©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin Activates Nuclear Factor B in Mammalian Cells through a Raf-1-mediated Pathway (*)

(Received for publication, July 10, 1995; and in revised form, August 10, 1995)

France Bertrand Carole Philippe (2) Pierre Jacques Antoine Laurent Baud (2) André Groyer (1) Jacqueline Capeau Gisèle Cherqui (§)

From the  (1)From INSERM U.402, Laboratoire de Biologie Cellulaire, Faculté de Médecine Saint-Antoine, 27, rue Chaligny and INSERM U.142, Hôpital Saint-Antoine, 75571 Paris CEDEX 12, and (2)INSERM U.64, Hôpital Tenon, 75970 Paris Cedex 20, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We examined the effect of insulin on nuclear factor kappaB (NF-kappaB) activity in Chinese hamster ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr and Tyr autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-kappaB. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-kappaB: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with IkappaB degradation and/or NF-kappaBbulletIkappaB complex dissociation; 3) was independent of both PMA-sensitive and atypical () protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-kappaB DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-kappaB-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-kappaB in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.


INTRODUCTION

Insulin exerts a wide array of biological effects including regulation of growth and gene expression. The cytoplasmic events implicated in the transduction of insulin signals from the membrane insulin receptor to the transcriptional machinery in the nucleus are beginning to be understood. Studies using transfected cells overexpressing human insulin receptors and/or various proteins of insulin signaling pathways have shown that, after binding to its receptor, insulin activates insulin receptor tyrosine kinase activity and triggers tyrosine phosphorylation of at least two major substrates, IRS-1 and Shc(1) . Once phosphorylated, insulin receptor substrates interact with several proteins including the Grb2bulletSos complex which activates the Ras-Raf-1-MAP kinase (^1)pathway (1) . In contrast, little is known at present as concerns the nuclear transcription factors which are specifically activated by insulin to regulate gene expression.

The nuclear factor-kappaB (NF-kappaB) was originally described as being present in the cytosol of most cell types as an inactive heterodimer composed of 50-kDa (p50, NF-kappaB1) and 65-kDa (p65, Rel A) subunits and bound to one of the IkappaB inhibitor proteins(2, 3, 4) . Activation of NF-kappaB involves phosphorylation and degradation of IkappaB. This results in the dissociation of the NF-kappaBbulletIkappaB complex, a process which can be blocked by inhibitors such as pyrrolidine dithiocarbamate (PDTC), aspirin, and sodium salicylate(5, 6) . Thereafter, the active NF-kappaB p50/p65 heterodimer translocates to the nucleus, where it directly binds to its cognate DNA sequences. NF-kappaB is a pleiotropic activator which participates in the induction of a wide variety of cellular genes including genes encoding for signaling proteins. Activation of this factor can be achieved in many cell types by agonists of immune and inflammatory responses and also by mitogens(7, 8, 9) . In this regard, a recent paper (10) reported that the sequence of events triggered by insulin to induce maturation of Xenopus oocytes involves NF-kappaB activation. This finding prompted us to examine whether insulin was able to activate NF-kappaB in mammalian cells and, if so, to examine the signal transduction pathway involved. To this end, we used parental Chinese hamster ovary (CHO) cells and CHO cells overexpressing either wild-type human insulin receptors or kinase-defective insulin receptors mutated at Tyr and Tyr, two autophosphorylation sites playing a crucial role in receptor activation (11, 12, 13, 14, 15) . Our study demonstrates that insulin activates NF-kappaB in mammalian cells through a pathway which requires insulin receptor tyrosine kinase and Raf-1 kinase activities.


EXPERIMENTAL PROCEDURES

Reagents

[-P]ATP (10 Ci/mmol), [alpha-P]dCTP (3000 Ci/mmol), ECL detection kit, Hybond N membranes, and hyperfilms-MP were from Amersham Corp. [20-^3H]Phorbol 12,13-dibutyrate (PDBu, 18 Ci/mmol) and acetyl coenzyme A [acetyl-^3H], CAT assay grade were obtained from DuPont NEN. Insulin was purchased from Novo Laboratories and IGF-1 from Calbiochem. Rabbit polyclonal antibodies against p50 and p65 subunits of NF-kappaB were from Santa Cruz Biotechnology, Inc. MAP kinase R2 antibody, [Ser]PKC- pseudosubstrate peptide (PRKRQGSVRRRV), and PKC- inhibitor peptide (SIYRRGARRWRKL) were purchased from Upstate Biotechnology, Inc. [Ser]PKC-alpha pseudosubstrate peptide (RFARKGSLRQKNV) was from Life Technologies, Inc. NF-kappaB consensus oligonucleotide was from Genosys Biotechnologies and Oct-1 consensus oligonucleotide from Promega. Reverse transcriptase and Taq DNA polymerase were obtained from Perkin-Elmer and DNA polymerase 1 Klenow fragment from New England Biolabs. Other reagents were purchased from Sigma.

Cell Lines

The three different CHO cell lines (generous gift from Dr. E. Clauser, INSERM U.36, Paris) used in this study have been previously described(12, 13, 14) . These include the parental cell line (CHO), the CHO cell line transfected with a plasmid coding for the native form of the human insulin receptor (CHO-R), and the CHO cell line expressing human insulin receptors in which the 2 tyrosines at positions 1162 and 1163 have been replaced with phenylalanine residues by directed mutagenesis (CHO-Y2). Cells were grown in Ham's F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS). Prior to stimulation with insulin, cells were growth arrested at confluence by a 24-h incubation with serum-free Ham's F-12 medium.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were prepared from parental and transfected CHO cells by the method of Dignam et al.(16) with minor modifications, after treatment with or without insulin, IGF-1, or PMA, in the absence or presence of various agents. After two washings in 5 ml of ice-cold phosphate-buffered saline (PBS), cells were harvested in a 1.5-ml tube and centrifuged at 2,000 times g for 5 min in a microcentrifuge. The cell pellet was resuspended in 400 µl of buffer A (10 mM HEPES/KOH, pH 7.9, 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.1% Triton X-100), supplemented with the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 2 µM aprotinin, and 1.0 µg/ml of antipaïn, pepstatin, benzamidine, and leupeptin. After homogenization in a tight-fitting Dounce homogenizer, cell lysates were maintained 10 min on ice and centrifuged at 2,000 times g for 10 min. The nuclear pellet was then washed in buffer A without Triton X-100, resuspended in 100 µl of buffer B (20 mM HEPES/KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, and the mixture of protease inhibitors described above). After 30 min at 4 °C under constant agitation, nuclear debris were centrifuged at 13.000 times g for 5 min, and the supernatant (nuclear extract) was distributed into 15-µl aliquots which were stored at -80 °C until analysis by EMSA. Protein was determined by using the Bio-Rad reagent according to the manufacturer's instructions. Where indicated, nuclear extracts were incubated for 30 min at 4 °C with antibodies against p50 or p65 subunits of NF-kappaB before the binding reaction. The double-stranded NF-kappaB probe: 5`-AGCTTCAGAGGGGACTTTCCGAGAGG-3`; 3`-AGTCTCCCCTGAAAGGCTCTCCAGCT-5` (17) was annealed and end-labeled by using the DNA polymerase 1 Klenow fragment in the presence of 50 µCi of [alpha-P]dCTP and other unlabeled dNTPs at 20 µM each in a 50-µl reaction mixture containing 10 mM Tris-HCl, pH 7.9, 10 mM MgCl(2), 1 mM DTT, and 1 mM EDTA. Unincorporated nucleotides were removed by filtration through a Nuctrap column (Stratagène). Binding reactions were carried out in a 20-µl binding reaction mixture (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10% glycerol, 0.2% Nonidet P-40, and 3 µg of poly(dI-dC)) containing 5 µg of nuclear proteins and 0.5-2 ng of the NF-kappaB probe (40,000-80,000 counts/min). In some experiments, the binding reaction mixture also contained a large excess of unlabeled oligonucleotides containing either the NF-kappaB or the OCT-1 (immunoglobulin enhancer octanucleotide factor-1) consensus sequences. Samples were incubated at room temperature for 25 min and fractionated by electrophoresis on a 6% non-denaturing polyacrylamide gel in TAE buffer (7 mM Tris, pH 7.5, 3 mM sodium acetate, 1 mM EDTA), which had been pre-electrophoresed for 1 h at 80 V. Gels were run at 160 V for 2.5 h. Following electrophoresis, gels were transferred to 3MM paper (Whatman Ltd.), dried in a gel dryer under vacuum at 80 °C, and exposed to x-ray Hyperfilm-MP at -20 °C using an intensifying screen. Where indicated, results were quantified by laser scanning densitometry of the autoradiographs.

Down-regulation of Protein Kinase C (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 as described previously(12) .

Amplification of PKC- mRNA by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated by the method of Chirgwin et al.(18) from CHO-R cells and both colonic Caco-2 cells and hepatoma BC1 cells as controls. Before reverse transcription, RNA was treated at 68 °C for 10 min and cooled on ice. Two µg of RNA was reverse transcribed for 1 h at 37 °C to cDNA in a 20-µl reaction mixture containing 500 µM dNTPs, 10 mM DTT, RNasin at 1 unit/µl, 5 µM random hexamer, and reverse transcriptase at 10 units/µl. Then 5 µl of the reverse transcription reaction was used in a PCR reaction volume of 25 µl containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl(2), 0.001% gelatin), 2 units of Taq DNA polymerase, and two pairs of primers, each at 0.5 µM. PKC- primers [5`-TCCGTCAAAGCCTCCCATGTT-3` (sense) and 5`-ACGGGCTCGCTGGTGAACTGT-3` (antisense)] were designed to generate a 228-base pair fragment (from nucleotides 1425-1653) in the catalytic domain. beta-Actin-specific primers designed to amplify a 316-base pair fragment were chosen as reported elsewhere(19) . To prevent evaporation, 70 µl of mineral oil was layered on top of each sample. A thermocycler (Perkin-Elmer) was set to the following cycle parameters: 1 min at 94 °C for denaturation, 30 s at 61 °C for primer annealing, and 1.5 min at 72 °C for elongation. This cycle was repeated 30 times. Following amplification, samples (10 µl) were electrophoresed on 2% agarose gels, transferred to Hybond N membranes, and hybridized to a [-P]ATP-labeled PKC- specific probe (generous gift from Y. Nishizuka, Kobe University, Japan) or to a 30-mer oligonucleotide beta-actin probe(19) . Hybridization was carried out with buffer containing 6 times SPE (0.2 M Na(2)HPO(4), pH 7.6, 3.6 M NaCl, 20 mM) EDTA, 5 times Denhardt's solution, 0.5 mg/ml heparin, and 0.2% SDS. Membranes were then washed with 6 times SPE, 0.1% SDS at 55 °C for 30 min, and autoradiographed (Hyperfilms-MP).

In Situ PKC- Activity Assay

The effect of insulin on PKC- activity was evaluated by using a recently described (20) in situ assay. CHO-R and Caco-2 cells were distributed to Eppendorf microcentrifuge tubes (20,000 cells/tube) in serum-free medium. After incubation in the presence or absence of insulin or PMA, the medium was removed and replaced with 40 µl of permeabilization solution (137 mM NaCl, 5.4 mM KCl, 0.3 mM sodium phosphate, 0.4 mM potassium phosphate, 1 mg/ml glucose, 20 mM HEPES, pH 7.2, 50 µg/ml digitonin, 10 mM magnesium chloride, 25 mM beta-glycerophosphate) containing 150 µM -peptide (pseudosubstrate site in PKC- substituting Ser for Ala) together with or without 300 µM PKC- inhibitor peptide. The reactions were initiated by addition of 100 µM [-P]ATP and were terminated after 10 min at 30 °C with 15 µl of 25% trichloroacetic acid (w/v). Samples were left for at least 10 min on ice and then centrifuged at 5,000 times g for 5 min. Supernatants (50 µl) were spotted on 2 times 2-cm phosphocellulose squares (Whatman P-81) which were washed batchwise in three changes (500 ml each) of 75 mM phosphoric acid and one change of 75 mM sodium phosphate (pH 7.5). Once dried, the P-81 papers were counted for bound radioactivity. Assays were performed in duplicate and tubes containing no -peptide were included in each assay to estimate the ``background'' phosphorylation of basic cell components which were not precipitated by trichloroacetic acid and which adhered to P-81 paper. The same in situ assay was used to measure the activity of classical and novel PKCs, except that the permeabilization solution contained 2.5 mM CaCl(2) and that reactions were carried out with the [Ser]PKC-alpha substrate peptide.

MAP Kinase Western Blotting

Serum-deprived CHO-R cells were incubated with or without 10M insulin, in the absence or presence of 0.25 mM 8-bromo-cAMP. After three washings with ice-cold PBS, cells were lysed and nuclear fractions prepared as above were examined for lactate dehydrogenase activity which was used as a cytosolic marker. These fractions were then analyzed by Western blotting using an anti-rat MAP kinase R2 antibody (1:1000) and a goat anti-rabbit IgG conjugated to horseradish peroxidase. ECL detection was monitored with reagents from Amersham, as recommended by the manufacturer. Quantification was performed by scanning densitometry of the autoradiographs.

Transient Transfection

CHO-R cells were transfected using the calcium phosphate precipitation method. Briefly, CHO-R cells (5 times 10^5 cells/60 mm-dish) were seeded 24 h before transfection in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10% FCS. Medium was renewed 4 h prior to transfection. Cells were transfected with 5 µg of either (Igkappa)3-conaluc, a reporter plasmid which contains three copies of the immunoglobulin kappa chain enhancer kappaB site upstream of the minimal conalbumin promoter fused to the luciferase reporter gene or its control counterpart conaluc (generous gift from Dr. A. Israel, Institut Pasteur, Paris, France). In some experiments, cells were co-transfected with 5 µg of RSV-C4beta, a plasmid that expresses the Raf-1 dominant negative mutant Raf-C4 (generous gift from Dr. U. R. Rapp, Institute of Medical Radiobiology and Cell Biology, Wurzburg, Germany). One µg of a pRSV-CAT was always included as a monitor for transfection efficiency. Transfections were carried out in the presence of a constant amount of DNA (11 µg/60-mm dish). After transfection, cells were incubated for 36 h in Ham's F-12 medium containing 0.3% FCS, then for 12 h in the presence or absence of 10M insulin and harvested for luciferase and CAT assays.

Luciferase and CAT Assays

The cell monolayers were rinsed twice with Ca- and Mg-free PBS, covered with 0.7 ml of chilled lysis buffer (25 mM Tris, pH 7.8, with H(3)PO(4), 10 mM MgCl(2), 1 mM EDTA, 1 mM DTT, 1% Triton X-100, and 15% glycerol), maintained on ice for 30 min and scraped. The lysates were centrifuged for 10 min at 13,000 times g. Luciferase activity was assayed in lysis buffer containing 50 mM ATP and 100 µM luciferin, using a monolight 2010 luminometer (Berthold). CAT activity was measured by the diffusion-based CAT assay(21) . Correction for differences in transfection efficiency between plates within an experiment was performed by normalizing the luciferase activity to the CAT activity in each extract. All transfections were performed three times in triplicate.


RESULTS AND DISCUSSION

Insulin Activates NF-kappaB in CHO-R Cells through Its Own Receptors

Insulin activated NF-kappaB in CHO-R cells in a time-dependent manner. The effect of the hormone was detected at 1 h, reached a maximum at 6 h, and was no longer detected at 24 h (Fig. 1A). Similarly, Baldwin et al.(7) reported that serum induction of NF-kappaB in BALB/c3T3 cells culminated at 6 h and was no longer detected at 24 h. The hormone increased the formation of two NF-kappaB complexes with a major effect on the upper complex designated as band 1 and a minor effect on the lower complex designated as band 2 (Fig. 1A). Insulin activated NF-kappaB in a concentration-dependent manner (Fig. 1B). The effect of the hormone regularly increased from 10M t0 10M (Fig. 1B). At the latter concentration, the effect of insulin was equivalent to that elicited by 10M IGF-1 (Fig. 1B). Two observations reported in our previous (14) and present papers argue for insulin activating NF-kappaB through its own receptors. First, insulin was potent to activate NF-kappaB at 10 and 10M, two concentrations at which insulin was unable to displace the binding of I-labeled IGF-1 to IGF-1 receptors in CHO-R cells(14) . Second, insulin activation was related to insulin receptor number. This is supported by the finding that the sensitivity to insulin exhibited by CHO-R cells for NF-kappaB activation (Fig. 1B) was markedly decreased in parental CHO cells (Fig. 2). In contrast, these cells proved to be fully responsive to IGF-1 as well as to PMA, a well-known inducer of NF-kappaB activity in several cell types(2, 3, 5) (Fig. 2). The above results therefore indicate that both insulin and IGF-1 are good inducers of NF-kappaB in CHO-R cells and that insulin activates this transcription factor in the insulin receptor overexpressing cell line through its own receptors.


Figure 1: Activation of NF-kappaB in CHO-R cells by insulin and IGF-1. Serum-starved CHO-R cells were incubated in FCS-free medium with or without 10M insulin for the indicated times (A) or with or without the indicated concentrations of insulin or IGF-1 for 6 h (B). Nuclear extracts were prepared and assayed for NF-kappaB activity, as described under ``Experimental Procedures.'' Autoradiographs from the concentration experiments were quantified at the level of band 1 by laser scanning densitometry. The autoradiographs are representative of three (A and B for IGF-1) or seven (B for insulin) experiments.




Figure 2: Activation of NF-kappaB in CHO cells by insulin, IGF-1, and PMA. Serum-deprived CHO cells were incubated for 6 h in FCS-free medium with or without the indicated concentrations of insulin, or IGF-1, or PMA. Nuclear extracts were prepared and assayed for NF-kappaB activity, as described under ``Experimental Procedures.'' This is a representative experiment independently performed three times.



Characterization of Insulin-induced NF-kappaBbulletDNA Complexes

The specificity of bands 1 and 2 was assessed in competition experiments. Both bands disappeared in the presence of the unlabeled oligonucleotide containing the NF-kappaB-binding site whereas they remained unchanged in the presence of the unlabeled oligonucleotide containing the unrelated OCT-1 consensus sequence (Fig. 3A). We further characterized the insulin-induced DNAbulletprotein complexes by using antibodies directed against the p50 and p65 NF-kappaB subunits. Incubation of nuclear extracts (30 min at 4 °C) from insulin-treated cells (6 h, 10M) with anti-p50 antibody abolished band 2 with a concomitant supershift, and reduced the amount of band 1, suggesting the presence of p50 in the two protein complexes (Fig. 3B). In contrast, the anti-p65 antibody had no effect on band 2, suggesting that p65 was absent from the band 2 DNAbulletprotein complex. However this antibody, like the p50 antibody, markedly reduced the intensity of band 1, indicating that band 1 corresponds to a DNAbulletprotein complex composed of both p50 and p65 NF-kappaB subunits (Fig. 3B). These experiments enabled us to identify the major and minor DNAbulletprotein NF-kappaB complexes induced by insulin as the p50/p65 heterodimer (band 1) and p50 homodimer (band 2), respectively. This finding is of particular interest since the p50/p65 heterodimer is known as the main effective and also the major inducible form of NF-kappaB(22) , whereas the p50 homodimer has been reported to occur as constitutive factor in nuclei of certain cell types(2, 3, 23) .


Figure 3: Characterization of insulin-induced NF-kappaBbulletDNA complexes. Nuclear extracts prepared from control or insulin-stimulated (6 h, 10M) CHO-R cells were assayed for NF-kappaB activity either in the absence (lanes 1 and 2) or presence of unlabeled oligonucleotides containing the NF-kappaB (lane 3) or the OCT-1 (lane 4) consensus sequences (A) or after a 30-min incubation without (lane 1) or with anti-p50 (lane 2) or anti-p65 (lane 3) antibodies (B). EMSA was performed as described under ``Experimental Procedures.'' This is a representative experiment independently performed three times.



Insulin Activates NF-kappaB-dependent Luciferase Reporter Gene Expression

To determine whether insulin enhances NF-kappaB-driven reporter gene expression, CHO-R cells were transfected transiently with (Igkappa)3-conaluc, a reporter plasmid in which the activity of the minimal conalbumin promoter fused to the luciferase reporter gene may be enhanced by three copies of the immunoglobulin kappa chain enhancer kappaB site(24) . When transiently transfected CHO-R cells were treated for 12 h with 10M insulin, a 3.2 ± 0.5-fold increase in normalized luciferase activity was observed as compared to the activity measured in unstimulated cells (Fig. 4B). Under identical conditions, insulin had no effect on normalized luciferase activity in CHO-R cells which had been transfected with the control conaluc plasmid (Fig. 4A). This argues for NF-kappaB-binding sites being specifically activated by insulin treatment. Together, these experiments support the conclusion that insulin stimulation of NF-kappaB DNA binding activity in CHO-R cells (Fig. 1) results in activation of NF-kappaB-mediated gene transcription (Fig. 4B).


Figure 4: Insulin induction of NF-kappaB-mediated luciferase reporter gene activity in CHO-R cells. CHO-R cells were co-transfected by the CaPO(4) method with 5 µg of either control (conaluc A) or test ((Igkappa)3-conaluc, B) plasmids, in the absence or presence of the expression plasmid for the Raf-1 dominant negative mutant Raf-C4. All transfections included 1 µg of pRSV-CAT plasmid as a monitor for transfection efficiency and were carried out at a constant amount of DNA (11 µg/dish). After transfection, cells were maintained for 36 h in culture medium containing 0.3% FCS and then for 12 h in the absence or presence of 10M insulin. Forty-eight h post-transfection, cell extracts were prepared and assayed for luciferase and CAT activities as described under ``Experimental Procedures.'' The results, presented as normalized luciferase activity (ratio of NF-kappaB-luciferase activity to the CAT activity measured in each extract) are the means ± S.E. from three independent experiments, each performed in triplicate.



Insulin Activation of NF-kappaB Requires Insulin Receptor Tyrosine Kinase Activity

To investigate the role of the insulin receptor tyrosine kinase in the activation of NF-kappaB by insulin, we studied the effect of insulin in CHO-Y2 cells expressing insulin receptors made kinase defective by mutation at Tyr and Tyr, two major autophosphorylation sites of the insulin receptor tyrosine kinase domain playing a crucial role in receptor activation(25) . Nuclear extracts from CHO-Y2 cells which had been treated with 10M insulin for various time periods (0-6 h) or with graded concentrations (0-10M) of insulin for 6 h were tested for NF-kappaB activity. As shown in Fig. 5, the effect of insulin in CHO-Y2 cells was dramatically reduced as compared to that observed in CHO-R cells (Fig. 1). Similar reduction was previously reported for insulin receptor tyrosine kinase activity(14) . This finding further argues for an effect of insulin being mediated by insulin and not IGF-1 receptors, since we previously reported that CHO-Y2 cells expressed the same number of IGF-1 receptors as CHO-R cells(13) . Most importantly, this finding shows that insulin receptor autophosphorylation sites Tyr and Tyr play a pivotal role in insulin activation of NF-kappaB and therefore indicate that this process takes place among the multiple insulin-stimulated pathways requiring the integrity of the insulin receptor tyrosine kinase activity(25) . Otherwise, the finding that the activation of NF-kappaB by insulin was lower in CHO-Y2 cells than in CHO cells most probably reflects a dominant negative effect exerted by the overexpressed mutated insulin receptors on endogenous insulin receptors or their downstream targets, as has been previously found for other insulin-responsive pathways(12, 14) .


Figure 5: Loss of NF-kappaB activation by insulin in CHO-Y2 cells. Experiments described in Fig. 1, A and B, with insulin were performed in CHO-Y2-cells. Maximal insulin activation of NF-kappaB in CHO-R cells is given as a control. This is a representative experiment independently performed three times.



Insulin Activation of NF-kappaB Involves a Post-translational Mechanism

To approach the mechanism whereby insulin induced NF-kappaB in CHO-R cells, nuclear extracts were prepared from CHO-R cells which had been treated for 6 h with 10M insulin in the presence or absence of the protein synthesis inhibitor cycloheximide (10 µg/ml). This cycloheximide treatment was found to inhibit 90-95% of basal and insulin-stimulated protein synthesis in CHO-R cells, in accordance with previous results(14) . Cycloheximide alone increased NF-kappaB activity in CHO-R cells (Fig. 6A), as previously reported in other cell types(5, 7, 26) . The mechanism underlying this increase is unknown but may involve inhibition of IkappaB synthesis, as has been suggested by others(5, 8, 26) . Most importantly, we observed that activation of NF-kappaB by insulin was preserved in the presence of cycloheximide, indicating that this process did not require de novo protein synthesis (Fig. 6A). This finding, together with the finding that NF-kappaB activation by insulin occurred within the first hour of treatment, argue for a post-translational effect of insulin on NF-kappaB activity. This may proceed from the ability of the hormone to promote dissociation of the NF-kappaBbulletIkappaB complex.


Figure 6: Effect of cycloheximide and inhibitors of NF-kappaBbulletIkappaB complex dissociation on NF-kappaB activation by insulin in CHO-R cells. Nuclear extracts were prepared from CHO-R cells which had been incubated for 6 h with or without 10M insulin in the absence or presence of 10 µg/ml cycloheximide (CHX) (A) or 0.1 mM PDTC (B) or 5 mM aspirin or 10 mM sodium salicylate (NaSal) (C). EMSA was performed as described under ``Experimental Procedures.'' This is a representative experiment independently performed three times.



To test this hypothesis, we examined the effect of: 1) PDTC, a thiol compound scavenging reactive oxygen intermediates and, thereby, impeding the release of IkappaB from NF-kappaB(5) ; and 2) aspirin and sodium salicylate, two drugs interfering with a pathway that leads to IkappaB phosphorylation and/or degradation or both(6) . As shown in Fig. 6B, PDTC (0.1 mM) completely abolished maximal insulin activation of NF-kappaB, as indicated by the complete disappearance of band 1 and band 2 DNA-binding complexes in nuclear extracts prepared from CHO-R cells treated with insulin (6 h, 10-^7M). PDTC was reported to cause similar inhibition in Jurkat cells exposed to various NF-kappaB activators including lipopolysaccharide, tumor necrosis factor-alpha, interleukin-1, and PMA(5) . As shown in Fig. 6C, sodium salicylate (10 mM) and aspirin (5 mM) were also potent to inhibit insulin induction of NF-kappaB in CHO-R cells. In accordance with these results, sodium salicylate and aspirin were recently shown to inhibit activation of NF-kappaB by lipopolysaccharide, tumor necrosis factor-alpha, and phytohemagglutinin plus PMA in Jurkat cells(6) . The mechanisms involved in sodium salicylate or PDTC inhibitory effects in CHO-R cells could be identical to those previously described in the above studies, i.e. impairment of a pathway leading to IkappaB degradation and/or NF-kappaBbulletIkappaB complex dissociation.

Insulin Activation of NF-kappaB Appears to be Independent of PMA-sensitive and PMA-insensitive PKC Isoforms

Because PKC is a known inducer of NF-kappaB (2, 3, 4) and PMA activates NF-kappaB in CHO cells (Fig. 2), we next examined PKC involvement in insulin activation of NF-kappaB. We thus tested the effect of insulin (6 h, 10M) on NF-kappaB activity in CHO-R cells which 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(12) . As shown in Fig. 7A, the activation of NF-kappaB by insulin was similar in untreated and PMA-treated CHO-R cells, indicating that insulin signaling of NF-kappaB activity was mediated by a pathway which was independent of PMA-sensitive PKCs, i.e. classical and novel PKC isoforms. However, PKC-, an atypical PMA-insensitive PKC isoform(27) , was recently reported to induce phosphorylation of IkappaB in vitro(28) and also to be involved in insulin-induced maturation of Xenopus oocytes, a Ras-mediated process associated with NF-kappaB activation(10) . We therefore evaluated the level of expression of this PKC isoform in CHO-R cells by the RT-PCR technique. The data indicated that PKC- was detectable in CHO-R cells but at a very low level as compared to that observed in control Caco-2 and BC1 cells (Fig. 7B). Since this finding raised the possibility that PKC- may be involved in insulin stimulation of NF-kappaB activity in CHO-R cells, we next examined whether insulin was able to increase PKC- activity under conditions where it activated NF-kappaB in CHO-R cells. To this end, we took advantage of a recently described in situ PKC- activity assay(20) . In this assay, the phosphorylation of the PKC- peptide, the most efficient substrate for PKC-(29) , was measured in digitonin-permeabilized cells either in the presence or in the absence of the PKC- inhibitor pseudosubstrate peptide. As could be expected from the results presented in Fig. 7B, the level of PKC- activity in CHO-R cells (0.9 nmol of P transferred to PKC- peptide/10^5 cells) was far lower than that measured in Caco-2 cells (3.5 nmol of P transferred to PKC- peptide/10^5 cells). Insulin (10M) had no effect on PKC- activity (90-100% of control) in CHO-R cells, whatever the time of incubation examined (30 min, 1, 2, and 6 h). Similarly, PMA (1.62 times 10M, 15 min) was ineffective on PKC- activity in CHO-R cells (110% of the control value), as previously shown in other cell types(27) . In contrast, PMA (1.62 times 10M) caused a marked increase in PKC activity (130 and 200% of the control value at 5 and 15 min, respectively) when the same permeabilization procedure was run out with the PKC-alpha peptide, a good substrate for classical and novel PKCs (29) . Taken as a whole, the above results argue against the involvement of PMA-sensitive PKC isoforms in insulin activation of NF-kappaB in CHO-R cells and do not favor the hypothesis that the PMA-insensitive PKC- isoform may play a role in this process.


Figure 7: Effect of a long-term exposure of CHO-R cells to PMA on NF-kappaB activation by insulin (A)and PKC- mRNA expression in CHO-R cells (B). A, nuclear extracts prepared from control or PMA-treated (24 h, 2.5 µM) CHO-R cells which had been incubated for 6 h in the absence or presence of 10M insulin were assayed for NF-kappaB activity as described under ``Experimental Procedures.'' This is a representative experiment independently performed three times. B, total RNA (2 µg) from CHO-R cells or from Caco-2 or BC1 cells was reverse transcribed to cDNA and amplified by PCR as described under ``Experimental Procedures.'' Amplified PKC- cDNA was detected by hybridization to a specific PKC- cDNA probe. Level of beta-actin mRNA was evaluated in these assays as a control. Ethidium bromide staining of PKC- and beta-actin bands is given in the bottom panel.



Insulin Activation of NF-kappaB Is Inhibited by 8-Bromo-cAMP, an Agent Interfering with Growth Factor Activation of Raf-1 Kinase

The recent study of Li and Sedivy (30) reported the ability of Raf-1 kinase to activate NF-kappaB by dissociating the NF-kappaBbulletIkappaB complex. Moreover, several papers provided evidence that, in mammalian cells, the Ras-Raf-1 pathway mediated the activation of NF-kappaB by various stimuli or inducers(31, 32, 33) . We judged these findings of particular interest since we (34) and others(35, 36) previously reported the capacity of insulin to activate this pathway in CHO-R cells. We therefore investigated whether insulin activation of NF-kappaB in these cells was affected when inhibiting Raf-1 kinase by 8-bromo-cAMP. This cell-permeable cAMP analogue is an activator of protein kinase A, a kinase which, in some cell types(37, 38) , inhibits Ras-dependent activation of Raf-1 kinase by phosphorylating the enzyme on its kinase domain(39) . As a preliminary to this experiment, we examined insulin activation of MAP kinase, a downstream substrate of Raf-1, by evaluating MAP kinase nuclear translocation in CHO-R cells(40) .

Insulin (6 h, 10M) increased the amount of immunoreactive MAP kinase associated with the nuclear fraction (225% of the value measured in the nuclear fraction from control cells), indicating the ability of the hormone to induce a long term MAP kinase translocation in the nucleus of CHO-R cells. Treatment of CHO-R cells with 8-bromo-cAMP (0.25 mM) prevented the insulin-induced increase in nuclear immunoreactive MAP kinase (92% of the control value). In view of these data, we investigated the effect of 8-bromo-cAMP on NF-kappaB activation by insulin. Fig. 8shows that 8-bromo-cAMP (0.25 mM) did not increase the DNA binding activity of NF-kappaB, making unlikely in vivo activation of this transcription factor by protein kinase A in CHO-R cells, in contrast to what has been found in in vitro experiments using cytosolic fractions from 70 Z/3 cells(41) . This compound inhibited insulin activation of NF-kappaB in a concentration-dependent manner, with the maximal inhibition being observed at 0.25 mM (Fig. 8). In contrast, even at a 2-fold higher concentration (0.5 mM), 8-bromo-cGMP poorly modified insulin-stimulated NF-kappaB DNA-binding activity in CHO-R cells (Fig. 8), demonstrating the specificity of 8-bromo-cAMP inhibitory effect. These experiments showed that: 1) under conditions where insulin induced the maximal stimulation of NF-kappaB activity, it activated Raf-1 kinase, as assessed by MAP-kinase nuclear translocation, a process shown to be associated with growth factor signaling(40) ; and 2) 8-bromo-cAMP activation of protein kinase A inhibited insulin stimulation of both Raf-1 kinase and NF-kappaB DNA binding activity. Such findings argue for the notion that Raf-1 kinase mediates the activation of NF-kappaB by insulin in CHO-R cells.


Figure 8: Effect of 8-bromo-cAMP on NF-kappaB activation by insulin in CHO-R cells. Nuclear extracts were prepared from CHO-R cells which had been incubated for 6 h with or without 10M insulin in the absence or presence of the indicated concentrations of 8-bromo-cAMP or 8-bromo-cGMP. EMSA was performed as described under ``Experimental Procedures.'' This is a representative experiment independently performed three times.



Insulin Activation of NF-kappaB Is Blocked in CHO-R Cells Expressing a Raf-1 Dominant Negative Mutant

To strengthen the above hypothesis, we examined the activation by insulin of NF-kappaB-mediated luciferase gene expression in CHO-R cells transfected with the Raf-1 dominant negative mutant Raf-C4. This mutant encodes the cysteine-rich amino-terminal regulatory domain of Raf-1 but lacks the kinase domain and a serine/threonine region which is believed to function as a regulatory phosphorylation site(42) . In these experiments, CHO-R cells were transfected with the (Igkappa)3-conaluc reporter plasmid (5 µg), in the presence or absence of the expression plasmid for Raf-C4 (5 µg). A pRSV-CAT (1 µg) was included as a monitor for transfection efficiency. Co-transfection of Raf-C4 in CHO-R cells completely abolished the effect of insulin on the kappaB enhancer activity: the significant insulin-induced increase (3.2 ± 0.5-fold) in normalized luciferase activity measured in CHO-R cells transfected with (Igkappa)3-conaluc (Fig. 4B) was no longer observed in cells which had been co-transfected with Raf-C4 (0.90 ± 0.07-fold) (Fig. 4B). These results provide clear evidence that the pathway whereby insulin activates NF-kappaB-mediated gene expression in CHO-R cells involves Raf-1 kinase.

The question of whether insulin activation of NF-kappaB in these cells is a MAP kinase-mediated process cannot be answered by the results presented here. A recent paper (33) studying activation of NF-kappaB by hypoxia concluded to the involvement of Ras and Raf-1 but not of MAP kinase in this process. Whether or not this may be the case for insulin activation of NF-kappaB remains to be determined. As well, it would be interesting to examine whether, in other cell lines, a pathway bifurcating from Ras and involving PKC- may, in parallel to the Raf-1 kinase pathway, participate in NF-kappaB activation by insulin. In this regard, both Raf-1 and PKC- were reported to initiate parallel pathways downstream of Ras for regulation of mouse fibroblast proliferation(43) .

In conclusion, our study provides the first evidence that insulin specifically activates NF-kappaB in mammalian cells. This process involves a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.


FOOTNOTES

*
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche contre le Cancer (Villejuif, France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-40-01-13-56; Fax: 33-1-40-01-14-99.

(^1)
The abbreviations used are: MAP kinase, mitogen-activated protein kinase; NF-kappaB, nuclear factor kappaB; CHO, Chinese hamster ovary; PDTC, pyrrolidine dithiocarbamate; FCS, fetal calf serum; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; DTT, dithiothreitol; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate; RT-PCR, reverse transcriptase-polymerase chain reaction; IGF-1, insulin growth factor-1.


ACKNOWLEDGEMENTS

We thank Betty Jacquin for secretarial assistance and Hélène Robin for technical assistance. We are greatly indebted to Dr. Eric Clauser for providing us with the transfected CHO cell lines, to Dr. Alain Israel for the anti-p50 and anti-p65 NF-kappaB antibodies and the (Igkappa)3-conaluc plasmid, to Dr. Y. Nishizuka for the PKC- probe, and to Dr. U. R. Rapp for the Raf-1 dominant negative mutant Raf-C4.


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