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 Grb2
Sos
complex which activates the Ras-Raf-1-MAP kinase (
)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-
B
(NF-
B) was originally described as being present in the cytosol of
most cell types as an inactive heterodimer composed of 50-kDa (p50,
NF-
B1) and 65-kDa (p65, Rel A) subunits and bound to one of the
I
B inhibitor proteins(2, 3, 4) .
Activation of NF-
B involves phosphorylation and degradation of
I
B. This results in the dissociation of the NF-
B
I
B
complex, a process which can be blocked by inhibitors such as
pyrrolidine dithiocarbamate (PDTC), aspirin, and sodium
salicylate(5, 6) . Thereafter, the active NF-
B
p50/p65 heterodimer translocates to the nucleus, where it directly
binds to its cognate DNA sequences. NF-
B 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-
B
activation. This finding prompted us to examine whether insulin was
able to activate NF-
B 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-
B 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), [
-
P]dCTP (3000 Ci/mmol), ECL
detection kit, Hybond N
membranes, and hyperfilms-MP
were from Amersham Corp. [20-
H]Phorbol
12,13-dibutyrate (PDBu, 18 Ci/mmol) and acetyl coenzyme A
[acetyl-
H], 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-
B 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-
pseudosubstrate peptide
(RFARKGSLRQKNV) was from Life Technologies, Inc. NF-
B 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
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
, 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
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
, 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
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-
B before the binding reaction. The
double-stranded NF-
B 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 [
-
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
, 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-
B 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-
B 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
[
H]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
, 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.
-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
-actin probe(19) . Hybridization
was carried out with buffer containing 6
SPE (0.2 M
Na
HPO
, pH 7.6, 3.6 M NaCl, 20
mM) EDTA, 5
Denhardt's solution, 0.5 mg/ml
heparin, and 0.2% SDS. Membranes were then washed with 6
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
-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
g for 5 min.
Supernatants (50 µl) were spotted on 2
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
and that reactions were carried out with the
[Ser
]PKC-
substrate peptide.
MAP Kinase Western Blotting
Serum-deprived CHO-R
cells were incubated with or without 10
M
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
10
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
(Ig
)3-conaluc, a reporter plasmid which contains three copies of
the immunoglobulin
chain enhancer
B 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-C4
, 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 10
M 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
PO
, 10 mM
MgCl
, 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
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-
B in CHO-R Cells through Its
Own Receptors
Insulin activated NF-
B 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-
B in
BALB/c3T3 cells culminated at 6 h and was no longer detected at 24 h.
The hormone increased the formation of two NF-
B 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-
B in a
concentration-dependent manner (Fig. 1B). The effect of
the hormone regularly increased from 10
M t0 10
M (Fig. 1B). At
the latter concentration, the effect of insulin was equivalent to that
elicited by 10
M IGF-1 (Fig. 1B). Two observations reported in our previous (14) and present papers argue for insulin activating NF-
B
through its own receptors. First, insulin was potent to activate
NF-
B at 10
and 10
M, 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-
B
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-
B 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-
B 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-
B in CHO-R cells by insulin and IGF-1. Serum-starved CHO-R
cells were incubated in FCS-free medium with or without 10
M 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-
B
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-
B 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-
B activity, as described under ``Experimental
Procedures.'' This is a representative experiment independently
performed three times.
Characterization of Insulin-induced NF-
B
DNA
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-
B-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
DNA
protein complexes by using antibodies directed against the p50
and p65 NF-
B subunits. Incubation of nuclear extracts (30 min at 4
°C) from insulin-treated cells (6 h, 10
M) 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
DNA
protein complex. However this antibody, like the p50 antibody,
markedly reduced the intensity of band 1, indicating that band 1
corresponds to a DNA
protein complex composed of both p50 and p65
NF-
B subunits (Fig. 3B). These experiments enabled
us to identify the major and minor DNA
protein NF-
B 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-
B(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-
B
DNA complexes. Nuclear extracts prepared from control or
insulin-stimulated (6 h, 10
M) CHO-R cells
were assayed for NF-
B activity either in the absence (lanes 1 and 2) or presence of unlabeled oligonucleotides
containing the NF-
B (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-
B-dependent Luciferase Reporter
Gene Expression
To determine whether insulin enhances
NF-
B-driven reporter gene expression, CHO-R cells were transfected
transiently with (Ig
)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
chain enhancer
B site(24) . When transiently
transfected CHO-R cells were treated for 12 h with 10
M 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-
B-binding sites being specifically activated by insulin
treatment. Together, these experiments support the conclusion that
insulin stimulation of NF-
B DNA binding activity in CHO-R cells (Fig. 1) results in activation of NF-
B-mediated gene
transcription (Fig. 4B).
Figure 4:
Insulin induction of NF-
B-mediated
luciferase reporter gene activity in CHO-R cells. CHO-R cells were
co-transfected by the CaPO
method with 5 µg of either
control (conaluc A) or test ((Ig
)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
10
M 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-
B-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-
B Requires Insulin Receptor
Tyrosine Kinase Activity
To investigate the role of the insulin
receptor tyrosine kinase in the activation of NF-
B 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 10
M insulin for various
time periods (0-6 h) or with graded concentrations
(0-10
M) of insulin for 6 h were
tested for NF-
B 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-
B 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-
B 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-
B 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-
B in CHO-R cells is given as a control.
This is a representative experiment independently performed three
times.
Insulin Activation of NF-
B Involves a
Post-translational Mechanism
To approach the mechanism whereby
insulin induced NF-
B in CHO-R cells, nuclear extracts were
prepared from CHO-R cells which had been treated for 6 h with
10
M 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-
B
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 I
B synthesis, as has been suggested by
others(5, 8, 26) . Most importantly, we
observed that activation of NF-
B 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-
B activation by insulin
occurred within the first hour of treatment, argue for a
post-translational effect of insulin on NF-
B activity. This may
proceed from the ability of the hormone to promote dissociation of the
NF-
B
I
B complex.
Figure 6:
Effect of cycloheximide and inhibitors of
NF-
B
I
B complex dissociation on NF-
B 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 10
M 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 I
B from
NF-
B(5) ; and 2) aspirin and sodium salicylate, two drugs
interfering with a pathway that leads to I
B phosphorylation and/or
degradation or both(6) . As shown in Fig. 6B,
PDTC (0.1 mM) completely abolished maximal insulin activation
of NF-
B, 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-
M). PDTC was
reported to cause similar inhibition in Jurkat cells exposed to various
NF-
B activators including lipopolysaccharide, tumor necrosis
factor-
, 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-
B in CHO-R cells. In accordance with these results, sodium
salicylate and aspirin were recently shown to inhibit activation of
NF-
B by lipopolysaccharide, tumor necrosis factor-
, 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 I
B degradation
and/or NF-
B
I
B complex dissociation.
Insulin Activation of NF-
B Appears to be Independent
of PMA-sensitive and PMA-insensitive PKC Isoforms
Because PKC is
a known inducer of NF-
B (2, 3, 4) and
PMA activates NF-
B in CHO cells (Fig. 2), we next examined
PKC involvement in insulin activation of NF-
B. We thus tested the
effect of insulin (6 h, 10
M) on NF-
B
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
[
H]PDBu to whole cells(12) . As shown in Fig. 7A, the activation of NF-
B by insulin was
similar in untreated and PMA-treated CHO-R cells, indicating that
insulin signaling of NF-
B 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 I
B in vitro(28) and also to be involved in
insulin-induced maturation of Xenopus oocytes, a Ras-mediated process
associated with NF-
B 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-
B activity in CHO-R cells, we next
examined whether insulin was able to increase PKC-
activity under
conditions where it activated NF-
B 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
cells) was far lower than that measured in
Caco-2 cells (3.5 nmol of
P transferred to PKC-
peptide/10
cells). Insulin (10
M) 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
10
M, 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
10
M) 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-
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-
B 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-
B 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 10
M insulin were
assayed for NF-
B 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
-actin
mRNA was evaluated in these assays as a control. Ethidium bromide
staining of PKC-
and
-actin bands is given in the bottom
panel.
Insulin Activation of NF-
B 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-
B
by dissociating the NF-
B
I
B complex. Moreover, several
papers provided evidence that, in mammalian cells, the Ras-Raf-1
pathway mediated the activation of NF-
B 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-
B 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, 10
M)
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-
B activation by insulin. Fig. 8shows that 8-bromo-cAMP (0.25 mM) did not
increase the DNA binding activity of NF-
B, 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-
B 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-
B 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-
B 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-
B DNA binding activity. Such findings argue for the notion that
Raf-1 kinase mediates the activation of NF-
B by insulin in CHO-R
cells.
Figure 8:
Effect of 8-bromo-cAMP on NF-
B
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
10
M 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-
B 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-
B-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 (Ig
)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
B enhancer activity: the significant insulin-induced increase (3.2
± 0.5-fold) in normalized luciferase activity measured in CHO-R
cells transfected with (Ig
)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-
B-mediated gene expression in CHO-R cells involves Raf-1
kinase.The question of whether insulin activation of NF-
B 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-
B 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-
B 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-
B 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-
B in mammalian cells. This process involves a
post-translational mechanism requiring both insulin receptor tyrosine
kinase and Raf-1 kinase activities.