoride, 5 mM EDTA, 40 mM
-glycerophosphate, 0.2 mM sodium
orthovanadate and supplemented with the following protease inhibitors:
phenylmethylsulfonyl fluoride (0.1 mM), leupeptin (1 mg/ml), and benzamidine (1 mM). After removal of insoluble
material by centrifugation (12,000 × g, 15 min at 4 °C), equal amounts (150 µg) of protein were immunoprecipitated overnight at 4 °C with 1 µg of the ERK1 (C-16) polyclonal MAPK antibody, then mixed with 30 µl of protein A-Sepharose and incubated for a further hour. Immune complexes were collected by centrifugation at 12,000 × g (4 °C, 15 min), washed four times in
Triton X-100 lysis buffer and once with kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol, 10 mM
p-nitrophenyl phosphate). Final pellets were resuspended in
40 µl of kinase buffer containing 10 µg of myelin basic protein and
50 µM ATP (3 µCi of [
-32P]ATP). After
a 30-min incubation at 30 °C, the reaction was stopped by adding 40 µl of 2 × Laemmli's buffer. The samples were treated at
95 °C for 5 min and separated by SDS-PAGE on 12% (w/v)
polyacrylamide gels (29/1). The gels were then stained with Coomassie
Blue, dried, and subjected to autoradiography.
Western Blot Analysis-- Cell lysates prepared as above were separated by SDS-PAGE on 12% (w/v) polyacrylamide gels and electrotransferred onto Hybond-ECL nitrocellulose membranes in 25 mM Tris, 192 mM glycine. Membranes were blocked in Tris-buffered saline (20 mM Tris-HCl (pH 7.5), 137 mM NaCl) containing 0.1% Tween 20 and 3% non-fat dry milk for 30 min at room temperature. The blots were then incubated overnight at 4 °C in blocking solution with the MAPK R2 (1:1,000) or c-Rel (1:500) antibodies. Thereafter, gels were washed in Tris-buffered saline and incubated with either horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000) or goat anti-mouse IgG (1:10,000) in blocking solution for 1 h. The blots were visualized by the Amersham ECL system.
Statistical Analysis-- Results are given as the means ± S.E. for the indicated numbers of independently performed experiments. Differences between the mean values were evaluated by Student's t test.
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RESULTS |
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Insulin Inhibits Apoptosis in CHO-R Cells through Its Own
Receptors--
CHO-R cells maintained in SFM undergo apoptosis as
indicated by characteristic nucleosomal DNA fragmentation starting at
15 h and continuing for 24 h after serum deprivation (Fig.
1A). The ability of insulin to
inhibit apoptosis was studied by incubating the cells in SFM in the
presence of increasing concentrations (109-10
7 M) of the hormone.
Insulin reduced apoptosis in CHO-R cells in a
concentration-dependent manner (Fig. 1B) with a
slight effect being detected at 10
9 M and
submaximal and maximal protective effects being observed at
10
8 and 10
7 M, respectively.
Note that in CHO-R cells the inhibition of apoptosis by
10
8 M insulin was equivalent to that elicited
by IGF-I at 10
8 M, a concentration reported
to be maximally effective on this process in different cell types (29,
32). As compared with CHO-R cells, parental CHO cells exhibited
decreased sensitivity to insulin for inhibition of apoptosis (Fig.
2A). When these experiments were repeated in CHO-Y2 cells which overexpress tyrosine
kinase-deficient IRs mutated at Tyr1162/1163
autophosphorylation sites, we observed that, at 10
8 and
10
7 M, insulin had no protective effect at
all against apoptosis (Fig. 2B).
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Role of NF-B in Insulin Antiapoptotic Signaling--
We
previously reported that insulin activates NF-
B in CHO-R cells (2).
In view of recent findings from several groups (18, 20-22) indicating
that NF-
B activation reduces programmed cell death in different cell
types, we investigated the role of this transcription factor in insulin
antiapoptotic signaling. To this end, we tested the effect of LLnL
(5 × 10
5 M), a calpain I inhibitor, and
Z-AL (3 × 10
5 M), a potent and
selective proteasome inhibitor, both of which were shown to inhibit
NF-
B activity by preventing I
B-
degradation (42, 43). At the
maximally effective concentrations employed, LLnL and Z-AL almost
completely abrogated basal and insulin-stimulated NF-
B
transcriptional activity in CHO-R cells, as determined by the
luciferase assay (Table I). Under these
conditions, LLnL and Z-AL had no appreciable effect on the extent of
apoptosis induced by a 24-h serum deprivation (Fig.
4). In contrast, LLnL and Z-AL markedly
decreased the protective effect exerted by insulin against this process
with the latter being more potent than the former (Fig. 4).
NH4Cl (2 × 10
3 M), a
nonspecific lysosomal protease inhibitor which had no effect on
insulin-stimulated NF-
B-mediated luciferase activity (Table I), did
not modify the inhibitory effect of insulin on apoptosis (Fig. 4).
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Role of Raf-1 but Not of MAPK in Insulin Activation of NF-B and
Insulin Antiapoptotic Signaling--
We (2) and others (3) recently
reported that the activation of NF-
B by insulin involved a
Raf-1-mediated pathway. This is confirmed in the present paper since
the dominant-negative Raf-1 mutant Raf-C4 (39) abolished insulin
stimulation of NF-
B-mediated luciferase activity (Table I). To
investigate the role of Raf-1 in insulin antiapoptotic signaling, CHO-R
cells were transiently transfected with the pCMV5/lacZ
reporter plasmid together with pCMV or the RSV-C4
expression vector
coding for Raf-C4. Fifteen hours after transfection, the medium was
removed and cells were incubated for a further 24 h in SFM in the
presence or absence of 10
7 M insulin prior to
evaluating the percentage of apoptotic cells by scoring
-galactosidase transfectants as healthy or apoptotic. As shown in
Fig. 6A, Raf-C4 did not
appreciably modify the percentage of apoptosis induced by a 24-h serum
deprivation as assessed by the closely similar percentages of apoptotic
cells determined in CHO-R cells cotransfected with
pCMV5/lacZ and Raf-C4 (35 ± 5%) and in those
cotransfected with pCMV5/lacZ and pCMV (34 ± 4%). In
contrast, Raf-C4 markedly reduced the protective effect exerted by
insulin since the percentage of apoptotic cells among insulin-treated
CHO-R cells cotransfected with pCMV5/lacZ and Raf-C4 was
significantly increased (24 ± 3%) as compared with the
percentage of apoptotic cells determined in insulin-treated cells
cotransfected with pCMV5/lacZ and pCMV (6.0 ± 1.5%).
These results indicate that insulin antiapoptotic signaling, like
insulin activation of NF-
B, involves a Raf-1-mediated pathway.
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Role of PI 3-Kinase in Insulin Antiapoptotic Signaling but Not in
Insulin Activation of NF-B--
Because we found that: (i) the
inhibition of insulin-stimulated NF-
B activity by proteasome
inhibitors or I
B-
(A32/36) markedly reduced but not totally
prevented insulin protection from apoptosis (Figs. 4 and 5A)
and (ii) that overexpressed c-Rel only partially mimicked the insulin
protective effect (Fig. 5C), we examined the possible
implication of another pathway in insulin antiapoptotic signaling. We
tested the involvement of PI 3-kinase since this kinase has recently
emerged as an essential intermediary in the signal transduction pathway
used by growth factors (26, 29, 32) and cytokines (31) to protect cells
from apoptosis. For this purpose, we tested LY294002, a potent PI
3-kinase inhibitor (44). Table I shows that a 24-h treatment with a
3 × 10
5 M concentration of LY294002 did
not modify either basal or insulin-stimulated NF-
B-mediated
luciferase activity in CHO-R cells. Similarly, LY294002 (3 × 10
5 M, 24 h) did not affect the extent
of apoptosis induced by a 24-h serum deprivation (Fig.
7A) in these cells. In
contrast, LY294002 (1-3 × 10
5 M,
24 h) caused a concentration-dependent inhibition of
the protective effect exerted by insulin (10
7
M) against apoptosis (Fig. 7A). This finding
together with the lack of effect of LY294002 on insulin stimulation of
NF-
B activity indicated that the PI 3-kinase inhibitor reduced
insulin antiapoptotic signaling independently of NF-
B. Finally,
since we observed that insulin protection against apoptosis was only
partially abolished by the proteasome inhibitor Z-AL (Figs. 4 and
7B) or LY294002 (Fig. 7), we tested the combined effects of
these compounds on this process. As shown in Fig. 7B, the
profile of DNA degradation observed in serum-deprived CHO-R cells
treated with insulin in the presence of a 3 × 10
5
M concentration of LY294002 and Z-AL was closely similar to
that induced by a 24-h serum deprivation in control cells, indicating that inhibition of both the PI 3-kinase and the NF-
B pathways completely abolished the protective effect against apoptosis exerted by
insulin in CHO-R cells.
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DISCUSSION |
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The present study was designed to examine the ability of insulin
to exert a specific antiapoptotic function in mammalian cells and to
examine the role of NF-B in this function. To this end, we used
parental CHO cells and CHO transfectants that overexpress wild-type
human IRs or IRs made kinase-defective by mutation at Tyr1162/1163, two autophosphorylation sites playing a
crucial role in receptor activation (33-36). Using these cell lines,
we recently demonstrated that insulin is able to activate NF-
B
through a pathway that requires both IR tyrosine kinase and Raf-1
kinase activities (2).
In contrast to several studies which observed the antiapoptotic
function of insulin exclusively at high concentrations which are known
to activate IGF-I receptors (23-29), the present study reports that
insulin is able to produce a concentration-dependent inhibition of apoptosis in CHO-R cells in a low range of insulin concentrations (109-10
7 M). The
finding that at 10
9 M insulin only slightly
inhibited apoptosis is probably due to the high rate of degradation of
the hormone observed in CHO-R cells (35). Several observations indicate
that insulin triggered antiapoptotic signaling through its own
receptors. First, insulin was potent to inhibit apoptosis at
10
8 M, a concentration at which insulin was
unable to displace the binding of 125I-labeled IGF-I to
CHO-R cells (35). Second, the sensitivity of parental CHO cells to
insulin for inhibition of apoptosis was markedly reduced as
compared with that exhibited by CHO-R cells, indicating that this
inhibition is related to IR number. Third, at 10
8 and
10
7 M, insulin had no effect at all in CHO-Y2
cells overexpressing tyrosine kinase-deficient IRs mutated at
Tyr1162/1163 autophosphorylation sites. This finding which
most probably reflects a dominant-negative effect of overexpressed
mutated human IRs on endogenous IRs (34, 35), indicates that the
antiapoptotic capacity of insulin requires the presence of the IR 1162 and 1163 tyrosine residues. In addition, this finding shows that there is a marked difference between the antiapoptotic function of insulin which is suppressed by mutation of two of the major autophosphorylation sites in the IR kinase domain and the antiapoptotic function of IGF-I
which was reported to be unaffected by mutation of the corresponding autophosphorylation sites in the IGF-I receptor kinase domain (32,
45).
The role of NF-B in insulin antiapoptotic signaling was investigated
by two complementary approaches: (i) inhibiting NF-
B activity by the
proteasome inhibitors LLnL and Z-AL or by I
B-
(A32/36)
expression; and (ii) inducing a deliberate activation of this
transcription factor by transient or stable expression of the c-Rel
subunit of NF-
B. Noteworthy, we established that neither maximally
effective concentrations of LLnL or Z-AL nor expression of the
dominant-negative I
B-
(A32/36) mutant did modify the extent of
apoptosis induced by a 24-h serum deprivation in CHO-R cells,
indicating no intrinsic apoptotic effect of NF-
B inhibition in this
cell type. Such a result could be explained by the low level of
constitutive NF-
B activity expressed in CHO-R cells (2). Consistent
with this explanation, Wu et al. (19) observed that NF-
B
inhibition by the protease inhibitor
N-tosyl-L-chloromethyl ketone induced apoptosis
in WEHI 231 cells which express constitutive nuclear NF-
B, whereas
it failed to induce this process in NIH 3T3 fibroblasts which exhibit
an extremely low level of nuclear NF-
B. Interestingly, we
demonstrated that both the proteasome inhibitors and I
B-
(A32/36)
expression markedly reduced the protective effect of insulin against
apoptosis, as assessed by the following: (i) DNA degradation was
reproducibly observed in the lysates from CHO-R cells treated with
insulin in the presence of LLnL or Z-AL whereas it was undetectable in
the lysates from CHO-R cells treated with insulin only; and (ii) the
percentage of apoptotic cells in insulin-treated CHO-R cells
cotransfected with pCMV5/lacZ and I
B-
(A32/36) was
significantly increased as compared with that percentage evaluated in
insulin-treated CHO-R cells cotransfected with pCMV5/lacZ
and pCMV. Accordingly, we observed that expression of c-Rel in CHO-R
cells, which mimicked the insulin-induced increase in NF-
B activity,
protected these cells against apoptosis as assessed by the
significantly decreased number of apoptotic cells counted after a 24-h
serum deprivation in c-Rel transfectants (Rel-3 clone) as compared with
the number counted in control CHO-R cells. In agreement with this
finding, recent papers reported that transfection of a c-Rel expression vector in HeLa or WEHI 231 cells significantly reduced TNF-
- or
anti-IgM-induced apoptosis (19, 22). Altogether, the experiments reported here strongly argue for the antiapoptotic function exerted by
insulin in CHO-R cells being mediated, at least in part, through the
activation of NF-
B. The mechanisms by which insulin-activated NF-
B may reduce apoptosis in CHO-R cells are completely unknown. In
this regard, Liu et al. (22) reported that the activation of
NF-
B by TNF-
reduced TNF-
-induced apoptosis in HeLa cells and
proposed a role for NF-
B in the induction of as yet unknown antiapoptotic genes.
Consistent with the idea that the activation of NF-B by insulin is
an intermediary step in insulin antiapoptotic signaling, both processes
were found to be mediated by a common pathway involving Raf-1 kinase
but not the ERK1 and ERK2 isoforms of MAPK. Indeed we observed that
transient expression of the dominant-negative Raf-1 mutant Raf-C4 in
CHO-R cells abolished insulin-stimulated NF-
B activity and markedly
reduced insulin antiapoptotic signaling. This is supported by the loss
of the insulin-induced increase in NF-
B-mediated luciferase activity
in CHO-R cells expressing Raf-C4 and by the increased percentage of
apoptotic cells observed in these cells in the presence of insulin as
compared with the percentage determined in insulin-treated cells
transfected with the vector alone. The involvement of Raf-1 in
ligand-induced NF-
B activation has been previously reported by
several studies including ours (2, 3, 46-48). However, the question of
whether Raf-1 itself is responsible for the phosphorylation of
I
B-
gave rise to controversial results (49-51). In this regard,
it is worth noting that thus far several kinases have been reported to
be involved in the phosphorylation of I
B-
and activation of
NF-
B. These include: PKC
(50), the double-stranded RNA-activated
p86 protein kinase (52), casein kinase II (53), MEKK1 (54),
p90rsk (55) and CHUK (for conserved
helix-loop-helix ubiquitous kinase) (56) which is identical to IKK
(for I
B-
kinase) (57). In light
of these data, it was recently suggested that multiple independent NF-
B activation pathways exist, each requiring distinct I
B-
kinases (55). On the other hand, the finding reported here that Raf-1
kinase activity is required for insulin antiapoptotic signaling is
consistent with the notion that Raf-1 probably plays a critical role in
the promotion of cell survival, as supported by several recent studies
(58-60). Conversely, Raf-1 has been found to induce apoptosis in
fibroblasts (61), indicating the ability of this kinase to exert
opposite effects depending on the cell context.
In contrast to Raf-1, MAPK failed to be involved in insulin activation
of NF-B and insulin antiapoptotic signaling. We indeed found that
the stable expression of a MAPK antisense which almost completely
suppressed MAPK expression and activity in CHO-R cells, did not affect
insulin-stimulated NF-
B activity nor insulin protection from
apoptosis, as assessed by the persistence of the insulin-induced increase in NF-
B-mediated luciferase activity and the absence of DNA
degradation in insulin-treated CHO-R cells depleted of MAPK. The
involvement of Raf-1 but not MAPK in insulin activation of NF-
B is
consistent with the result of Koong et al. (62) who observed
that a dominant-negative mutant of Raf-1 but not the dominant-negative
mutants of ERK1 and ERK2 abrogated NF-
B activation by hypoxia in NIH
3T3 cells. However, a role for MAPK in the activation of a
B-dependent promoter by PKC
and TNF-
was
subsequently reported in COS cells (63). These heterogenous observations raise the possibility that the role of MAPK in NF-
B activation may be related to the stimulus applied and/or the cell type
studied. This also appears to be the case for the role of MAPK in the
suppression of programmed cell death. In accordance with the present
finding arguing for no implication of MAPK in insulin antiapoptotic
signaling, MAPK was found to have no role in cell survival promoted by
insulin (30), erythropoietin (64), or v-abl (65). Conversely, MAPK
activation prevented apoptosis induced by nerve growth factor
withdrawal in PC12 cells (66) and was required for the suppression of
ceramide-induced apoptosis by sphingosine 1-phosphate in HL-60 cells
(67). In addition, the notion that the role of MAPK in ligand-induced
inhibition of apoptosis is cell-type specific is illustrated by the
finding that MAPK was required for IGF-I antiapoptotic signaling in
PC12 cells (29) but not in neurons (68) or cerebellar granule cells (69).
Although the activation of NF-B by insulin was demonstrated to be a
critical step in insulin antiapoptotic signaling, it only partially
accounted for the effect of the hormone on this process. This is
supported by the finding that the inhibition of apoptosis by insulin
was markedly reduced but not totally abrogated by NF-
B inhibitors
and was not fully mimicked by the overexpression of the NF-
B c-Rel
subunit. We thus tested the involvement of PI 3-kinase which is known
to be activated by insulin and was recently shown to play a role in the
promotion of cell survival by IGF-I through a mechanism involving the
activation of Akt (28, 32). In CHO-R cells, LY290042 appreciably
reduced the antiapoptotic capacity of insulin but had no effect on
insulin-stimulated NF-
B activity, pointing to a role of PI 3-kinase
in the antiapoptotic function of insulin but not in the activation of
NF-
B by the hormone. This finding argued for two independent
pathways mediating the antiapoptotic function of insulin. In
support of this notion, we observed that the treatment of CHO-R cells
with LY290042 in combination with Z-AL completely abrogated the
protection exerted by insulin against apoptosis, indicating that PI
3-kinase and NF-
B play separate roles in insulin antiapoptotic
signaling. Similar to what was observed here for insulin, the
antiapoptotic functions of either IGF-I (29) or interleukin-3 (70)
were reported to be mediated by two independent pathways.
In conclusion, our study provides evidence for the ability of insulin
to trigger antiapoptotic signaling in mammalian cells through the
activation of its own receptors. Our results moreover indicate that
insulin antiapoptotic signaling requires the integrity of the IR
tyrosine kinase and is mediated by both a pathway that includes Raf-1
kinase and leads to NF-B activation and a pathway that is dependent
on PI 3-kinase activity. In addition to the activation of these
pathways, the antiapoptotic function exerted by insulin in CHO-R
cells may require the inhibition of other signaling pathways and in
particular that involving the p38 MAP kinase, as has been recently
reported for PC12 cells and Rat-1 fibroblasts (30). Further studies
will be needed to examine this point.
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ACKNOWLEDGEMENTS |
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We thank Betty Jacquin for secretarial
assistance. We are greatly indebted to Prof. E. Clauser for the CHO
transfected cells, Dr. A. Israel for the (Ig)3-conaluc plasmid and
the dominant-negative I
B-
(A32/36) mutant, Dr. Nancy Rice for the
c-rel expression plasmid, and Dr. U. R. Rapp for the Raf-1
dominant-negative mutant Raf-C4.
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FOOTNOTES |
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* This work was supported by the Ligue Nationale contre le Cancer (Nanterre, France) and the Association pour la Recherche sur le Cancer (Villejuif, France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from Ligue Nationale contre le Cancer.
To whom correspondence should be addressed. Tel.:
33-1-40-01-13-56; Fax: 33-1-40-01-14-99; E-mail:
cherqui{at}st-antoine.inserm.fr.
1
The abbreviations used are: TNF, tumor necrosis
factor; CHO, Chinese hamster ovary; ERK, extracellular signal-regulated
kinase; FCS, fetal calf serum; IGF-I, insulin-like growth factor I; IR, insulin receptor; LLnL,
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; MAPK, mitogen-activated protein kinase; NF-B, nuclear factor
B;
PI 3-kinase, phosphatidylinositol 3-kinase; SFM, serum-free medium;
Z-AL, Z-Ile-Glu-(OtBu)-Ala-leucinal; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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