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Endocrinology Vol. 141, No. 3 922-931
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin-Mediated Cell Proliferation and Survival Involve Inhibition of c-Jun N-terminal Kinases through a Phosphatidylinositol 3-Kinase- and Mitogen-Activated Protein Kinase Phosphatase-1-Dependent Pathway1

Christele Desbois-Mouthon, Axelle Cadoret, Marie-Jose Blivet-Van Eggelpoël, France Bertrand, Martine Caron, Azeddine Atfi, Gisele Cherqui and Jacqueline Capeau

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN EXERTS, via a specific transmembrane receptor, a wide range of biological responses affecting glucose, lipid, and protein metabolism as well as cell proliferation and survival (1, 2). Insulin binding to its receptor results in rapid activation of the receptor tyrosine kinase activity that mediates the tyrosine phosphorylation of a variety of endogenous substrates, including IRS-1, IRS-2, and Shc. Substrate phosphorylation generates docking sites for several molecules, such as phosphatidylinositol 3-kinase (PI 3-K) and the adapter Grb2 that links Sos, a guanine nucleotide exchange factor for Ras. These early signaling events result in the activation of the PI 3-K and Ras/extracellular-regulated kinases (ERKs) pathways.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
pcDNA3/Neo-JNK1 (a gift from S. Gutkind) and pcDNA3/Neo-JNK1 mutant (Ala and Phe substituted at Thr183 and Tyr185, respectively; a gift from R. J. Davis) have been described previously (34). Wild-type and mutant JNK1 complementary DNA (cDNA) were subcloned between the HindIII and ApaI sites of pcDNA3/Zeo (Invitrogen, Carlsbad, CA) for generating zeocin-resistant stable cell lines. The expression vectors for constitutively active Akt/PKB (pSG5-gagPKB) and dominant negative myristoylated and kinase-dead (K179A) Akt/PKB (pcDNA3-myrPKB-KD) were gifts from B. M. Burgering. pcDNA1/Neo/MKP-1 plasmid was a gift from G. L’Allemain. The antisense full-length MKP-1 construct was obtained by subcloning the MKP-1 cDNA between the HindIII and XbaI sites of pcDNA3/Hygro(-) (Invitrogen).

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 [{gamma}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Long term insulin inhibition of JNKs in different cell lines
We recently reported that insulin (10 nM) exerted a long term inhibitory effect on JNK activity in CHO-IR cells overexpressing human IRs (33). To strengthen the significance of this observation, we now investigated the effect of insulin in parental CHO cells transfected with an empty vector and human skin fibroblasts. As shown in Fig. 1AGo, 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 1BGo 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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  		Figure 1. Insulin inhibition of JNKs in different cell lines. A, CHO-IR cells and parental CHO cells transfected with an empty vector were treated for 2 h with increasing concentrations of insulin. Three hundred micrograms of total cell lysate were used for immunoprecipitating JNKs (upper panel). Kinase activity in immunoprecipitates was determined by examining the phosphorylation level of recombinant GST-c-Jun protein as described in Materials and Methods. The lower panel shows that JNK1 expression was not modified in these experiments, as assessed by Western blotting with a polyclonal anti-JNK1 antibody. Results are representative of two independent experiments. B, Human skin fibroblasts were incubated in the presence or absence of insulin (100 nM) for 2 h. JNK activity and JNK1 expression were examined as described above. Results are representative of two independent experiments.

 
Role of PI 3-K in insulin inhibition of JNK activity
As a first step to identify the signaling components potentially involved in insulin-mediated JNK inhibition, we evaluated the role of PI 3-K in this process. To this end, we examined the effects of two different chemical inhibitors of PI 3-K, wortmannin and LY294002, on JNK activity in control and insulin-treated CHO-IR cells. In these assays, cells were preincubated for 1 h with or without wortmannin (100 nM) or LY294002 (10 µM) and then treated for an additional 2 h in the absence or presence of insulin (10 nM). The catalytic activity of endogenous JNKs was evaluated in vitro by examining the phosphorylation of recombinant GST-c-Jun in anti-JNK1 immunoprecipitates. In accordance with our previous results (33), Fig. 2Go shows that insulin inhibited JNK activity by about 50% in control cells. In contrast, cell pretreatment with wortmannin (Fig. 2Go, A and C) or LY294002 (Fig. 2Go, 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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  		Figure 2. Effects of the PI 3-K inhibitors, wortmannin and LY294002, on insulin inhibition of JNKs. CHO-IR cells were pretreated with wortmannin (Wort; 100 nM; A), LY294002 (LY; 10 µM; B), or vehicle (Cont) for 1 h and then incubated with or without insulin (10 nM) for an additional 2 h. Three hundred micrograms of cell lysate were used for evaluating JNK activity as described in Materials and Methods. Results are representative of three independent experiments (A and B) or are the mean ± SEM (C). *, P < 0.01; {dagger}, P < 0.01 (compared with control cells without or with insulin, respectively).

 
Insulin inhibition of JNKs involves insulin induction of MKP-1 through a PI 3-K-dependent pathway
JNKs have been demonstrated to be inactivated in vitro and in vivo by a growing family of dual specificity phosphatases (17, 18, 19, 20, 21, 22). To examine whether in our cell system, insulin inhibition of JNKs was phosphatase dependent, cells were pretreated with sodium orthovanadate, an inhibitor of tyrosine and dual specificity phosphatases. Vanadate (500 µM) prevented JNK inhibition by insulin, whereas it had no effect on the basal level of JNK activity (Fig. 3AGo), 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. 3BGo).



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  		Figure 3. Effects of sodium orthovanadate and actinomycin D on insulin inhibition of JNKs. CHO-IR cells were pretreated with or without sodium orthovanadate (Van; 500 µM; A) or actinomycin D (AcD; 5 µg/ml) or vehicle (Cont; B) for 1 h and then incubated in the presence or absence of insulin (10 nM) for an additional 2 h. Three hundred micrograms of cell lysate were used for evaluating JNK activity as described in Materials and Methods. Results are representative of two independent experiments.

 
We focused on MKP-1 because MKP-1 expression has been shown to be stimulated by insulin in CHO cells (37). We studied the effect of insulin on MKP-1 expression and JNK activity in CHO-IR cells for different time periods. We observed that the time course for insulin-induced MKP-1 expression correlated with the time course for insulin-induced JNK inhibition, as the effect of insulin on both MKP-1 expression and JNK activity was detected at 30 min (Fig. 4AGo).



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  		Figure 4. Role of MKP-1 as an effector of PI 3-K to mediate insulin inhibition of JNKs. A, CHO-IR cells were incubated with or without insulin (10 nM) for different time periods. Fifty micrograms of cell lysate were analyzed by Western blot using a polyclonal anti-MKP-1 antibody (upper panel), and 300 µg cell lysate were used for evaluating JNK activity (lower panel) as described in Materials and Methods. B, CHO-IR cells (8.0 x 105 cells/dish) were transiently transfected with 5 µg pcDNA3/Hygro or pcDNA3/Hygro containing the antisense MKP-1 cDNA. Fifteen hours posttransfection, the medium was removed, and cells were allowed to recover for 8 h and serum deprived for 17 h. Cells were then incubated with or without insulin (10 nM) for 2 h and lysed. Fifty micrograms of cell lysate were tested for MKP-1 expression by Western blot analysis (left panel), and 300 µg cell lysate were used for evaluating JNK activity (right panel) as described in Materials and Methods. C, CHO-IR cells were pretreated with wortmannin (Wort; 100 nM), LY294002 (LY; 10 µM), or vehicle (Cont) for 1 h and then incubated with or without insulin (10 nM) for an additional 2 h. Fifty micrograms of cell lysate were analyzed by Western blot using a polyclonal anti-MKP-1 antibody. Results are representative of three independent experiments.

 
To determine whether the stimulation of MKP-1 expression by insulin played a role in insulin inhibition of JNKs, CHO-IR cells were transiently transfected with an antisense full-length MKP-1 construct or with the corresponding empty vector. After a 2-h incubation with or without insulin, transfected cells were examined for MKP-1 expression (Fig. 4BGo, left panel) and JNK activity (Fig. 4BGo, 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. 2Go), 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. 4CGo), 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. 5AGo). 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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  		Figure 5. Effect of the stable overexpression of wild-type and mutant JNK1 on insulin stimulation of cell proliferation. A, CHO-IR cells (5 x 105 cells/dish) were stably transfected with 15 µg pcDNA3/Zeo (Zeo), pcDNA3/Zeo/wild-type JNK1 (WT1, WT2), or pcDNA3/Zeo/mutant JNK1 (Mut1, Mut2). Ten micrograms of cell extract obtained from each cell line were submitted to Western blot analysis with a polyclonal anti-JNK1 antibody. B, CHO-IR cells (3 x 105 cells/well) overexpressing wild-type JNK1 (left panel) or mutant JNK1 (right panel) were incubated in the absence or presence of insulin (10 and 100 nM) for 20 h. [Methyl-3H]thymidine (1 µCi/well) was added for the last 3 h of the incubation. The basal values of [methyl-3H]thymidine incorporated into DNA were 378 ± 21, 342 ± 31, and 333 ± 55 cpm/µg protein for Zeo, WT1, and WT2 cells, respectively (left panel), and 294 ± 44, 300 ± 28, and 276 ±13 cpm/µg protein for Zeo, Mut1, and Mut2 cells, respectively (right panel). Results are the mean ± SEM of three or four independent experiments performed in duplicate. *, P < 0.01; {dagger}, P < 0.01 compared with Zeo cells treated with 10 nM or 100 nM insulin, respectively.

 
We first examined the effect of the overexpression of wild-type or mutant JNK1 on basal and insulin-stimulated DNA synthesis. The basal values of [methyl-3H]thymidine incorporation for the clones overexpressing wild-type or mutant JNK1 were closely similar to those determined in control cells (see Fig. 5BGo). 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. 5BGo, 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. 5BGo, 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. 6Go, 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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  		Figure 6. Effect of the stable overexpression of mutant JNK1 on insulin stimulation of ERK activity. Total cell lysates (15 µg) from control and insulin-stimulated (100 nM, 10 min) Zeo, Mut1, and Mut2 cells were analyzed by Western blot using an anti-active-ERK antibody that recognizes the active phosphorylated form of ERK1 (pp44) and ERK2 (pp42; upper panel). To correct for differences in protein loading, blots were stripped and reprobed with a polyclonal anti-ERK1 (p44) antibody that cross-reacted with ERK2 (p42; lower panel). Results are representative of three independent experiments.

 
Role of JNK inhibition through MKP-1 expression in the antiapoptotic function of insulin
We recently demonstrated that insulin potently inhibited serum withdrawal-induced apoptosis in CHO-IR cells (6). As the activation of JNKs has been implicated in apoptosis signaling initiated by serum withdrawal (27, 40, 41), we wondered whether the inhibition of JNKs induced by insulin served its antiapoptotic function. We performed DNA fragmentation assays in Zeo, Mut1, and Mut2 cells to evaluate the extent of apoptosis induced by 24-h serum deprivation in the absence or presence of insulin. Figure 7AGo 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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  		Figure 7. Effect of the overexpression of wild-type JNK1, mutant JNK1, or MKP-1 on insulin protection against serum withdrawal-induced apoptosis. A, CHO-IR cells overexpressing mutant JNK1 (Mut1, Mut2; 9 x 105 cells/dish) were cultured for 24 h in SFM supplemented, or not, with 100 nM insulin. DNA fragmentation was evaluated as described in Materials and Methods and compared with that obtained in cells transfected with the empty vector (Zeo). The gel is representative of two independent experiments. B, CHO-IR cells (2.5 x 105 cells/well) were transiently transfected with 4 µg of an empty vector (pcDNA3/Neo) or an expression vector containing the wild-type JNK1 cDNA together with 1 µg of the ß-galactosidase vector (pCMV5/lacZ). Fifteen hours posttransfection, the medium was removed, and cells were incubated for an additional 24 h in SFM in the presence or absence of 100 nM insulin. Evaluation of the number of apoptotic cells was performed by ß-galactosidase staining as described in Materials and Methods. Results are the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.001; {dagger}, P < 0.001 (compared with control cells without or with insulin, respectively). C, CHO-IR cells (2.5 x 105 cells/well) were transiently transfected with 4 µg pcDNA3/Hygro or pcDNA3/Hygro containing the antisense MKP-1 cDNA together with 1 µg of the ß-galactosidase vector (pCMV5/lacZ). Fifteen hours posttransfection, the medium was removed, and cells were incubated for an additional 24 h in SFM in the presence or absence of 100 nM insulin. Evaluation of the number of apoptotic cells was determined by ß-galactosidase staining as described in Materials and Methods. Results are the means of two independent experiments performed in duplicate.

 
Because the clones overexpressing wild-type JNK1 could not be maintained in culture, the effect of overexpressed wild-type JNK1 on apoptosis was evaluated in transient transfection experiments. CHO-IR cells were cotransfected with pcDNA3/Neo or the wild-type JNK1 plasmid and a plasmid containing the ß-galactosidase gene (pCMV5/lacZ). Transfected cells were then incubated for 24 h in SFM in the absence or presence of 100 nM insulin. After fixation and staining, the percentage of apoptotic cells was evaluated by scoring blue ß-galactosidase transfectants as healthy or apoptotic, as judged by blebbing of the membranes and shrinkage of the cell bodies. As shown in Fig. 7BGo, 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. 7CGo). 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 1Go). 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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  		Table 1. Effect of the overexpression of mutant JNK1 on insulin stimulation of protein and glycogen syntheses

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We recently demonstrated that in CHO-IR cells, insulin exerted a biphasic effect on JNK activity, with a rapid and transient activation phase followed by a sustained inhibition phase (33). The present study was designed to delineate the mechanisms by which insulin inhibited JNKs and to analyze the role of this inhibition in insulin signaling of cell proliferation and survival.

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 {gamma}-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-{alpha}- 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-{kappa}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.


   Acknowledgments
 
We are greatly indebted to Dr. S. Gutkind for the wild-type JNK1 plasmid, to Dr. R. J. Davis for the mutant JNK1 plasmid, to Dr. B. M. Burgering for the Akt/PKB plasmids, and to Dr. G. L’Allemain for the MKP-1 plasmid. We thank Luis Chan for technical assistance, Martine Auclair for providing us with human skin fibroblasts, and Betty Jacquin for secretarial support.


   Footnotes
 
1 This work was supported by grants from the Ligue Nationale Contre le Cancer (Comité de Paris) and the Assocation pour la Recherche sur le Cancer. Back

Received September 14, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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