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INSERM U-402, Faculté de Médecine Saint-Antoine (C.D.M., A.C., M.J.B.V.E., F.B., M.C., G.C., J.C.), and INSERM U-482 (A.A.), Hôpital Saint-Antoine, 75571 Paris, France
Address all correspondence and requests for reprints to: Dr. Christèle Desbois-Mouthon, INSERM U-402, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75571 Paris Cedex 12, France. E-mail: desbois{at}st-antoine.inserm.fr
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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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The molecular events initiated by insulin to regulate cell proliferation and survival have been the subject of intense investigations. To date, several lines of evidence indicate that PI 3-K plays a critical role in insulin signaling of these two processes (3). Indeed, the blockage of PI 3-K by chemical inhibitors or overexpression of dominant negative mutants inhibited both the mitogenic (4, 5) and antiapoptotic (6, 7) effects of insulin in various cell types. PI 3-K has been shown to interact with the Ras signaling pathway to mediate the proliferative effect of insulin (8, 9). To mediate the protective effect exerted by insulin against apoptosis, PI 3-K has been demonstrated to activate Akt/PKB (10), a serine/threonine kinase that phosphorylates and thereby inactivates components of the cell death machinery, such as the Bcl-2 family member Bad (11), the glycogen synthase kinase-3 (12), the protease caspase 9 (13), and the transcription factor FKHR (14).
c-Jun N-terminal kinases (JNKs) are serine/threonine kinases that belong to the mitogen-activated protein kinase (MAPK) family (15, 16). JNKs are regulated by an upstream kinase cascade and are directly phosphorylated at Thr183 and Tyr185 by the dual specificity MAPK kinases SEK1/MKK4 and MKK7. Activated JNKs phosphorylate transcription factors such as c-Jun, activating transcription factor-2, and Elk1 (17). Inactivation or attenuation of JNK signaling can be accomplished by dephosphorylation of JNKs on the regulatory phosphothreonine and phosphotyrosine residues, and an expanding family of dual specificity phosphatases, including MAPK phosphatase-1 (MKP-1/CL100) (17, 18, 19, 20), MKP-2/TYP1 (18, 20, 21), and M3/6 (22) has been involved in this process.
The activation of JNKs was initially shown to be promoted by stress- and inflammation-related stimuli and to be associated with cell growth arrest and apoptosis (23, 24, 25, 26, 27). However, it was also reported that JNKs were not involved in apoptosis or even that they were protective (23, 24, 25, 28, 29). In addition, some studies revealed that growth factors and oncogenes induced a sustained activation of JNKs to mediate their effects on proliferation and transformation (23, 24, 25, 30, 31). Insulin was initially reported to produce a rapid activation of JNKs, which was associated with the phosphorylation of c-Jun and the activation of the activating protein-1 transcription complex (32). Recently, we provided evidence that this activation was transient and followed by a sustained inhibition phase (33). However, the signaling pathway and the physiological significance of long term JNK inhibition remain unknown. In the present study we focused on the role of PI 3-K and MKP-1 as potential mediators of insulin inhibition of JNKs, and we examined the relevance of this inhibition to insulin-mediated cell proliferation and survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and transfections
CHO-IR cells overexpressing human insulin receptors (IRs) have
been described previously (33). CHO-IR cells and parental CHO cells
transfected with an empty vector (pcDNA3/Neo) were routinely maintained
in Ham’s F-12 medium supplemented with 10% FCS and 300 µg/ml
geneticin (Life Technologies, Inc., SARL, Cergy Pontoise,
France). Human skin fibroblasts were cultivated in DMEM containing 10%
FCS (Life Technologies, Inc., SARL). In most experiments
cells were preincubated in serum-free medium (SFM) for 24 h. In
some assays cells were preincubated in the presence of wortmannin,
LY294002, actinomycin D, or sodium orthovanadate (Sigma-Aldrich Corp., Saint Quentin Fallavier, France). Cells that did not
receive inhibitors received vehicles (dimethylsulfoxide) for wortmannin
and LY294002, methanol for actinomycin D). To establish permanent
transfectants, cells were plated in 100-mm culture dishes (5 x
105 cells/dish) and transfected with 15 µg
pcDNA3/Zeo, pcDNA3/Zeo/wild-type JNK1, or pcDNA3/Zeo/mutant JNK1 by
using Transfast (Promega Corp., Madison, WI). After
48 h, cultures were split into complete medium supplemented with
300 µg/ml Zeocin (Cayla, France) and maintained until clone
formation. Clones were isolated using cloning cylinders and were tested
for transgene expression by Western blot analysis. For transient
transfections, cells plated in six-well plates (2.5 x
105 cells/well) or 100-mm dishes (8.0 x
105 cells/dish) were transfected by using
Transfast or the calcium phosphate precipitation method as previously
described (35). The total amount of DNA was kept constant with
pcDNA3.
JNK assay
For assaying JNK activity, cells were rinsed twice with ice-cold
PBS and scraped on ice in lysis buffer containing 20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1
mM EGTA, 1% (vol/vol) Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM ß-glycerophosphate, 1
mM Na3VO4, 1
mM phenylmethylsulfonylfluoride, and 1 µg/ml leupeptin.
Lysates were clarified by centrifugation at 13,000 rpm for 10 min, and
supernatants (300 µg protein) were incubated for 1 h with 2 µg
anti-JNK1 antibody (C-17, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) and for 2 additional h with 25 µl protein A/G
Plus-agarose (Santa Cruz Biotechnology, Inc.). Beads were
then pelleted by quick centrifugation, and immune complexes were washed
twice with lysis buffer and twice with kinase buffer [25
mM Tris-HCl (pH 7.5), 5 mM
ß-glycerophosphate, 2 mM dithiothreitol, 0.1
mM Na3VO4, and
10 mM MgCl2]. The kinase reaction
was initiated by resuspending the pelleted beads in 30 µl kinase
buffer plus 2 µg glutathione-S-transferase (GST)-c-Jun
(New England Biolabs, Inc., Beverly, MA), 5 µCi
[
-33P]ATP (Amersham Pharmacia Biotech France SA, Les Ulis, France), and 20
µM unlabeled ATP. The reaction was terminated
after 20 min at 30 C by the addition of Laemmli sample buffer.
Substrate phosphorylation was examined by 12% SDS-PAGE followed by
autoradiography.
Western blot analysis
Ten to 50 µg cell lysates prepared as described above were
subjected to electrophoresis on 12% SDS-polyacrylamide gels and
electroblotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) for 1.5 h at 50 V. After blocking in 3%
nonfat dry milk (Bio-Rad Laboratories, Inc., Hercules,
CA), the membranes were probed for 1 h at room temperature with
anti-JNK1 (1:1,000; C-17, Santa Cruz Biotechnology, Inc.),
anti-active ERK (1:20,000; Promega Corp.), anti-ERK1
(1:1,000; C-16, Santa Cruz Biotechnology, Inc.), or
anti-MKP-1 (1:500; M-18, Santa Cruz Biotechnology, Inc.)
antibody. Immune complexes were visualized by chemiluminescent
detection (New England Biolabs, Inc.).
Measurement of DNA synthesis
Cells seeded in six-well plates (3 x
105 cells/well) were incubated in the absence or
presence of insulin (10 and 100 nM) for 20 h.
[Methyl-3H]thymidine (1
µCi/well; Amersham Pharmacia Biotech) was added for the
last 3 h of the incubation period. The amount of radioactivity
incorporated into DNA was determined as previously described (36).
Evaluation of cell number
Cell number was estimated using a colorimetric assay in which
the reduction of a tetrazolium salt
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT),
Sigma-Aldrich Corp.) by mitochondrial dehydrogenases of
living cells is detected. Cells plated on six-well plates (3 x
105 cells/well) were maintained for 24 h in
the absence or presence of 100 nM insulin. The medium was
removed, and MTT (250 µg/ml) was added to each well for 4 h at
37 C. Cells were dissolved in dimethylsulfoxide and shaken for 15 min.
An aliquot of each sample was transferred to a 96-well plate and read
in an enzyme-linked immunosorbent assay reader at 540 nm.
Analysis of apoptosis
The extent of serum withdrawal-induced apoptosis was evaluated
in stable and transient transfectants by the DNA fragmentation and the
ß-galactosidase assays, respectively. Analysis of DNA fragmentation
was performed as previously reported (6). Briefly, cells were collected
by centrifugation, washed twice in ice-cold PBS and lysed in 1%
Nonidet P-40, 20 mM EDTA, and 50 mM Tris-HCl
(pH 7.5). After centrifugation, the supernatants were incubated with 5
mg/ml ribonuclease A and 1% SDS (wt/vol) for 2 h at 56 C. Then
2.5 mg/ml proteinase K were added, and the incubation was continued for
at least 2 h at 37 C. DNA fragments were precipitated in the
presence of ethanol and 10 M ammonium acetate at -20 C
overnight and then analyzed by electrophoresis on 1% agarose gels. For
ß-galactosidase assays, CHO-IR cells were transiently transfected
with 2 µg pcDNA3/Neo or pcDNA3/Neo/wild-type JNK1 together with 0.5
µg pCMV5/lacZ. Fifteen hours posttransfection, the medium
was removed, and cells were incubated for an additional 24 h in
SFM in the absence or presence of 100 nM insulin.
Cells were fixed in 1% formol-0.8% glutaraldehyde at 4 C for 10 min,
washed twice with PBS, and stained with
5-bromo-4-chloro-3-indolyl-ß-D-galactoside (0.8
mg/ml), 4 mM
K3Fe(CN)6, 4
mM
K4Fe(CN)6, and 2
mM MgCl2. Quantification of
apoptosis was performed by scoring ß-galactosidase transfectants as
healthy or apoptotic.
Measurement of protein and glycogen syntheses
For measurement of protein synthesis, cells plated in six-well
plates (4 x 105 cells/well) were incubated
for 3 h with L-[35S]methionine
(15 µCi/well; Amersham Pharmacia Biotech) in the absence
or presence of 100 nM insulin. After two washes with
ice-cold PBS, cells were incubated for 30 min at 4 C in 5%
trichloroacetic acid, rinsed twice with ice-cold 80% ethanol,
air-dried, and solubilized in 20% KOH. The amount of radioactivity
incorporated into protein was determined by liquid scintillation
counting. For glycogen synthesis, cells plated in six-well plates
(4 x 105 cells/well) were incubated for 30
min in the absence or presence of insulin (10 nM), and
D-[U-14C]glucose (2 µCi/ml;
Amersham Pharmacia Biotech) was added for an additional
2 h. The amount of radioactivity incorporated into glycogen was
determined as previously reported (36).
Statistical analysis
Results are given as the mean ± SEM for the
indicated number 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, parental CHO cells
responded to the inhibitory effect of insulin on JNK activity, but with
a decreased sensitivity compared with CHO-IR cells, as this inhibition
was observed at 100 nM insulin in the former vs.
10 nM in the latter. This finding indicates that
insulin inhibition of JNKs is related to the number of IRs expressed at
the cell surface, similar to what was recently found for the
antiapoptotic effect of insulin (6). Figure 1B shows that insulin also
inhibited JNK activity in human skin fibroblasts. This finding argues
for the relevance of JNK inhibition by insulin in normal nontransfected
cells.
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shows that insulin inhibited JNK activity by about 50% in control
cells. In contrast, cell pretreatment with wortmannin (Fig. 2, A and C)
or LY294002 (Fig. 2, B and C) completely abrogated insulin-induced
inhibition of JNKs under conditions where each drug had no effect on
basal JNK activity. These findings are consistent with the idea that
insulin mediates JNK inhibition through a PI 3-K-dependent pathway.
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indicating that insulin-mediated JNK inhibition occurred through the
activation and/or the induction of a phosphatase(s). Consistent with
the latter hypothesis, the transcription inhibitor actinomycin D (5
µg/ml) completely reversed insulin inhibition of JNKs, whereas it did
not appreciably modify basal JNK activity (Fig. 3B).
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).
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, left panel) and JNK activity (Fig. 4B, right
panel). Compared with control cells, cells transfected with the
antisense MKP-1 construct exhibited an approximately 50% decrease in
basal and insulin-stimulated MKP-1 expression. When we examined the
activation state of endogenous JNKs, we observed that the inhibition of
JNK activity exerted by insulin in control cells was partially reverted
in MKP-1-depleted cells, suggesting a role for MKP-1 as a mediator of
the inhibitory effect of insulin on JNKs. The fact that MKP-1 depletion
did not appreciably modify the JNK activity in the absence of insulin
suggests that basal JNK activity escapes MKP-1 regulation, probably due
to the very low level of MKP-1 expressed in CHO cells in the absence of
a specific inducer.
This finding together with the above result (Fig. 2), showing a role
for PI 3-K in insulin inhibition of JNKs, led us to examine whether in
CHO-IR cells insulin stimulated MKP-1 expression through a PI
3-K-dependent pathway, as shown recently in vascular smooth muscle
cells (38). We tested the effects of wortmannin (100 nM)
and LY294002 (10 µM) on insulin stimulation of MKP-1
expression in CHO-IR cells and observed that both inhibitors abolished
this process (Fig. 4C), indicating that PI 3-K lies upstream of MKP-1
in the signaling pathway initiated by insulin to repress JNK
activity.
Role of JNK inhibition in insulin stimulation of cell
proliferation
At this point of the study, we sought to examine the role of JNK
inhibition in insulin-mediated cell proliferation and survival. To this
end, CHO-IR cells were stably transfected with an expression vector
conferring zeocin resistance and containing the cDNA for the human
wild-type or mutant JNK1. Mutant JNK1 cDNA encodes an inactive enzyme
mutated on Thr183 and
Tyr185 that acts as a dominant negative mutant of
JNK1 (34, 39). After characterization of the zeocin-resistant clones by
immunoblotting with an anti-JNK1 antibody, four clones were selected
for further experiments (Fig. 5A). These
are WT1 and WT2, two clones exhibiting a 2- to 3-fold increase in the
amount of wild-type JNK1 compared with control cells (Zeo cells), and
Mut1 and Mut2, two clones displaying a 6- to 7-fold overexpression of
mutant JNK1.
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). The overexpression of wild-type JNK1 reduced by about 40% the
stimulation of DNA synthesis elicited by insulin (10 and 100
nM) in WT1 and WT2 cells (Fig. 5B, left
panel). It must be noted that WT1 and WT2 cells could not be
maintained in culture for a long time because after a few passages
cells detached from culture dishes, probably due to the ability of
overexpressed JNK1 to reduce growth factor-mediated proliferation and
to initiate apoptosis (see below). The overexpression of mutant JNK1 in
Mut1 and Mut2 cells increased insulin stimulation of DNA synthesis by
about 60% compared with that in Zeo cells (Fig. 5B, right
panel). Consistent with this finding, insulin (100
nM) proved to be more potent to increase the Mut1
cell number than the Zeo cell number (38 ± 3% vs.
15 ± 1% over basal), as assessed by the MTT assay.
Activation of ERKs has been demonstrated to be of importance in the
stimulation of cell proliferation by insulin (1, 2). Thus, we wondered
whether the increase in the stimulatory effect of insulin on DNA
synthesis and cell number observed in cells overexpressing mutant JNK1
could result from increased insulin stimulation of ERK activity. As
shown in Fig. 6, insulin (100
nM, 10 min) stimulated the phosphorylation of
p44ERK1 and p42ERK2 to a
similar extent in Zeo, Mut1, and Mut2 cells, excluding the possibility
that the stimulatory effect of mutant JNK1 on insulin-induced cell
proliferation could result from enhanced ERK activation by insulin.
Together, these results are consistent with the idea that JNKs play an
antiproliferative role in CHO-IR cells and that their inhibition by
insulin contributes to the proliferative effect of the hormone.
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shows that
Zeo cells maintained in SFM exhibited a marked oligonucleosomal DNA
degradation typical of apoptosis. The addition of insulin (100
nM, 24 h) strongly reduced DNA degradation in these
cells, thus confirming our previous data (6). This effect was mimicked
by overexpression of mutant JNK1, as in the absence of insulin, Mut1
and Mut2 cells exhibited a ladder of DNA fragmentation similar to that
observed in control cells maintained in the presence of insulin. This
indicates that overexpressed mutant JNK1, like insulin, protected CHO
cells against serum withdrawal-induced apoptosis.
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, the percentage of apoptotic cells, which amounted to 37
± 1% in control cells maintained in SFM in the absence of insulin,
fell to 9 ± 1% in the presence of insulin, indicating that
insulin inhibited apoptosis in CHO-IR cells by about 75%, in
accordance with our earlier report (6). The transient expression of
wild-type JNK1 significantly increased the extent of serum
withdrawal-induced apoptosis, as the percentage of apoptotic cells in
JNK1 transfectants cultured for 24 h in SFM in the absence of
insulin was 55 ± 1%. Moreover, the transient expression of
wild-type JNK1 markedly reduced the ability of insulin to protect cells
against apoptosis. Indeed, the percentage of apoptotic cells was
44 ± 2% in cells transfected with wild-type JNK1 cultured in the
presence of insulin, indicating that insulin inhibited apoptosis by
only 20% in these cells. In addition, we observed that the transient
expression of the antisense MKP-1 construct in CHO-IR cells did not
alter the extent of apoptosis in the absence of insulin, but abrogated
the ability of insulin to protect these cells against apoptosis (Fig. 7C). Together, these data strongly argue that JNK1 acts as a
proapoptotic kinase in CHO cells and that insulin inhibition of JNK
activity through MKP-1 induction contributes to the protective effect
exerted by the hormone against serum withdrawal-induced apoptosis.
Role of JNK inhibition in insulin stimulation of protein and
glycogen syntheses
Finally, we sought to determine whether insulin inhibition of JNKs
was specifically implicated in insulin signaling of cell proliferation
and survival or whether this inhibition could also contribute to
insulin signaling of metabolic processes. We observed that
overexpression of mutant JNK1 was without effect on insulin stimulation
of protein and glycogen syntheses, as the hormone (100 nM)
increased protein synthesis by about 1.3-fold and glycogen synthesis by
about 2.8-fold in control, Mut1, and Mut2 cells (Table 1). These data suggest that JNK
inhibition is required for insulin signaling of cell proliferation and
survival, but not for insulin regulation of protein and glycogen
syntheses in CHO-IR cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We show that insulin inhibition of JNK activity is mediated through PI 3-K activation, as this inhibition was reversed by cell preincubation with the PI 3-K inhibitors, wortmannin and LY294002. Considered together with our previous results showing that insulin activates JNKs through a PI 3-K-dependent pathway (33), this finding indicates that PI 3-K mediates both the short term stimulatory and long term inhibitory effects of insulin on JNKs. Of interest, insulin-like growth factor I (IGF-I) was recently reported to inhibit anisomycin-induced JNK activation through a PI 3-K-dependent pathway in human embryonic kidney 293 cells (42). However, this effect differed from that exerted by insulin, as it was very rapid and reached a maximum after a 5-min treatment with IGF-I.
In addition to PI 3-K, insulin inhibition of JNKs required the de novo synthesis of a phosphatase(s) capable of dephosphorylating JNKs, as supported by the experiments conducted in the presence of orthovanadate or actinomycin D. Several studies have reported that ERKs and JNKs serve as substrates for the dual specificity phosphatase MKP-1 (17, 18, 19, 20, 43). MKP-1 is an immediate-early gene product that is potently induced in response to both mitogenic and stress stimuli. Recently, insulin stimulation of MKP-1 expression has been evidenced in CHO and vascular smooth muscle cells (37, 44), and it has been suggested that this stimulation could result in a negative feedback signal attenuating ERK activation. In the present study we provide evidence that in CHO-IR cells, insulin stimulation of MKP-1 expression is involved in the inhibitory effect of insulin on JNK activity. This is supported by the findings that insulin inhibited JNKs under conditions where it stimulated MKP-1 protein expression and, more importantly, that the selective decrease in endogenous MKP-1 expression induced by using an antisense cDNA strategy partially reversed the inhibitory effect of insulin on JNK activity. Together with abrogating insulin inhibition of JNKs, wortmannin and LY294002 blocked insulin stimulation of MKP-1 protein expression in CHO-IR cells. This indicates that PI 3-K is an effector of insulin involved in insulin induction of MKP-1 expression and JNK inhibition.
The stable overexpression of wild-type JNK1 reduced the effect of insulin on DNA synthesis in CHO-IR cells, and, reciprocally, the overexpression of dominant negative JNK1 enhanced DNA synthesis in response to insulin. The latter finding was presumably due to inhibition of JNK signaling, as it could not be attributed to an increased ability of insulin to stimulate ERK activity, a process that plays a preponderant role in insulin regulation of DNA synthesis. It therefore seems reasonable to propose that JNKs exert an antiproliferative action in CHO-IR cells and that their inhibition by insulin contributes to insulin stimulation of cell proliferation. As a recent report by Dong and colleagues (26) suggested that the hyperproliferation exhibited by T cells from Jnk1-/- mice could result from their resistance to apoptosis, the question of whether the antiproliferative effect of JNKs in CHO-IR cells could be linked to their proapoptotic function remains to be elucidated.
Several studies have suggested that JNKs play a critical role in the
apoptotic signaling pathways initiated by various stimuli, including UV
and -irradiation, ceramide treatment, and growth factor withdrawal
(23, 24, 25, 26, 27, 40, 41). In the present study the DNA fragmentation assays
performed in stable mutant JNK1 transfectants showed that
overexpression of mutant JNK1 greatly decreased the extent of serum
withdrawal-induced apoptosis and mimicked the protective effect exerted
by insulin in control CHO-IR cells. In accordance with these findings,
the ß-galactosidase assays performed in transient wild-type JNK1
transfectants showed that overexpressed wild-type JNK1 increased the
sensitivity of CHO-IR cells to serum withdrawal-induced apoptosis and
reduced insulin protection against apoptosis. Together, these results
suggest that JNKs mediate growth factor withdrawal-induced apoptosis in
CHO-IR cells and point to a role of JNK inhibition in the antiapoptotic
function of insulin.
Only a few studies have addressed the implication of JNKs in the signaling of metabolic processes. Thus, it has been reported that JNK activation by anisomycin and RO31–8220 in NIH-3T3 cells enhanced the phosphorylation of the translation initiation factor eIF4E (45). In addition, it has been suggested that JNK activation could mediate insulin stimulation of glycogen synthase in skeletal muscle (46). The data obtained in CHO-IR cells overexpressing mutant JNK1 do not support a role for the JNK signaling pathway in insulin regulation of glycogen and protein syntheses. Taken as a whole, our findings, instead, suggest that in these cells JNKs are selectively involved in the insulin regulation of cell proliferation and survival.
Both PI 3-K and MKP-1 have been shown to play a physiological role in
the promotion of cell survival. PI 3-K is an important mediator of
survival signals triggered by various growth factors, including insulin
and IGF-I (3, 6, 7, 47, 48, 49). Up to now, the molecular mechanisms by
which this kinase controls growth factor-mediated cell survival were
shown to involve Akt/PKB activation and subsequent
phosphorylation/inhibition of proapoptotic molecules such as Bad (11),
glycogen synthase kinase-3 (12), caspase 9 (13), and FKHR (14). MKP-1
has recently emerged as a phosphatase endowed with a cytoprotective
function against tumor necrosis factor-
- and UV-induced apoptosis,
which involves JNK inhibition (50, 51, 52). In agreement with these
findings, our study provides evidence that an antisense MKP-1 construct
abolished the protection exerted by insulin against serum
withdrawal-induced apoptosis through its ability to reverse insulin
inhibition of JNKs.
We recently reported that the antiapoptotic function exerted by insulin
in CHO-IR cells was mediated by two pathways, with one involving Raf-1
and leading to nuclear factor-
B activation and the other requiring
PI 3-K activation (6). We show here that the latter pathway leads to
JNK inhibition through the induction of MKP-1 expression. These
findings point to a novel mechanism by which PI 3-K regulates cell
survival in response to insulin. In addition, we observed that the
transient expression of a dominant negative mutant of Akt/PKB
(myrPKB-KD) or of a constitutively active Akt/PKB (gagPKB) in CHO-IR
cells did not affect or mimic insulin-induced MKP-1 expression and JNK
inhibition. These preliminary results do not support a role for Akt/PKB
in the insulin signaling pathway leading to MKP-1 induction and JNK
inhibition. Further studies using other Akt/PKB constructs will be
required to verify this hypothesis. In this regard, recent studies
(53, 54, 55) reporting discrepant results concerning the role of Akt/PKB in
insulin inhibition of phosphoenolpyruvate carboxykinase gene
transcription point to the necessity of using a panel of Akt/PKB
constructs for evaluating the involvement of this kinase in an insulin
biological response.
In conclusion, our study provides the first evidence that the activation of the PI 3-K/MKP-1 signaling pathway induced by insulin results in long term inhibition of JNKs, which contributes to insulin-mediated proliferation and survival.
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Acknowledgments |
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Footnotes |
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Received September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
in the antiapoptotic function of insulin. J Biol Chem 273:2931–2938B in
mammalian cells through a Raf-1-mediated pathway. J Biol Chem 270:24435–24441 radiation. J Biol Chem 271:31929–31936 in rat
mesangial cells. J Biol Chem 273:4027–4034 in rat mesangial cells.
J Biol Chem 273:10362–10366
do not
prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene
transcription. J Biol Chem 274:21305–21312This article has been cited by other articles:
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A. Imasato, C. Desbois-Mouthon, J. Han, H. Kai, A. C. B. Cato, S. Akira, and J.-D. Li Inhibition of p38 MAPK by Glucocorticoids via Induction of MAPK Phosphatase-1 Enhances Nontypeable Haemophilus influenzae-induced Expression of Toll-like Receptor 2 J. Biol. Chem., November 27, 2002; 277(49): 47444 - 47450. [Abstract] [Full Text] [PDF]
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Q. Xu, T. Konta, A. Furusu, K. Nakayama, J. Lucio-Cazana, L. G. Fine, and M. Kitamura Transcriptional Induction of Mitogen-activated Protein Kinase Phosphatase 1 by Retinoids. SELECTIVE ROLES OF NUCLEAR RECEPTORS AND CONTRIBUTION TO THE ANTIAPOPTOTIC EFFECT J. Biol. Chem., October 25, 2002; 277(44): 41693 - 41700. [Abstract] [Full Text] [PDF]
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J.-M. Ricort and M. Binoux Insulin-like Growth Factor-binding Protein-3 Activates a Phosphotyrosine Phosphatase. EFFECTS ON THE INSULIN-LIKE GROWTH FACTOR SIGNALING PATHWAY J. Biol. Chem., May 24, 2002; 277(22): 19448 - 19454. [Abstract] [Full Text] [PDF]
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M. Potente, U. R. Michaelis, B. Fisslthaler, R. Busse, and I. Fleming Cytochrome P450 2C9-induced Endothelial Cell Proliferation Involves Induction of Mitogen-activated Protein (MAP) Kinase Phosphatase-1, Inhibition of the c-Jun N-terminal Kinase, and Up-regulation of Cyclin D1 J. Biol. Chem., May 3, 2002; 277(18): 15671 - 15676. [Abstract] [Full Text] [PDF]
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 J. C. Wolfman, T. Palmby, C. J. Der, and A. Wolfman Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38 Mol. Cell. Biol., March 1, 2002; 22(5): 1589 - 1606. [Abstract] [Full Text] [PDF]
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 R. Paumelle, D. Tulasne, C. Leroy, J. Coll, B. Vandenbunder, and V. Fafeur Sequential Activation of ERK and Repression of JNK by Scatter Factor/Hepatocyte Growth Factor in Madin-Darby Canine Kidney Epithelial Cells Mol. Biol. Cell, November 1, 2000; 11(11): 3751 - 3763. [Abstract] [Full Text]
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, P < 0.01 (compared with control cells without
or with insulin, respectively).
