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Endocrinology Vol. 142, No. 6 2669-2676
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulin-Induced c-Jun N-Terminal Kinase Activation Is Negatively Regulated by Protein Kinase C {delta}1

Katsutaro Morino, Hiroshi Maegawa, Toshiki Fujita, Noriko Takahara, Katsuya Egawa, Atsunori Kashiwagi and Ryuichi Kikkawa

Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan

Address all correspondence and requests for reprints to: Hiroshi Maegawa, Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga, 520-2192, Japan. E-mail: maegawa{at}belle.shiga-med.ac jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of protein kinase C (PKC) in insulin-induced c-Jun N-terminal kinase (JNK) activation in rat 1 fibroblasts expressing human insulin receptors. Insulin treatment led to increased SAPK/ERK kinase 1 (SEK1) phosphorylation, and then stimulated JNK activity in a dose- and time-dependent manner, as measured either by a solid-phase kinase assay using glutathione S-transferase (GST)-c-Jun fusion protein as a substrate, or by quantitation of the levels of phosphorylated JNK by Western blotting using anti-phospho-JNK antibody. Insulin-induced JNK activation was potentiated by either preincubating cells with 2 nM GF109203X (PKC inhibitor) or down-regulation of PKC by overnight treatment with 100 nM tetradecanoyl phorbol acetate. In contrast, brief preincubation with 100 nM tetradecanoyl phorbol acetate inhibited the insulin- induced JNK activation. Furthermore, we found that 5 µM rottlerin, a PKC{delta} inhibitor, enhanced insulin-induced JNK activation, but a PKCß inhibitor, LY333531, had no effect. Consistent with these findings, overexpression of PKC{delta} led to decreased insulin-induced JNK activation, whereas overexpression of PKCß had no effect. Although overexpression of wild-type PKC{delta} attenuated insulin-induced JNK activation, a kinase-dead PKC{delta} mutant did not cause such attenuation. Finally, we found that the magnitude of insulin-induced JNK activation was inversely correlated with the expression level of PKC{delta} among different cell lines. In conclusion, the expression of PKC{delta} may negatively regulate insulin-induced JNK activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN BINDING STIMULATES tyrosine autophosphorylation of the insulin receptor and activates the intrinsic tyrosine kinase activity of the receptor, leading to the phosphorylation of insulin receptor substrates (IRSs) and Shc. These phosphorylated proteins then bind various Src homology 2 domain-containing signaling molecules, which in turn propagate the signals that underlie the many biological effects of insulin (1, 2). The two best-characterized insulin signaling pathways are the linkage of the insulin receptor to the activities of extracellular signal-regulated kinases (ERK), which constitutes one class of the mitogen-activated protein kinases (MAPKs), and the phosphatidylinositol (PI)3-kinase pathway.

c-Jun N-terminal kinase (JNK), also known as a stress-activated protein kinase, is a serine/threonine-specific protein kinase that is shown to be stimulated in cells in response to various environmental stresses or proinflammatory cytokines such as tumor necrosis factor-{alpha} (3, 4). Subsequent studies have revealed that several growth factors, such as epidermal growth factor (EGF), nerve growth factor, and platelet-derived growth factor (PDGF), also activate JNK (5, 6, 7).

Insulin is shown to stimulate JNK activation, resulting in the activation of skeletal muscle glycogen synthase in vivo (8). Thus, insulin-induced JNK activation is thought to be important in the biological actions of insulin. However, other studies indicate that insulin fails to activate JNK in skeletal muscle and other cell lines (9, 10). Therefore, it still remains controversial whether insulin can stimulate JNK activity. To understand this discrepancy, it is speculated that JNK activation by insulin is inhibited by another signaling molecule, which is also activated by insulin. Because insulin activates protein kinase C (PKC) (11, 12) and that PKC-dependent pathway modulates JNK activation in some cell types (13, 14, 15), it is thus important to assess whether insulin can stimulate JNK activity and how PKC pathway can regulate JNK activation in many cell types. In the present study, we found that insulin was able to stimulate JNK activity and the PKC{delta} negatively regulated the insulin-induced JNK activation. Furthermore, the magnitude of JNK activation by insulin was inversely correlated with cellular expression level of PKC{delta} among many cell types. Thus, the content of PKC{delta} might be one of key determinants to regulate cellular JNK activation by insulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Purified human insulin and PKCß-specific inhibitor LY333531 were gifts from Eli Lilly & Co. (Indianapolis, IL). Selective PKC inhibitor GF109203X (bisindolyl maleimide) and PKC{delta}-specific inhibitor rottlerin were purchased from Calbiochem (San Diego, CA). Lipofectamine was obtained from Life Technologies (Grand Island, NY). [{gamma}-32P]ATP was obtained from NEN Life Science Products (Boston, MA). Anti-JNK1, anti-PKC ({alpha}, ß1, ß2, {delta}, {epsilon}, {gamma}, and µ), and anti-MAPK phosphatase (MKP)-1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antiphosphotyrosine antibody (PY69) was also from Santa Cruz Biotechnology, Inc. Anti-phospho-specific antibodies against ERK, Akt, SEK1, c-Jun, pan-PKC, and PKC{delta} were obtained from New England Biolabs, Inc. (Beverly, MA). Antiactivated JNK antibody was obtained from Promega Corp. (Madison, WI). Protein G Sepharose and glutathione-Sepharose were purchased from Pharmacia PL Biochemical (Uppsala, Sweden). Aprotinin, phenylmethylsulfonyl fluoride (PMSF) and tetradecanoyl phorbol acetate (TPA) were purchased from Sigma (St. Louis, MO). All other reagents were of analytical grade from Nakarai Chemicals (Kyoto, Japan).

Cell culture
Rat 1 fibroblasts that overexpress human insulin receptor (HIRc cells) were provided by Dr. J. M. Olefsky (University of California, San Diego, CA) (16) and maintained in DMEM supplemented with 10% FCS. L6 myoblasts and 3T3L1 preadipoctyes were purchased from American Type Culture Collection (Rockville, MA), and allowed to differentiate by the standard procedures. HepG2 and Fao hepatoma cells were grown and maintained in DMEM with 10% FCS.

Measurement of the activities of JNK
JNK activity was measured by a solid-phase kinase assay as described (17). GST fusion protein expression vector pGEX2T-c-Jun (1–79) was provided by Dr. M. Kalin (University of California, San Diego, CA), and GST fusion protein was purified from Escherichia coli. To prepare the sample for the assay, cells were lysed in a buffer containing 25 mM HEPES (pH 7.5), 0.3 M NaCl, 1.5 nM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 20 mM ß glycerophosphate, 1 mM vanadate, 0.1% Triton X-100, 1 mM PMSF, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. The cell lysates were centrifuged at 12,000 x g for 30 min, and protein concentration was determined by using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). For the solid-phase kinase assay, cell lysates were incubated with GST-c-Jun fusion protein bound to glutathione-Sepharose beads at 4 C for 3 h. The beads were recovered by centrifugation at 10,000 x g for 10 sec and then washed three times with a buffer containing 20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100, and once with kinase buffer [20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM ß-glycerophosphate, 10 mM p-nitrophenyl phosphate, 0.1 mM vanadate, 2 mM dithiothreitol]. The beads were then incubated with 30 µl of kinase buffer containing 20 mM unlabeled ATP and 5 mCi [{gamma}-32P]ATP at 30 C for 20 min. The reaction was terminated by the addition of 30 µl 3x Laemmli sample buffer and boiling at 100 C for 5 min. Phosphorylated proteins were resolved by 12% SDS-PAGE, followed by autoradiography. The relative kinase activities were quantified by Instant Imager Electronic Autoradiography (Packard, Meriden, CT).

Western blotting
Western blot analysis was performed as described (18). Cells were harvested and lysed in ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM PMSF, 50 µM aprotinin, 5 µg/ml leupeptin, and 2 mM benzamidin. After centrifugation at 15,000 rpm at 4 C for 20 min, the supernatant (30 µg protein) was resolved by 10% SDS-PAGE, electrotransferred to Immobilon P (Millipore Corp., Bedford, MA), and blotted with the indicated antibodies. Bound antibodies were detected with horseradish peroxidase-conjugated anti-IgG and visualized with an Enhanced Chemi-Luminescence detection system (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK).

Effects of PKC inhibitors and an activator on insulin-induced JNK activation
Cells were preincubated with either 1–10 nM GF109203X, 2.5–10 µM rottlerin, or 10–100 nM TPA for 10 min, and stimulated with 100 nM insulin, and then the insulin-induced JNK activation was assessed at the indicated times by either the solid-phase kinase assay or quantitation of phosphorylated JNK by Western blotting using antiactivated-JNK antibody.

Expression of PKC isoform in HIRc cells
Expression plasmids encoding human PKC{alpha}, ß1, ß2, and {delta} were provided by Dr. A. Reifel Miller (Lilly Research Laboratories, Indianapolis, IN) (19). The expression plasmid encoding PKCµ was provided by Dr. F.-J. Johannes (University of Stuttgart, Stuttgart, Germany). Cells were transfected with 1 µg of each expression plasmid by the lipofectamine method. After cells were cultured for 72 h, they were used in the experiments to measure JNK activity.

Generation of adenovirus encoding wild-type PKC{delta} and PKC{delta} with a mutant ATP binding site
A QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used for the site-directed mutagenesis. An oligonucleotide in which the lysine residue at amino acid 376 of human PKC{delta} was changed to arginine (20) was used as the primer in the in vitro mutagenesis reaction. The mutant complementary DNA, encoding a kinase-defective PKC{delta} and a complementary DNA encoding a wild-type human PKC{delta} were cloned into pAdTrack-CMV (21). Plasmids for the AdEasy system were provided by Dr. T.-C. He. (The Johns Hopkins Oncology Center, Baltimore, MD). After plasmids were subjected to the AdEasy system, the resulting plasmids were recombined with pAdEasy-1 in BJ5183 cells and recombinant plasmids were selected on kanamycin. Recombinant adenovirus was produced and amplified in HEK-293 cells. We assessed the effects of wild-type or mutated PKC{delta} on insulin-induced JNK activation by the adenovirus gene transfer technique (22).

Statistics
The data are expressed as mean ± SE, unless otherwise stated. Scheffé’s multiple comparison test was used to determine the significance of any differences among more than three groups and unpaired Student’s t test was used to determine the significance of differences between two groups. P less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin-induced JNK activation in rat 1 fibroblasts expressing HIRc
We found that insulin stimulated JNK activity in a dose- and time-dependent manner, as measured by a solid-phase kinase assay using GST-c-Jun (1–79) fusion protein as a substrate in HIRc cells, as shown in Fig. 1AGo. We also found that insulin stimulated phosphorylation of endogenous c-Jun in whole cells detected by Western blotting using anti-phospho-c-Jun antibody. Furthermore, we found that insulin simultaneously increased the expression of c-Jun protein (Fig. 1BGo). Consistently, we observed the insulin-induced phosphorylation of JNK detected by Western blotting using antiactivated JNK antibody. Moreover, we found that insulin induced phosphorylation of SEK1, one of the JNK kinases, detected by Western blotting using an anti-phospho-SEK1 antibody (Fig. 1BGo). Thus, insulin activated JNK activity at least through activation of SEK1. As shown in Fig. 1BGo, the phosphorylation of SEK1 continued to be high up to 60 min after insulin stimulation. However, the increased JNK activity and the magnitude of phosphorylation of JNK returned to the basal levels within 60 min after insulin stimulation, suggesting that the deactivation processes of the JNK and SEK1 kinases might be different.



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  		Figure 1. Insulin induced JNK activation in HIRc cells. Insulin-stimulated JNK activation was measured by the methods described in Materials and Methods. A, The dose-response (0–100 nM) and time course (0–60 min) of insulin-induced JNK activation measured by a solid-phase kinase assay using GST-c-Jun (1–79) as substrate. B, Time course of the phosphorylation levels of SEK1, JNK, and c-Jun as determined by Western blotting using specific antibodies against corresponding phosphorylated forms are shown. Cells were incubated with 100 nM insulin for the indicated times (0–60 min) and cell lysates were prepared. The protein content of each lane was assessed by reblotting with specific antibody against corresponding protein. Each Western blot is representative of four independent experiments.

 
Selective PKC inhibitor enhanced insulin-induced JNK activation
In the present study, we found that the GF109203X, a selective PKC inhibitor, enhanced insulin-induced JNK activation in HIRc cells. As shown in Fig. 2AGo, GF109203X (1–10 nM) enhanced insulin-induced JNK activation in a dose-dependent manner. This activation reached a level 2-fold greater than that of insulin alone at 10 min, and the maximal effect was observed at 30 min (Fig. 2BGo). We next assessed the enhancement of insulin-induced JNK phosphorylation by GF109203X using antiactivated JNK antibody and found that insulin-induced JNK phosphorylation was also increased by the pretreatment with 1–2 nM GF109203X (Fig. 2CGo), suggesting that PKCs might have a negative role in insulin-induced JNK activation.



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  		Figure 2. Effect of GF109203X on insulin-induced JNK activation. A, Dose-response of GF109203X on insulin-induced JNK activation. Cells were pretreated with GF109203X (1–10 nM). Ten minutes later, HIRc cells were stimulated with 100 nM insulin for 10 min and JNK activities were measured by a solid-phase kinase assay. Each column represents the mean + SEM of four separate experiments. *, P < 0.05; **, P < 0.01 vs. insulin-treated alone. B, Time course of GF109203X effect on insulin-induced JNK activation. Insulin alone ({circ}) and insulin + GF109203X (•). Each plot shows the mean of two independent experiments. C, GF109203X enhanced insulin-induced JNK phosphorylation detected by Western blotting using anti-phospho-JNK antibody. Protein content of each lane was assessed by reblotting with specific antibodies against corresponding proteins. Each Western blot is representative of four independent experiments.

 
PKC activator-inhibited JNK activation by insulin
We next tested the effect of TPA, a PKC activator, on insulin-induced JNK activation. Preincubating cells with 1–100 nM TPA inhibited insulin-induced JNK activation in a dose-dependent manner when measured by the solid-kinase assay, as shown in Fig. 3AGo. Furthermore, insulin-induced phosphorylation of JNK was also attenuated by pretreating cells with TPA in a dose-dependent manner (Fig. 3BGo), whereas TPA slightly increased JNK phosphorylation in the absence of insulin as reported (4). In contrast, overnight treatment with 100 nM TPA enhanced insulin-induced JNK activation (Fig. 3AGo). In contrast, insulin increased phosphorylation of PKCs as assessed by Western blotting using either antiphosphorylated pan-PKC or anti-phospho-PKC{delta} antibody (Fig. 3CGo).



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  		Figure 3. Effects of TPA on insulin-induced JNK activation. A, After 10 min of pretreating cells with 1–100 nM TPA, HIRc cells were stimulated with 100 nM insulin for 10 min and JNK activity was measured by solid-phase kinase assay. After incubating cells overnight with 100 nM TPA, cells were stimulated with 100 nM insulin for 10 min and JNK activity was measured (right lane). Each column represents the mean + SE of four independent experiments. *, P < 0.05 vs. insulin treated alone. B, TPA (0–100 nM) inhibited insulin-induced JNK phosphorylation detected by Western blotting with anti-phospho-JNK antibody. C, Insulin-induced phosphorylation of pan-PKC and PKC{delta} assessed by Western blotting using either antiphosphorylated pan-PKC or anti-PKC{delta} antibody, respectively. Cells were incubated with insulin (0–10 nM) for 10 min, and cell lysates were prepared. Each Western blot is representative of three independent experiments.

 
We next quantitated the content of PKC isoforms by Western blotting after overnight TPA treatment, and found that PKCß and {delta} were significantly down-regulated, whereas PKC{alpha}, {gamma}, and µ were almost unchanged in HIRc cells (data not shown). Therefore, PKCß and {delta} might be candidate molecules for regulators of insulin-induced JNK activity in HIRc cells.

PKC{delta} is a candidate molecule to inhibit JNK activation by insulin
As shown in Fig. 4AGo, preincubating cells with a PKC{delta}-specific inhibitor, rottlerin (2.5–10 µM), enhanced JNK activation by insulin, whereas LY333531, a PKCß-specific inhibitor, had no effect (data not shown).



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  		Figure 4.
 
We next assessed the effects of a transient overexpression of PKC{alpha}, ß1, ß2, and {delta} in HIRc cells on insulin-induced JNK activation using lipofectamine method. Although we failed to detect overexpression of PKC{alpha} because of the high amount of endogenous PKC{alpha}, we successfully overexpressed significant amounts of PKCß1 and {delta} as shown in Fig. 4BGo by lipofectamine method. Overexpression of the PKC{delta} isoform alone led to the attenuation of insulin-induced JNK activation by 42.0% (P < 0.01), whereas PKCß1 did not. Overexpression of PKCß2 had no effect on insulin-induced JNK activation (data not shown). We observed that overexpression of PKC{delta} also decreased the insulin-induced phosphorylation of JNK by 41.9 ± 7.9% (P < 0.01) as shown in Fig. 4CGo.

Using adenovirus-mediated gene transfer, overexpression of wild-type PKC{delta} in HIRc cells had no effects on insulin-induced phosphorylation states of insulin receptor, IRS-1, ERK, and Akt as shown in Fig. 4DGo. However, overexpression of wild-type PKC{delta} led to the attenuation of the insulin-induced JNK activation. In contrast, overexpression of a kinase-defective PKC{delta} failed to suppress insulin-induced JNK activation. These results strongly indicate that the inhibitory effect on insulin-induced JNK activation is specific for the PKC{delta} in HIRc cells.

To investigate whether PKC{delta} inhibited JNK kinase directly, we measured the direct physical interaction between JNK and PKC{delta} protein by the immunoprecipitation method, but we were not able to observe any direct association (data not shown).

Insulin increased the expression of MKP-1
To clarify a mechanism how PKC{delta} inhibited insulin-induced JNK activation, we next assessed the content of MKP-1 protein by Western blotting, because MKP-1 is a phosphatase that specifically dephosphorylates MAP kinase (23). As shown in Fig. 1AGo, we observed significant insulin-induced JNK activation within 10 min after insulin stimulation, but MKP-1 protein levels were the same as the basal level at that time (Fig. 5Go), although the MKP-1 protein content was increased after 30 min of insulin stimulation as shown in Fig. 5Go. In contrast, MKP-2 content was unchanged by insulin stimulation. Furthermore, pretreating cells with GF109203X had no effect on the MKP-1 content and overexpression of PKC{delta} decreased the MKP-1 content (data not shown). These findings suggest that the inhibition of the PKC{delta} on insulin-induced JNK activation may not be through the increment of MKP-1.



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  		Figure 5. Insulin increased expression of MKP-1 protein. Cells were incubated with 100 nM insulin for the indicated times (0–60 min) and cell lysates were prepared. MKP-1 expression levels were determined by Western blotting using anti-MKP-1 antibody. Each bar represents the mean + SE of four experiments. *, P < 0.05 vs. the basal state. MKP-2 levels were unchanged by insulin stimulation.

 
Effect of PKC{delta} on insulin-induced SEK1 phosphorylation
To clarify whether the PKC{delta} isoform acted upstream of JNK, we tested SEK1 phosphorylation by Western blotting with anti-phospho-SEK1 antibody. Pretreating cells with 10 nM GF109203X enhanced insulin-induced SEK1 phosphorylation as shown in Fig. 6AGo. In contrast, pretreating cells with 100 nM TPA decreased the phosphorylation level of SEK1 by insulin as well as JNK phosphorylation (Fig. 6BGo). Furthermore, overexpression of the PKC{delta} also attenuated insulin-induced SEK1 phosphorylation (Fig. 6CGo).



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  		Figure 6. Effect of PKC on insulin-induced SEK1 phosphorylation. A, Effect of GF109203X on insulin-induced SEK1 phosphorylation. Pretreating cells with GF109203X (1–10 nM) for 10 min, HIRc cells were incubated with 100 nM insulin for 10 min, and then, cell lysate was prepared. B, Effect of TPA on insulin-induced SEK1 phosphorylation. Pretreating cells with 100 nM TPA for 10 min attenuated insulin-induced phosphorylation of both SEK1 and JNK. C, Effect of the PKC{delta} overexpression on insulin-induced SEK1 phosphorylation. Overexpression of PKC{delta} isoform by lipofectamine method also inhibited insulin-induced SEK1 phosphorylation. Each column represents the mean + SEM of four independent experiments. *, P < 0.05 vs. insulin treated. Each Western blot is representative of three independent experiments.

 
Modification of insulin-induced JNK activation by expressed levels of PKC{delta}
To assess whether insulin could stimulate JNK activity in various other cell types, we measured the effect of insulin on JNK activation in HepG2, Fao hepatoma cells, L6 myocytes, and 3T3L1 adipocytes. As shown in Fig. 7AGo, the magnitude of JNK activation by insulin was varied among these cell lines, and the activation of JNK by insulin was inversely correlated with the expression levels of PKC{delta} isoform in those cells. In contrast, there was no relationship between JNK activation and its content among these cells. Furthermore, we observed that a PKC inhibitor potentiated insulin-induced JNK activation in HepG2 cells, which highly expressed PKC{delta}, but not in L6 myocytes with low expression of PKC{delta} (Fig. 7BGo).



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  		Figure 7. Insulin-induced JNK activation and expression levels of PKC{delta}. A, Insulin-induced JNK activation in HepG2, Fao, L6 Myocytes, and 3T3L1 adipocytes. Cells were incubated with 100 nM insulin for 10 min and cell lysates were prepared. Insulin-induced JNK phosphorylation were detected by Western blotting with anti-phospho-JNK antibody. The expression levels of PKC{delta} and JNK1 were also assessed by Western blotting. Same amount of cell lysate (40 µg) was applied to each lane. B, Effect of 10 nM GF109203X on insulin-induced JNK activation. HepG2 hepatoma cells and L6 myocytes were pretreated with 10 nM GF109203X. Ten minutes later, cells were stimulated with 100 nM insulin for 10 min and JNK activities were measured by Western blotting using anti-phospho-JNK antibody. Each Western blot is representative of four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin-induced JNK activation
JNK, known as stress-activated protein kinase, has been reported to be activated by insulin in skeletal muscle and some cell lines (8, 24). However, other studies show that insulin fails to activate JNK in skeletal muscle and other cell lines (9, 10). Thus, whether insulin can stimulate JNK activity is still controversial. In the present study, we found that insulin stimulated JNK activity using several independent methods in HIRc cells as well as various other cell lines such as HepG2 and Fao hepatoma cells, L6 myocytes, and 3T3L1 adipocytes. Moreover, we found that insulin-induced JNK activation was associated with SEK1 phosphorylation. Thus, these findings indicate that insulin is able to stimulate JNK activity in these cells.

Regarding the molecular mechanisms of insulin-induced JNK activation, PI3-kinase participates in the process of activation of the JNK initiated by receptor-like tyrosine kinases, including the EGF and PDGF receptors (3, 4). Furthermore, in Chinese hamster ovary cells overexpressing HIRc, wortmannin completely inhibited JNK activation by insulin (24). In the present study, PI3-kinase inhibitors (wortmannin and Ly294002) only partially inhibited insulin-induced JNK activation in HIRc cells (data not shown). In contrast, it has also been reported that insulin activates JNK through a p21ras-dependent pathway in rat 1 fibroblasts (25). Moreover, the requirement of SHP-2 for JNK activation is also reported (26). Thus, it is likely that there are redundant pathways in insulin-induced JNK activation among the different cell types and tissues.

PKC inhibited insulin-induced JNK activation
In the current study, we found that a PKC inhibitor potentiated insulin-induced JNK activation in HIRc cells. In contrast, TPA, a PKC activator attenuated the insulin-induced JNK activation. Furthermore, down-regulation of PKCs by overnight treatment with TPA enhanced JNK activation by insulin. These findings strongly suggest that PKCs inhibit insulin-induced JNK activation in HIRc cells. Consistently, a recent study has demonstrated that PKC inhibits the calcium-dependent JNK activation in rat liver epithelial cells (15). However, JNK is activated by a PKC-dependent pathway in some cell types (13, 14). Therefore, further investigation is needed to clarify a direct effect of PKC on JNK activation in many cell types.

Concerning the expression of PKC isoforms in HIRc cells, all isoforms except PKC{gamma} were detected and that PKCß1, ß2, and {delta} were significantly down-regulated, whereas PKC{alpha}, {gamma}, and µ were almost unchanged following overnight TPA treatment in HIRc cells (data not shown). However, GF109203X, which inhibits both conventional and novel PKCs, accelerated insulin-induced JNK activation; therefore, PKCß1, ß2, and {delta} were thought to be candidate PKCs. Furthermore, we found that phosphorylation of PKCs by insulin as assessed by Western-blotting using either anti-phospho pan-PKC or anti-phospho-PKC{delta} antibody (Fig. 3CGo).

To further clarify the involvement of specific PKC isoforms in the inhibition of JNK, we next examined the effects of isoform-specific PKC inhibitors on insulin-induced JNK activation. We found that preincubating cells with rottlerin, a PKC{delta}-specific inhibitor, but not a PKCß-specific inhibitor, LY333531, enhanced JNK activation by insulin. Furthermore, overexpression of PKC{delta} in HIRc cells using lipofectamine method, led to the attenuation of insulin-induced JNK activation by 58% (P < 0.01), whereas overexpression of PKCß did not affect JNK activity (Fig. 4BGo). Moreover, overexpression of wild-type PKC{delta} by adenovirus gene transfer inhibited insulin-induced JNK activation, but a kinase-defective mutant PKC{delta} did not, as shown in Fig. 4DGo. Because it has been reported that PDGF inhibited EGF-mediated JNK activation, apparently through PKCµ activation (27), we tested whether overexpression of PKCµ could affect JNK activation, but we did not find that PKCµ suppressed JNK activation by insulin in HIRc cells (data not shown). These results strongly indicate that the PKC{delta} isoform alone inhibits insulin-induced JNK activation in HIRc cells.

Regarding physiological roles of this inhibitory effect of PKC{delta} on insulin induced JNK activation, we speculate that insulin may stimulate both JNK and PKC simultaneously, and that activated PKC{delta} may inhibit insulin-induced JNK activation by a negative feedback mechanism, and cellular content of PKC{delta} may be one of key the determinants of how cellular JNK responses to insulin.

Insulin-induced MKP-1 expression
Because MKP-1 is an early immediate gene and believed to be one of the major phosphatases of the JNK family, we assessed whether insulin could reduce the content of MKP-1 protein. Within 10 min after insulin stimulation, we observed insulin-induced JNK activation, but did not in MKP-1 content in the current study. Furthermore, we found that the content of MKP-1 protein was significantly increased after 30 min of insulin stimulation in HIRc cells. Thus, we speculate that the activation of JNK by insulin is not mediated by MKP-1 inhibition.

Regarding the mechanism of insulin-induced MKP-1 expression, it has been reported that the ligand-induced phosphorylation of MKP-1 protein prevented its degradation (28). Thus, phosphorylation of MKP-1 as well as increased MKP-1 gene expression is induced by insulin, which might be responsible for insulin-induced MKP-1 expression. Concerning the possible association of MKP-1 with the inhibitory effect of PKC{delta}, Cook and colleagues (29) have reported that the selective PKC inhibitor Ro-31-8220 activates JNK by inhibiting MKP-1 expression. However, in their study, Ro-31-8220 inhibits MKP-1 expression in a PKC-independent pathway. Furthermore, we did not find any change in MKP-1 content in response to PKC inhibitors (GF109203X and rottlerin). We also found that TPA treatment for 30 min increased the MKP-1 content. Moreover, overexpression of PKC{delta} decreased MKP-1 content in the present study (data not shown). Thus, these findings suggests that MKP-1 is not involved in the inhibitory effect of PKC{delta}.

Effect of PKC on insulin-induced phosphorylation of SEK1
We further clarified whether PKC{delta} inhibited upstream of JNK activation and found that pretreating cells with GF109203X enhanced SEK1 phosphorylation and TPA decreased it (Fig. 6Go, A and B). Furthermore, overexpression of PKC{delta} led to attenuation of SEK1 phosphorylation as well as JNK activity. Thus, we speculate that PKC{delta} inhibits either JNK directly or upstream of JNK, such as SEK1. Nevertheless, we did not observe any physical association of PKC{delta} with JNK. Thus, it seems that PKC{delta} inhibits upstream of JNK. Further experiments are needed to document that insulin-induced JNK activation occurred via SEK1.

Modification of insulin-induced JNK activation by expressed levels of PKC{delta}
Finally, we found that insulin did stimulate JNK activation in various cell lines such as HepG2 and Fao hepatoma cells, L6 myocytes, and 3T3L1 adipocytes, even though the magnitude was varied among these cells. We also found that the responsiveness of JNK activity by insulin was inversely correlated with the expression levels of PKC{delta} isoform in each cell. Furthermore, we found that a PKC inhibitor also potentiated insulin-induced JNK activation in HepG2 cells, which highly expressed PKC{delta} isoform. However, PKC inhibitor failed to potentiate it in L6 myocytes, which expressed PKC{delta} protein at lesser extent. These findings suggest that cellular content of PKC{delta} may be one of key determinants how cellular JNK responses to insulin. In the present study, we identified a novel cross-talk between JNK and PKC{delta} in insulin signaling. Further investigation is needed to clarify its biological meanings of insulin-induced JNK activation and its cross-talk with PKC{delta}.


   Acknowledgments
 
We are grateful to Drs. D. Koya, T. Sugimoto, and K. Shi for their technical assistance and useful discussions. We are also grateful to Drs. J. M. Olefsky (University of California, San Diego, CA), A. Reifel Miller (Lilly Research Laboratories, Indianapolis, IL), F.-J. Johannes (University of Stuttgart, Stuttgart, Germany), and T.-C. He. (Johns Hopkins Oncology Center, Baltimore, MD) for the gift of specific cells and plasmids.


   Footnotes
 
1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan. Back

Received November 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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