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Endocrinology Vol. 139, No. 7 3133-3142
Copyright © 1998 by The Endocrine Society

ARTICLES

Insulin Induction of Protein Kinase C{alpha} Expression Is Independent of Insulin Receptor Tyr1162/1163 Residues and Involves Mitogen-Activated Protein Kinase Kinase 1 and Sustained Activation of Nuclear p44MAPK1

Pierre Jacques Antoine, France Bertrand, Martine Auclair, Jocelyne Magré, Jacqueline Capeau and Gisèle Cherqui

INSERM U-402, Institut Federatif de Recherche 65, Laboratoire de Biologie Cellulaire, Faculté de Médecine Saint-Antoine, 75571 Paris Cedex 12, France

Address all correspondence and requests for reprints to: Dr. Gisele Cherqui, INSERM U-402, IFR 65, Laboratoire de Biologie Cellulaire, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75571 Paris Cedex 12, France. E-mail: cherqui{at}st-antoine.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the effect of insulin on protein kinase C{alpha} (PKC{alpha}) expression and the implication of the mitogen-activated protein kinase kinase 1 mitogen-activated protein kinase (MAPK) pathway in this effect. PKC{alpha} expression was measured by quantitative RT-PCR and Western blotting using Chinese hamster ovary (CHO) cells overexpressing human insulin receptors of the wild type (CHO-R) or insulin receptors mutated at Tyr1162/1163 autophosphorylation sites (CHO-Y2). In CHO-R cells, insulin caused a time- and concentration-dependent increase in PKC{alpha} messenger RNA, with a maximum at 6 h and 10-8 M insulin. This increase involved a transcriptional mechanism, as it was not due to stabilization of PKC{alpha} messenger RNA and was associated with a similar increase in the immunoreactive PKC{alpha} level. Insulin induction of PKC{alpha} expression involved the MEK1-MAPK pathway, as it was 1) almost completely suppressed by the potent MEK1 inhibitor PD98059, 2) mimicked by the dominant-active MEK1 (S218D/S222D) mutant, and 3) associated with sustained MAPK activation. In CHO-Y2 cells in which the early phase of MAPK activation by insulin was lost and only the late and sustained phase of activation was observed, insulin signaling of PKC{alpha} expression was preserved and again involved the MEK1-MAPK pathway. Moreover, we show that in both CHO-R and CHO-Y2 cells, insulin stimulation of PKC{alpha} gene expression was associated with prolonged activation of nuclear p44MAPK. These results indicate that induction of PKC{alpha} gene expression by insulin is independent of Tyr1162/1163 autophosphorylation sites and correlates with sustained activation of p44MAPK at the nuclear level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN is a potent metabolic and growth-promoting hormone that stimulates a spectrum of physiological effects, including the expression of specific genes at the nuclear level. The initial steps in insulin action are the binding of insulin to its receptor and the subsequent activation of the receptor tyrosine kinase. This results in the phosphorylation of several substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which activate different signaling pathways. One of these pathways includes the membrane-associated Ras protein and a cascade of kinases comprised of Raf-1, mitogen-activated protein kinase kinase 1, and mitogen-activated protein kinases (MAPKs). This pathway is thought to convey the insulin signals that regulate cell growth, differentiation, and gene expression (1, 2, 3).

The interrelations between insulin signaling and protein kinase C (PKC) appear to be complex, as PKC was shown to play a dual role, positive and negative, in insulin action. Studies from different laboratories including ours initially reported that the stimulation of glucose transport by insulin involved the rapid activation of PKC by translocation of the enzyme from the cytoplasm to the membrane where it was degraded (4, 5, 6, 7). Conversely, other studies showed that PKC acted as a negative regulator of insulin signaling, as PKC inhibited the insulin receptor (IR) tyrosine kinase activity and antagonized a number of insulin’s actions (8, 9, 10, 11, 12, 13). Of particular interest, evidence was provided that in addition to triggering a rapid stimulation of PKC activity, insulin was able to increase PKC messenger RNA (mRNA) levels in rat adipocytes and rat skeletal muscle (14, 15). However, little is known at present about the signaling pathway involved in the stimulatory effect of insulin on PKC gene expression.

In this study, we investigated the roles of the IR Tyr1162/1163 autophosphorylation sites and the MEK1-MAPK pathway in the induction of PKC{alpha} gene expression by insulin. To this end, we used parental Chinese hamster ovary (CHO) cells, a cell type that mainly expresses the PKC{alpha} isoform, as well as various CHO transfectants. These include CHO cells overexpressing human wild-type IRs (CHO-R) or IRs mutated at Tyr1162/1163 (CHO-Y2), two residues shown to play a critical role in most (4, 16, 17), but not all (18, 19, 20), of the effects of insulin studied. These transfected cells provide an appropriate model for studying the effect of insulin on gene expression, as we previously showed that both CHO-R and CHO-Y2 cells retain a normal number of cell surface receptors after a prolonged treatment with insulin (21). In addition, we developed CHO-R transfectants expressing the dominant-active MEK1 (S218D/S222D) mutant (22) that are valuable in investigating the genes that are specifically targeted by the MEK1-MAPK pathway. Our results indicate that induction of PKC{alpha} gene expression by insulin is independent of IR Tyr1162/1163 autophosphorylation sites and correlates with a sustained activation of p44MAPK at the nuclear level. This is the first evidence that the gene coding for PKC{alpha} is a target for a specific MAPK isoform in mammalian cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
[{gamma}-32P]ATP (10 Ci/mmol), [{alpha}-32P]deoxy (d)-CTP (3000 Ci/mmol), the enhanced chemiluminescence (ECL) detection kit, Hybond N+ membranes, and Hyperfilms-MP were obtained from Amersham Corp. (Arlington Heights, IL). [20-3H]Phorbol 12,13-dibutyrate ([3H]PDBu; 18 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). Insulin was purchased from Novo Laboratories (Copenhagen, Denmark), and insulin-like growth factor I (IGF-I) was obtained from Calbiochem (La Jolla, CA). Rabbit polyclonal antibody against PKC{alpha} was purchased from Life Technologies (Grand Island, NY). The MAPK R2 antibody was purchased from Upstate Biotechnology (Lake Placid, NY); the immunoprecipitating ERK1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and the antiactive MAPK polyclonal antibody was purchased from Promega (Madison, WI). The 12CA5 monoclonal antibody that recognizes the HA epitope was purchased from Boehringer Mannheim (Indianapolis, IN). 5,6-Dichlorobenzimidazole riboside (DRB), 4ß-phorbol 12ß-myristate 13{alpha}-acetate (PMA), leupeptin, benzamidine, and phenylmethylsulfonylfluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO). The MEK1 inhibitor PD098059 was a generous gift from Parke-Davis (Detroit, MI).

Cell lines
The three different CHO cell lines (gifts from Prof. E. Clauser, INSERM U-36, Paris, France) used in this study have been previously described (4, 19, 21). These include the parental cell line (CHO) and the CHO cell lines overexpressing either wild-type human IRs (CHO-R) or Tyr1162/1163-mutated IRs (CHO-Y2). Cells were grown in Ham’s F-12 medium (Life Technologies) supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 mg/ml). Before experiments with effectors, cells were growth-arrested at confluence by incubation with serum-free Ham’s F-12 medium for 36 h.

Quantification of the PKC{alpha} mRNA
The amount of PKC{alpha} mRNA was determined by competitive PCR (23) using an internal control that was coamplified with target complementary DNA (cDNA) of PKC{alpha} using the same set of sense (5'-ATG GAT CAC ACT GAG AAG AGG-3') and antisense (5'-AAG GTT GTT GGA AGG TTG TTT-3') primers. These primers were chosen to avoid amplification from contaminating DNA template (and in a region presenting little homology with the genes coding for other PKC isoforms). The internal control used as a competitor was constructed from the 537-bp amplicons (from nucleotides 484-1000, access no. HSPKCA1/GenBank) for the natural PKC{alpha} produced with the primers described above. Thus, the 537-bp amplicon’s target cDNA was deleted of 200 bp using NcoI digestion and ligated to produce a 337-bp internal control that was subcloned into the PCR-TA cloning vector (Invitrogen, San Diego, CA). The PCR-TA vector containing the subcloned 337 bp was further used to produce large amounts of internal control.

Total RNA was isolated by the guanidinium thiocyanate method (24). Before RT, RNA was treated at 68 C for 10 min and cooled on ice. One microgram of RNA was reverse transcribed for 1 h at 42 C to cDNA in a 20-µl mixture containing 500 µM dNTPs, 10 mM dithiothreitol, 5 µM random hexamer, and reverse transcriptase at 10 U/µl. An aliquot (5 µl) from the previous RT reaction was submitted to PCR in the presence of 2 µl [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham), 0.05 fM internal control, 0.5 µM of each primer, and 2.5 U Tfl DNA polymerase using the buffer and conditions recommended by the supplier. After denaturation at 94 C for 10 min, amplification was carried out using 29 cycles of denaturation (94 C for 1 min), annealing (61 C for 1 min), and elongation (72 C for 1 min) followed by a final elongation step (72 C for 10 min). The PCR products were submitted to electrophoresis on a 6% polyacrylamide gel, transferred onto 3 MM paper (Whatman, Clifton, NJ), dried, and exposed to x-ray Hyperfilms-MP at -20 C using an intensifying screen. Results were quantified by scanning densitometry (NIH Image) of the autoradiographs.

Measurement of PKC{alpha} mRNA stability
The PKC{alpha} mRNA half-life was measured in control and insulin-treated cells using the RNA synthesis inhibitor DRB, as previously described (29).

Diethylaminoethyl (DEAE) purification of PKC{alpha}
Serum-deprived CHO-R cells treated with or without 10-8 M insulin for the indicated times were washed three times in 5 ml ice-cold PBS and then lysed in 800 µl buffer A [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM ß-glycerophosphate, and 1% Triton X-100] supplemented with 200 µM orthovanadate, 0.1 mM PMSF, and 1 µg/ml leupeptin. After centrifugation (13,000 x g, 30 min, 4 C) of the cell lysates, the supernatants were applied to a DEAE column that was washed with 3 ml buffer B [20 mM Tris-HCl (pH 7.5), 0.5 mM EGTA (pH 8), 2 mM EDTA (pH 8), and 2 mM dithiothreitol] supplemented with 2 mM PMSF and 10 µg/ml leupeptin. Elution was performed with 1 ml buffer B containing 100 mM NaCl. Protein was determined using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Western blotting
Cell lysates or DEAE-purified fractions (see above) or nuclear extracts prepared as previously described (26) were analyzed by SDS-PAGE on 12% (wt/vol) polyacrylamide gels and electrotransferred onto Hybond-ECL nitrocellulose membranes in 25 mM Tris and 192 mM glycine. Membranes were blocked in Tris-buffered saline (20 mM Tris-HCl, pH 7.5, and 137 mM NaCl) containing 0.1% Tween-20 and 3% nonfat dry milk for 30 min at room temperature. The blots were incubated overnight at 4 C in blocking solution with antirat MAPK R2 antibody (1:1000), antiactive rat MAPK antibody (25 ng/ml), or antirat PKC{alpha} antibody (1:1000) and then with a goat antirabbit IgG conjugated to the horseradish peroxidase. ECL detection was monitored with reagents from Amersham, as recommended by the manufacturer. Quantification was performed by scanning densitometry of the autoradiographs.

Down-regulation of PKC
CHO-R cells were incubated for 24 h in FCS-free Ham’s F-12 medium in the absence or presence of 2.5 µM PMA. Cells were then washed and assayed for specific binding of [3H]PDBu (10 nM) as previously described (4).

Immunocomplex MAPK assay
Total cell lysates prepared from CHO-R cells that had been treated for 12 min with or without 10-8 M insulin or 20% FCS were analyzed for MAPK activity using the immunocomplex MAPK assay recently described (27).

Stable transfections
Stable populations of CHO-R cells expressing the MEK1 (S218D/S222D) mutant were obtained using NHE1 as a selective marker and the H+ killing selection as reported previously (22). CHO-R cells (5 x 106 cells) in the exponential phase of growth were transfected by electroporation (250 µF, 270 V). Medium was renewed 6 h before transfection; then cells were pelleted, washed, resuspended in electroporation buffer (10 mM NaPO4, 250 mM sucrose, and 1 mM MgCl2, pH 7.45) at 107 cells/ml and transfected with 1 µg/106 cells of the pEAP expression vector (NHE1 cDNA) and 2 µg/106 cells of the pECE expression vector containing the MEK1 (S218D/S22D) mutant or the vector alone. Forty-eight hours after transfection, cells were subjected to an acid load selection that killed nontransfected cells (usually 90–95% of the cell population). Three additional acid load tests were performed to obtain a resistant population. Single clones selected from the population stably expressing the MEK1 (S218D/S22D) mutant were used in the study. The expression of transfected MEK1 (S218D/S22D) mutant construct was verified by immunoblotting using the HA-MEK1 antibody as primary detector.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin increases PKC{alpha} mRNA expression in a time- and concentration-dependent manner in CHO-R cells
To study the effect of insulin on PKC{alpha} mRNA expression, we established a quantitative RT-PCR assay. In this assay, the radioactivity of the amplified PKC{alpha} cDNA fragment was measured by densitometric scanning of the autoradiographs and was normalized to the radioactivity of an internal control deleted of 200 bp. As shown in Fig. 1AGo, insulin induced PKC{alpha} mRNA in a time-dependent manner. The effect of the hormone was detected at 2 h, reached a maximum of 100 ± 9% over the basal value (P < 0.01) at 6 h, and persisted until 24 h. Insulin (10-8–10-6 M) increased PKC{alpha} mRNA expression in a concentration-dependent manner, with the maximal increase (176 ± 21% over basal; P < 0.02) observed at 10-7 M (Fig. 1BGo). Similarly, IGF-I caused a concentration-dependent increase in PKC{alpha} mRNA expression, with the effect obtained at 10-8 M being almost equivalent to that observed with 10-7 M insulin. Compared with CHO-R cells, parental CHO cells exhibited a decreased sensitivity to insulin for inducing PKC{alpha} mRNA, which is consistent with the low number of IRs expressed in these cells (Fig. 2Go). These results indicate that insulin and IGF-I are good enhancers of PKC{alpha} mRNA expression in CHO-R cells and that the effects of insulin and IGF-I are mediated through their cognate receptors.



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  		Figure 1. Time- and concentration-dependent induction of PKC{alpha} mRNA by insulin in CHO-R cells. Serum-starved CHO-R cells were incubated in FCS-free Ham’s F-12 medium with or without 10-8 M insulin for the indicated times (A) or with or without the indicated concentrations of insulin or IGF-I for 6 h (B). Total RNA (1 µg) from CHO-R cells was reverse transcribed to cDNA and amplified by PCR, as described in Materials and Methods. Autoradiographs were quantified by laser scanning densitometry. Representative autoradiograms are shown in the left panels, and the results of the densitometric analysis of PKC{alpha} mRNA levels normalized to the internal control are shown in the right panels (mean ± SEM; n = 3).

 


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  		Figure 2. Decreased sensitivity of parental CHO cells to insulin for induction of PKC{alpha} mRNA. Serum-starved CHO cells were incubated in FCS-free Ham’s F-12 medium with or without the indicated concentrations of insulin for 6 h. Total RNA was prepared and submitted to RT-PCR, as described in Materials and Methods. The results (mean ± SEM; n = 3) are expressed as indicated in Fig. 1Go.

 
Insulin increases the expression of PKC{alpha}-immunoreactive protein in CHO-R cells
To assess the relevance of the insulin-induced increase in the PKC{alpha} mRNA level, we studied the effect of the hormone on the expression of PKC{alpha}-immunoreactive protein. To this end, cell lysates prepared from serum-deprived CHO-R cells that had been treated in the presence or the absence of 10-8 M insulin for different time periods (0–24 h) were purified by DEAE-52 chromatography and assayed for PKC{alpha} expression by Western blot analysis.

As shown in Fig. 3Go, insulin increased the level of PKC{alpha}-immunoreactive protein, with the maximal effect (80 ± 8% over basal; P < 0.01) being detected at 6 h and persisting up to 24 h. This effect was delayed compared with that on PKC{alpha} mRNA, which was detected at 2 h. It is noteworthy that the effects of 10-8 M insulin on the expression of PKC{alpha} mRNA (Fig. 1BGo) and PKC{alpha}-immunoreactive protein (Fig. 3Go) were similar (~100% over basal in both cases).



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  		Figure 3. Time-dependent induction of PKC{alpha}-immunoreactive protein by insulin in CHO-R cells. Total cell lysates prepared from CHO-R cells that had been treated with 10-8 M insulin for the indicated time periods were purified by DEAE-52 chromatography and analyzed (50 µg protein) by Western blotting using a specific PKC{alpha} antibody. The results (mean ± SEM; n = 3) were quantified as indicated in Fig. 1Go.

 
Insulin induces PKC{alpha} mRNA in CHO-R cells through a transcriptional mechanism
The insulin-induced increase in PKC{alpha} mRNA level reported above could reflect increased stability of the transcript and/or enhanced gene transcription. To address this issue, we compared the decline in PKC{alpha} mRNA expression in CHO-R cells after blockade of RNA synthesis by the potent inhibitor DRB (Fig. 4Go). In these assays, cells were treated for 2 h with or without 10-8 M insulin and then for different time periods up to 9 h with 25 µg/ml DRB. Figure 4Go shows that the decline in PKC{alpha} mRNA transcript observed in insulin-treated CHO-R cells was almost identical to that found in control cells, as assessed by the similar half-lives calculated in these two conditions (~5 h).



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  		Figure 4. Half-life of PKC{alpha} mRNA in control and insulin-treated CHO-R cells. Total RNA extracted from control or insulin-stimulated (10-8 M; 2 h) CHO-R cells that were subsequently treated with 25 µg/ml DRB for the indicated time periods was submitted to RT-PCR. The results (mean ± SEM; n = 3) were quantified as indicated in Fig. 1Go. Lozenges, Control; squares, insulin treated.

 
Insulin stimulation of PKC{alpha} gene expression is independent of insulin receptor Tyr1162 and Tyr1163 residues
We then investigated the role of the IR major Tyr1162/1163 autophosphorylation sites in insulin stimulation of PKC{alpha} gene expression. To this end, we studied the effect of the hormone in CHO-Y2 cells. As shown in Fig. 5Go, insulin increased the amount of PKC{alpha} mRNA by 107 ± 12% (P < 0.02) in the 10-8–10-6 M concentration range (Fig. 5BGo), and the kinetics of this effect were almost identical with those in CHO-R cells (Fig. 5AGo). This finding shows that IR mutation at Tyr1162/1163 does not affect the stimulation of PKC{alpha} gene expression.



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  		Figure 5. Concentration-dependent induction of PKC{alpha} mRNA by insulin in CHO-Y2 cells. Serum-starved CHO-Y2 cells were incubated in FCS-free Ham’s F-12 medium with or without the indicated concentrations of insulin for 6 h. Total RNA (1 µg) from CHO-Y2 cells was reverse transcribed to cDNA and amplified by PCR, as described in Materials and Methods. The results (mean ± SEM; n = 3) are expressed as indicated in Fig. 1Go.

 
Insulin stimulation of PKC{alpha} gene expression is mediated through a pathway independent of PMA-sensitive PKCs
We examined the cytoplasmic signaling molecules involved in the stimulation of PKC{alpha} gene expression by insulin. As we previously reported that insulin activated PKC in CHO-R cells (4), we first examined the involvement of PKC in this process. To this end, we studied the effect of insulin (10-8 M) on PKC{alpha} gene expression in CHO-R cells that had been treated for 24 h in the presence or absence of 2.5 µM PMA. This treatment was previously found to produce efficient down-regulation of PMA-sensitive PKC isoforms, as evaluated by measuring the specific binding of [3H]PDBu to whole cells (4). As shown in Fig. 6Go, the stimulation of PKC{alpha} mRNA expression induced by a 6-h treatment with 10-8 M insulin in CHO-R cells persisted in CHO-R cells depleted of PKC by a long term PMA treatment, which argues against a role of PMA-sensitive PKCs, i.e. classical and novel PKC isoforms in this process.



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  		Figure 6. Persistent insulin induction of PKC{alpha} gene in PKC-depleted CHO-R cells. Total RNA (1 µg), prepared from control or PMA-treated cells (24 h; 2.5 µM) CHO-R cells that were subsequently incubated for 6 h in the absence or the presence of 10-8 M insulin, was submitted to RT-PCR as described in Materials and Methods. The results (mean ± SEM; n = 3) are expressed as indicated in Fig. 1Go.

 
Insulin stimulation of PKC{alpha} gene expression is dependent on the MEK1-MAPK pathway in CHO-R cells and CHO-Y2 cells
We next investigated whether insulin stimulation of PKC{alpha} gene expression involved MEK1, the upstream activator of the p44MAPK and p42MAPK isoforms of MAPK. To this end, we used PD98059, a compound described as a potent MEK1 inhibitor in different cell types (28). To verify the efficacy of PD98059 in CHO-R cells, we examined the activity of MAPK in cells that had been treated for 1 h with 50 or 100 µM PD98059 and then stimulated by 10-7 M insulin or 20% FCS. As shown in Fig. 7AGo, MAPK activation by insulin or FCS in CHO-R cells was assessed by mobility shift-up of phosphorylated p42MAPK and p42MAPK and by increased phosphorylation of MBP in MAPK immunoprecipitates obtained with an antibody that recognizes p44MAPK and p42MAPK. The pretreatment of CHO-R cells with PD98059 decreased MAPK activation in a concentration-dependent manner, with total suppression of MAPK activity obtained at 100 µM. At this concentration, PD98059 decreased by about 95% the stimulation of PKC{alpha} gene expression induced by insulin (10-8 M) or IGF-I (10-9 M) in CHO-R cells, indicating a role for MEK1 in the effects of these agonists on this process (Fig. 7BGo).



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  		Figure 7. Inhibition of insulin or IGF-I induction of PKC{alpha} gene by the MEK1 inhibitor PD98059 in CHO-R cells. A, Serum-starved CHO-R cells were incubated for 1 h in the presence or absence of 50 or 100 µM PD98059 and then treated for 10 min with or without 10-7 M insulin or 20% FCS. Total cell extracts were prepared and were either immunoblotted (upper panel) or immunoprecipitated before being analyzed for MBP phosphorylation (lower panel). B, CHO-R cells were treated for 1 h with or without 100 µM PD98059 before stimulation for 6 h with or without 10-8 M insulin or 10-9 M IGF-I. Then, total RNA was extracted and submitted (1 µg) to RT-PCR analysis as described in Materials and Methods. The results (A: mean; n = 2; B: mean ± SEM; n = 3) are expressed as indicated in Fig. 1Go.

 
To strengthen this hypothesis, we examined whether the stable expression of the dominant-active MEK1 (S218D/S222D) mutant in CHO-R cells could mimic the insulin-induced increase in PKC{alpha} gene expression. Figure 8AGo shows overexpression of HA-MEK1 in an acid load-selected clone of CHO-R cells transfected with a plasmid coding for the S218D/S222D MEK1 mutant (22) in-frame with the HA epitope. In this clone, MAPK was constitutively activated, as indicated by the increased level of MBP phosphorylation compared with that in control CHO-R cells (Fig. 8AGo). Concomitant with constitutive MAPK activation, the dominant-active MEK1-transfected cells exhibited a 110 ± 10% increase (P < 0.01) in PKC{alpha} gene expression compared with that in control cells and no further increase in response to insulin (Fig. 8BGo). Taken together, these results strongly argue for a role for the MEK1-MAPK pathway in insulin stimulation of PKC{alpha} gene expression in CHO-R cells.



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  		Figure 8. Constitutively increased PKC{alpha} mRNA expression in a clone of CHO-R cells expressing a dominant active MEK1 (S218D/S22D) mutant. A, CHO-R cells transfected with the expression plasmid for the epitope-tagged HA-MEK1 S218D/S222D mutant were submitted to acid load selection as described in Materials and Methods. Control and transfected cells were rendered quiescent by serum starvation and then treated with or without 10-7 M insulin or 20% FCS for 10 min. Cell lysates were prepared and were assayed for HA-MEK1 expression by Western blotting using the 12CA5 antibody (upper panel) or were immunoprecipitated before being analyzed for MBP phosphorylation. B, Total RNA extracted from untreated or insulin-treated (10-8 M; 6 h) CHO-R cells or HA-MEK1-transfected cells was submitted to RT-PCR analysis as described in Materials and Methods. The results (mean ± SEM; A, n = 2; B, n = 3) were quantified as indicated in Fig. 1Go.

 
As insulin signaling of PKC{alpha} gene expression was preserved in CHO-Y2 cells expressing insulin receptors mutated at Tyr1162/1163, we also investigated the effect of PD98059 on this process in this cell line. As shown in Fig. 9Go, PD98059 efficiently inhibited insulin-stimulated PKC{alpha} gene expression in CHO-Y2 cells, indicating that mutated insulin receptors, like wild-type insulin receptors, initiate a signaling pathway involving the activation of MEK1 to increase PKC{alpha} gene expression.



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  		Figure 9. Inhibition of insulin or IGF-I induction of PKC{alpha} gene by PD98059 in CHO-Y2 cells. CHO-Y2 cells were treated for 1 h with or without 100 µM PD98059 before stimulation for 6 h with or without 10-8 M insulin or 10-9 M IGF-I. Then total RNA was extracted and submitted (1 µg) to RT-PCR analysis as described in Materials and Methods. The results (mean ± SEM; n = 3) are expressed as indicated in Fig. 1Go.

 
The ability of insulin to activate MEK1, the upstream activator of p44MAPK and p42MAPK, in CHO-Y2 cells was surprising, as a previous study reported that insulin was unable to trigger the early phase of MAPK activation in these cells (17). However, to our knowledge no study examined the later phase of MAPK activation, and this is why we determined the kinetics of MAPK activation by insulin between 30 min and 6 h in CHO-Y2 cells in comparison to CHO-R cells (Fig. 10Go).



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  		Figure 10. Kinetics of insulin stimulation of MAPK activity in CHO-R and CHO-Y2 cells. Cells were rendered quiescent by serum starvation and were then treated with 20% FCS (F) for 10 min or with or without 10-8 M insulin for the indicated time periods. Cell lysates were prepared and assayed for MAPK activity by Western blotting using an antiactive MAPK antibody as described in Materials and Methods. A reproductive experiment of three is shown.

 
In this assay, MAPK activity was determined with the use of an antibody that specifically recognizes the activated forms of MAPK, i.e. phosphorylated p44MAPK and p42MAPK. In CHO-R cells, the stimulation of MAPK by insulin was maximal at 30 min and persisted until 6 h. In contrast, in CHO-Y2 cells, the stimulation of MAPK by insulin was detected after 1 h and regularly increased up to 6 h, at which time the extent of MAPK phosphorylation was similar to that observed in CHO-R cells. This observation confirms the inability of insulin to trigger the rapid activation of MAPK in CHO-Y2 cells and indicates that this process is delayed in these cells. Considered together, the above findings show that insulin stimulation of PKC{alpha} mRNA expression is preserved in CHO-Y2 cells despite IR mutation at Tyr1162/1163 and loss of acute MAPK activation. They argue for insulin activating PKC{alpha} gene expression through a pathway that requires prolonged activation of MAPK.

Insulin stimulation of PKC{alpha} gene expression correlates with p44MAPK activation at the nuclear level
As long term activated MAPKs were detected in the nucleus (25), we examined whether insulin-induced PKC{alpha} gene expression correlated with insulin-induced nuclear MAPK activation. Nuclear fractions prepared from control and insulin-treated CHO-R and CHO-Y2 cells were immunoblotted with a MAPK antibody that recognizes p44MAPK and p42MAPK. Note that these nuclear fractions exhibited negligible contamination by cytosolic fractions, as judged by measuring the specific activity of lactate deshydrogenase in both fractions (0.08 IU/mg protein in nuclear fractions vs. 10 IU/mg protein in cytosolic fractions). As shown in Fig. 11Go, nuclear fractions prepared from CHO-R and CHO-Y2 cells that had been treated with 10-8 M insulin for 3 or 6 h displayed an increase in the amount of immunoreactive p44MAPK and, to a lesser extent, immunoreactive p42MAPK compared with controls. These results indicate that insulin promoted the long term activation of nuclear MAPK, probably through a translocation mechanism, regardless of whether Tyr1162/1163 was present in the IR. Furthermore, when the nuclear fractions from control and insulin-treated cells were immunoblotted with an antibody that recognizes phosphorylated p44MAPK and p42MAPK, only phosphorylated p44MAPK was detected in nuclear fractions prepared from insulin-treated CHO-R and CHO-Y2 cells, indicating that at the times studied, p44MAPK was the sole nuclear MAPK isoform that remained active. These results indicate that insulin-induced PKC{alpha} gene expression correlates with insulin-induced activation of p44MAPK at the nuclear level.



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  		Figure 11. Insulin stimulation of nuclear p42MAPK activation in CHO-R and CHO-Y2 cells. Serum-starved CHO-R cells and CHO-Y2 cells were treated with or without insulin 10-8 M for the indicated times. Nuclear fractions were prepared and were either immunoblotted with a specific anti- p42 and anti-p44 MAPK antibody (upper panel) or an anti-active MAPK antibody (lower panel) as described in Materials and Methods. The autoradiograph is representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was designed to examine the effect of insulin on PKC{alpha} expression and to define the role of the MEK1-MAPK pathway in this effect. Two observations argue for insulin increasing PKC{alpha} mRNA expression through its own receptors. First, insulin potently increased the amount of PKC{alpha} mRNA at 10-8 M, a concentration at which it was unable to compete with IGF-I for binding to IGF-I receptors in CHO-R cells (19). Second, the sensitivity to insulin exhibited by CHO-R cells was markedly decreased in parental CHO cells expressing a low level of IRs.

The kinetics of insulin stimulation of PKC{alpha} expression in CHO-R cells show that the effect of the hormone appeared at 2 h and persisted until 24 h. Previous studies examining the effect of insulin on PKC{alpha} mRNA levels reported discrepant results. In cultured adipocytes, Avignon et al. (14) observed a 40% increase in PKC{alpha} mRNA induced by insulin after 20 h when cells were cultured in the presence of 25 mM glucose. Otherwise, Ishizuka et al. (15) found that insulin maximally increased the PKC{alpha} mRNA level after 5 min in isolated adipocytes. In vivo studies performed on rat epididymal fat pads or gastrocnemius muscles revealed a maximal increase in PKC{alpha} mRNA 60 min after insulin injection and normalization of this level after 15–17 h (14). Therefore, the kinetics of insulin stimulation of PKC{alpha} gene expression appear to be dependent on the model studied. In addition, in these tissues, which express high levels of at least PKC{alpha}, -ß, and -{gamma}, complex alterations in the level of each isoform in response to insulin were observed, with possible cross-regulation between isoform expression to achieve total cellular PKC activity.

The insulin-induced increase in PKC{alpha} gene expression is the result of the transcriptional activation of the PKC{alpha} gene, as it was not due to stabilization of the transcript. This is assessed by the almost identical half-lives of PKC{alpha} mRNA determined in insulin-treated and control cells with the use of DRB. Of interest, the value of 5 h determined for the PKC{alpha} mRNA half-life in CHO-R cells is in good accordance with that previously determined in Caco-2 cells (6 h) (29).

Along with PKC{alpha} gene expression, insulin increased the level of PKC{alpha} immunoreactive protein in CHO-R cells. However, the effect of insulin was observed after a lag period of 6 h. As a possible explanation for this observation, one can invoke the ability of insulin to activate PKC in CHO-R cells as previously reported (4). Once activated, PKC translocates to the plasma membrane where it is actively proteolyzed (30). Therefore, the unaltered PKC protein level at early times after insulin stimulation may result from the fact that PKC{alpha} synthesis compensates for the degradation of activated PKC. At later times, PKC is no longer stimulated, and newly synthesized PKC{alpha} is detected. In accordance with the results reported here, the insulin stimulatory effects on PKC{alpha} mRNA and protein expression were not parallel in cultured adipocytes or adipose tissue (14).

Up until now, no information on the signaling pathway involved in insulin stimulation of PKC{alpha} gene expression has been made available. The results reported here show that insulin signaling of PKC{alpha} mRNA expression was preserved in CHO-Y2 cells that overexpress insulin receptors mutated at Tyr1162/1163. This argues against the implication of Tyr1162/1163 major receptor autophosphorylation sites in insulin-stimulated PKC{alpha} gene expression. Similar observations were previously reported for insulin stimulation of Glut-1 gene expression (19) and thymidine incorporation into DNA (20), two other long term effects of the hormone studied in these transfected CHO cells. In contrast, evidence was provided for Tyr1162/1163 involvement in the rapid activation of PKC by insulin (4), a process shown to be regulated by IRS-1 phosphorylation and phosphatidylinositol 3-kinase activation and to be involved in the short term activation of glucose transport (6). Therefore, it appears that insulin regulates PKC activity and PKC content in CHO cells through two distinct pathways that diverge at the level of the IR itself. On the one hand, the cytoplasmic pathway that triggers rapid PKC activation in CHO cells may involve IRS-1 and phosphatidylinositol 3-kinase activation, as was recently found by Standaert et al. for insulin-induced translocation of PKC{alpha} and PKCß in rat adipocytes (6). On the other hand, the cytoplasmic pathway mediating insulin signaling of PKC{alpha} gene expression was herein found to be independent of PKC and to involve delayed, but sustained, activation of MAPK, as discussed below.

We show here that PMA-sensitive PKCs failed to play a role in insulin-stimulated PKC{alpha} gene expression. In addition, a role for PKC{zeta} seems unlikely, because this PMA-insensitive PKC isoform is faintly expressed and fails to respond to insulin in CHO-R cells (26).

The role of the MEK1-MAPK pathway in insulin signaling of PKC{alpha} gene expression is supported by the following. At 100 µM, a concentration shown to maximally inhibit insulin-stimulated MAPK activity in CHO-R cells, the MEK1 inhibitor PD98059 almost completely abolished the insulin-induced increase in PKC{alpha} gene expression. CHO-R cells expressing the dominant-active MEK1 (S218D/S222D) mutant and exhibiting constitutive activation of MAPK, displayed constitutive increased PKC{alpha} gene expression and no longer responded to insulin. These findings clearly indicate that insulin signaling of PKC{alpha} gene expression requires activation of MEK1. Of interest, we observed that PD98059 blunted insulin stimulation of PKC{alpha} gene expression not only in CHO-R cells, which displayed rapid and sustained MAPK activation in response to insulin, but also in CHO-Y2 cells, in which rapid MAPK activation was previously shown to be altered (17). Therefore, we performed longer kinetic studies; we then found that in CHO-Y2 cells MAPK activation indeed occurred, but after a latency of 1 h, and we observed that this activation persisted for up to 6 h. Interestingly, the kinetics of MAPK activation in these cells paralleled those of PKC{alpha} expression, which strongly suggests that insulin signaling of PKC{alpha} gene expression requires sustained MAPK activation. In addition, we show that under conditions where insulin induced PKC{alpha} gene expression in CHO-R and CHO-Y2 cells, it increased the amount of immunoreactive MAPK in the nuclear fractions and produced a selective activation of p44MAPK in these fractions. Together these results indicate that the stimulation of PKC{alpha} gene expression by insulin correlates with sustained activation of p44MAPK in the nucleus.

Recent studies indicated that growth factor signaling of the MAPK cascade is more complicated than a simple linear activation of the MEK1-MAPK pathway. Thus, Grammer and Blenis (31) reported that p44MAPK and p42MAPK could be activated by MEK1-independent pathways in Swiss 3T3 fibroblasts (31). In our study performed on CHO transfectants, the use of the specific MEK1 inhibitor PD98059 and the dominant-active MEK1 enabled us to demonstrate the role of MEK1 in insulin induction of PKC{alpha} expression. Otherwise, recent studies using CHO transfectants indicated that insulin activation of several target proteins (STAT3, Shc66, and Phas1) required MEK1 activation, but was independent of MAPKs (32, 33, 34). Such a pathway seems unlikely for the induction of PKC{alpha} expression by insulin, because we observed that the kinetics of MAPK activation at the cytosolic and nuclear levels correlated with the kinetics of PKC{alpha} expression in CHO-Y2 cells, which strongly argues for the implication of MAPK in the effect of insulin. It is now recognized that early signaling of the Ras-MEK1-MAPK pathway by insulin involves the phosphorylation of Shc on tyrosine residues by activated IRs (35, 36). This raises the question of how Tyr1162/1163-mutated IRs are able to transduce the insulin signal to MAPK in CHO-Y2 cells. However, it must be emphasized that these mutated IRs (19) retain a significant, albeit reduced, tyrosine kinase activity toward endogenous (18) and exogenous (19) substrates. In addition, we previously showed that after a long term treatment with insulin, wild-type IRs are desensitized to the hormone, and their tyrosine kinase activity is reduced (19), whereas Tyr1162/1163-mutated IRs escape this desensitization (19). These data lead us to propose that the sustained MAPK activation observed in CHO-R and CHO-Y2 cells after a long term treatment with insulin results from the residual and persistent TK activity displayed by wild-type and Tyr1162/1163-mutated IRs, respectively.

In the past 15 yr, evidence has accumulated to suggest the existence of complex interrelations between insulin signaling and the PKC pathway. Insulin activates PKC, and, in turn, activated PKC inhibits a number of insulin-stimulated biological effects through an action at the receptor and/or the postreceptor level (8, 9, 10, 11, 12, 13). In light of these data, one may expect that increased PKC{alpha} expression will result in insulin resistance. This is indicated by several studies showing that in diabetic states characterized by hyperinsulinemia and hyperglycemia, insulin resistance is associated with overexpression and/or activation of PKC (37, 38, 39).

In conclusion, the present study reports that induction of PKC{alpha} gene expression by insulin in CHO cells is independent of IR Tyr1162/1163 autophosphorylation sites, involves the MEK1-MAPK pathway, and correlates with a sustained activation of p44MAPK at the nuclear level. It would be interesting to determine whether the other long term effects of insulin on stimulation of Glut1 expression (19) and DNA synthesis (20), which were shown to be independent of IR Tyr1162/1163 autophosphorylation sites, are also associated with the activation of this MAPK isoform in the nucleus. An important implication of our study is that prolonged activation by insulin increases the level of PKC{alpha} and hence provides new enzyme to replace the activated forms of PKC{alpha} that have been degraded. This mechanism could explain the sustained increase in the level of PKC{alpha} observed after prolonged stimulation and in situations of insulin resistance in diabetes.


   Acknowledgments
 
We are greatly indebted to Prof. E. Clauser for the CHO-transfected cells, and Dr. J. Pouysségur for the dominant active MEK1 construct.


   Footnotes
 
1 This work was supported by grants from the Ministère de la Recherche et de l’Espace, Naturalia et Biologia, and l’Aide aux Jeunes Diabétiques. Back

Received January 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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