(Received for publication, July 13, 1995; and in revised form, August 24, 1995)
From the
Insulin's effects primarily are initiated by insulin
binding to its plasma membrane receptor and the sequential tyrosine
phosphorylation of the insulin receptor and intracellular substrates,
such as insulin receptor substrate-1 (IRS-1). However, studies suggest
some insulin effects, including those at the nucleus, may not be
regulated by this pathway. The present study compared the levels of
insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation,
and phosphatidylinositol 3`-kinase activity to immediate early gene
c-fos and egr-1 mRNA expression in Chinese hamster
ovary (CHO) cells expressing only neomycin-resistant plasmid
(CHO), overexpressing wild type human insulin receptor
(CHO
) or ATP binding site-mutated insulin receptors
(CHO
). Insulin binding in CHO
cells was
markedly lower than that in other cell types. 10 nM insulin
significantly increased tyrosine phosphorylation of insulin receptor
and IRS-1 in CHO
cells. Phosphorylation of insulin
receptor and IRS-1 in CHO
and CHO
cells
was not detected in the presence or absence of insulin. Similarly,
insulin increased phosphatidylinositol 3-kinase activity only in
CHO
cells. As determined by Northern blot, nuclear
run-on analysis, and in situ hybridization, insulin induced
c-fos mRNA expression, through transcription, in CHO
cells but not in CHO
and CHO
cells, consistent with previous reports. In contrast, all three cell
types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that
insulin-induced egr-1 mRNA expression did not correlate with
the levels of insulin binding to insulin receptor or phosphorylation of
insulin receptor and IRS-1. These results suggest that different
mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin
receptor tyrosine kinase pathway and the latter by a yet to be
determined alternative signal transduction pathway.
Insulin's effects primarily are initiated by insulin
binding to its plasma membrane receptor and the sequential tyrosine
phosphorylation of the insulin receptor and intracellular substrates,
such as insulin receptor substrate-1 (IRS-1), ()IRS-2, or
Shc (reviewed in (1) ). These substrates bind to Src homology 2
domains of several cytoplasmic signal proteins through their tyrosine
phosphorylation sites. These proteins include the 85-kDa subunit of
phosphatidylinositol (PI) 3`-kinase, GRB-2, or Syp (tyrosine
phosphatase)(1) . Activation of these molecules and the
following activation of other intracellular molecules, such as
p21
, raf-1, mitogen-activated protein
kinase, or S6 kinase is believed to be responsible for many of
insulin's biological responses.
It is well known that insulin
affects nuclear events such as gene expression and cell growth
(reviewed in (2) ). One of insulin's effects on nuclear
events is the stimulation or inhibition of a number of genes,
specifically immediate early genes(3, 4) . The
immediate early genes are a large and diverse group, and the mechanisms
involved in their regulation are complex. The induction of c-fos transcription, one of the well-characterized immediate early
genes, by insulin or other growth factors is believed to require
receptor phosphorylation and p21 activation. For
instance, insulin induced c-fos mRNA accumulation in Chinese
hamster ovary (CHO) cells overexpressing human insulin receptor but not
in their parent cells(5) . Inhibition of p21
activity by dominant inhibitory mutants suppressed
insulin-induced activation of the c-fos promoter(6) .
However, Mundschau et al.(7) have shown that
induction of expression of the immediate early gene egr-1, but
not c-fos, c-myc, and JE, was independent of
platelet-derived growth factor receptor autophosphorylation using three
different conditions in which platelet-derived growth factor receptor
autophosphorylation was blocked. In addition, Eldredge et al.(8) reported that epidermal growth factor (EGF) induced
c-fos expression in the cells expressing kinase-deficient EGF
receptors. These results indicate the existence of another signaling
mechanism, which operates independently of growth factor receptor
tyrosine kinase activity and affects some, but not all, nuclear
responses to growth factor stimulation.
In the present study, we
tested the possibility of the existence of divergent pathways in
insulin signal transduction mechanisms regulating immediate early gene
expression. We utilized CHO cells stably transfected with only
neomycin-resistant plasmid (CHO), with genes for wild
type human insulin receptors (CHO
), or with ATP binding
site-mutated human insulin receptors in which alanine was substituted
for lysine at 1018 (CHO
) and examined the relationship
between the levels of insulin binding, insulin receptor and IRS-1
phosphorylation, and PI 3-kinase activity and immediate early gene
induction. The phosphorylation of insulin receptor and IRS-1 or the
activation of PI 3-kinase was found only in CHO
cells.
Induction of the c-fos gene required phosphorylation of
insulin receptor and IRS-1 as previously
reported(5, 9) . However, surprisingly,
insulin-induced egr-1 gene expression was observed in
CHO
and receptor tyrosine kinase negative cells to the
same extent as in CHO
cells as measured by Northern
blot, nuclear run-on, and in situ hybridization. The
expression levels stimulated by insulin were similar to the maximum
levels stimulated by serum, and similar dose curves were found in all
three cells. These findings suggest that insulin activates an
alternative or compensatory signal transduction pathway that is
independent of the receptor kinase and IRS-1 phosphorylation pathways.
Biotin-labeled probes for egr-1 (oligonucleotide) and c-fos (cDNA) were prepared as
described previously (17) . The thin sections were preincubated
for 15 min at 37 °C with 0.2 mg/ml proteinase K in 20 mM Tris-HCl, pH 7.4, 2 mM CaCl and washed in
water with 0.1% diethyl pyrocarbonate before being transferred to
2-µl drops of hybridization buffer (50% formamide, 10% dextran
sulfate, 0.8 mg/ml salmon sperm DNA, 0.8 mg/ml tRNA, and 2 µl of
biotin-labeled probe in a final volume of 50 µl of 2
SSC).
The sections were hybridized for 18-72 h at 37 °C. The
sections were washed once with 2
SSC at 37 °C, once with 1
SSC, and twice with PBS at room temperature. The sections were
then incubated for 60 min at room temperature with 1% ovalbumin, 0.2%
cold water fish skin gelatin, 0.02% Tween 20 in PBS. The sections were
incubated overnight at 4 °C with a 1:50 dilution of anti-biotin
antibody. The sections were washed in 10 mM Tris-HCl, pH 7.4,
in PBS and incubated for 60 min at room temperature with gold-labeled
protein A. The sections were washed and stained with 2% aqueous uranyl
acetate and examined in a JEOL 100 CX electron microscope.
Figure 1:
Effect of insulin on tyrosine
phosphorylation of IR and IRS-1 in CHO cell clones. CHO,
CHO
, and CHO
cells were incubated with
(+ lanes) or without (- lanes) 10 nM insulin for 1 min, and phosphotyrosine-containing proteins were
immunoprecipitated and subjected to SDS-polyacrylamide gel
electrophoresis and Western blot as described under ``Experimental
Procedures.'' IR, insulin receptor
-subunit; IRS-1, insulin receptor
substrate-1.
PI 3-kinase activity, one
of the well-known downstream effectors of insulin action, was measured
in all three cell types to determine if the IRS-1 pathway was activated
by insulin. Anti-PI 3-kinase antibody-associated PI 3-kinase activity
was measured in the cells treated with or without 17 nM insulin for 1 or 5 min. 17 nM insulin treatment for 5 min
increased the activity by 2-fold (4.2 ± 0.8 fmol/sample in
control, 8.9 ± 0.2 fmol/sample in insulin, p < 0.05)
in CHO cells but did not change significantly in
CHO
(6.5 ± 0.9 fmol/sample in control, 4.5
± 1.5 fmol/sample in insulin) or CHO
cells (7.1
± 2.0 fmol/sample in control, 6.8 ± 1.3 fmol/sample in
insulin). A similar 2-fold increase was observed with 1 min of insulin
treatment in CHO
cells but not in the other cell types.
These results confirmed CHO
cells had
phosphorylation-competent insulin receptors that phosphorylated one of
their major substrates, IRS-1, and activated PI 3-kinase, whereas
CHO
cells had insulin receptors that could not be
phosphorylated and could not activate their downstream substrates. In
CHO
cells, the number of insulin receptors was so low
that neither the phosphorylation of insulin receptor and IRS-1 nor the
activation of PI 3-kinase was detected. The reason that CHO
cells had even less phosphorylation (e.g. IGF-1
receptor) than CHO
cells might be the dominant negative
inhibition of native receptors by mutant receptors(18) .
Figure 2:
Effect of insulin or serum on immediate
early gene expression in CHO cell clones by Northern blot analysis.
CHO, CHO
, and CHO
cells
were incubated with no addition (C), 17 nM insulin (I), or 20% fetal bovine serum (S) for 60 min at 37
°C, and total RNA was isolated. 15 µg of RNA was subjected to
Northern blot with
P-labeled cDNA probe of c-fos, egr-1, and
-tubulin as described under
``Experimental Procedures.'' Similar results were obtained in
three other individual experiments.
Figure 3:
Insulin dose-dependent egr-1 mRNA
expression in CHO cell clones. CHO (
),
CHO
(
), and CHO
(
) cells
were incubated with 0-100 nM insulin for 60 min at 37
°C, and total RNA was isolated. 15 µg of RNA was subjected to
Northern blot with
P-labeled cDNA probe of egr-1 and
-tubulin as described under ``Experimental
Procedures'' and analyzed on a PhosphorImager using the ImageQuant
software (Molecular Dynamics). The quantitative data of egr-1 was standardized, divided by
-tubulin density, and expressed
as a ratio to control samples. The numbers are means of the four
individual experiments.
Figure 4:
Effect of insulin or serum on immediate
early gene transcription in CHO cell clones by nuclear run-on analysis.
CHO, CHO
, and CHO
cells
were incubated with no addition (C), 100 nM insulin (I), or 20% fetal bovine serum (S) for 25 min at 37
°C. The nuclei were isolated, and nuclear run-on analysis was
performed as described under ``Experimental
Procedures.''
We
next examined the effect of different doses of insulin on gene
expression. The level of egr-1 mRNA was quantified by
PhosphorImager and ImageQuant software, standardized by dividing by the
level of -tubulin mRNA, an insulin-insensitive gene, and expressed
as the ratio to the control as shown in Fig. 3. egr-1 mRNA showed a similar insulin dose-dependent increase in all three
cell types. The response in CHO
cells was not more
sensitive than other cell types. The fact that the lowest concentration
of insulin (1 nM) increased egr-1 mRNA level in all
three cell types suggests that the stimulation through IGF-1 receptor,
which has 100 times lower affinity for insulin than the insulin
receptor ((19) , and see ``Discussion''), is not
likely. In contrast, 1 nM insulin increased c-fos mRNA in CHO
cells but not in CHO
and
CHO
cells (data not shown).
Figure 5:
Effect of insulin on c-fos mRNA
expression in CHO cell clones visualized by in situ hybridization. CHO (A and B),
CHO
(C and D), and
CHO
(E and F) cells were incubated
with (B, D, and F) or without (A, C, and E) 17 nM insulin for 30 min. The
cells were fixed, embedded, and sectioned for in situ hybridization with biotinylated probes for c-fos as
described under ``Experimental Procedures.'' Magnification,
22,000; n, nucleus.
Figure 6:
Effect of insulin on egr-1 mRNA
expression in CHO cell clones visualized by in situ hybridization. CHO (A and B),
CHO
(C and D), CHO
(E and F) cells were incubated with (B, D, and F) or without (A, C, and E) 17 nM insulin for 30 min. The cells were fixed,
embedded, and sectioned for in situ hybridization with
biotinylated probes for egr-1 as described under
``Experimental Procedures.'' Magnification,
22,000; n, nucleus.
In the present study, we have found that insulin stimulation
of c-fos mRNA transcription occurs only in CHO cells but not in CHO
or CHO
cells,
suggesting that phosphorylation of the insulin receptor and IRS-1 and
its subsequent signaling cascade are necessary. On the other hand,
insulin stimulates egr-1 mRNA transcription to a similar level
of maximum stimulation by serum in all three CHO cell types, including
the cells expressing tyrosine kinase-defective insulin receptor. These
findings suggest that divergent pathways are involved in signal
transduction mechanisms in which insulin affects c-fos and egr-1 expression. The increase of egr-1 mRNA levels
in nuclear run-on analysis and the increase of gold-labeled egr-1 in the nucleus in in situ hybridization suggest that the
increase of egr-1 mRNA induced by insulin is, to a major
degree, through an increase at the transcriptional level.
Insulin-induced c-fos expression levels are low compared with
serum-induced c-fos expression, even in CHO
cells. This finding may be attributable to the fact that the IRS-1
phosphorylation level is low in CHO
cells, assuming
phosphorylation of insulin receptor and IRS-1 is essential for
insulininduced c-fos expression. In fact, a recent study
showed that transfection of IRS-1 increased the response of c-fos to insulin or IGF-1 in CHO cells(20) . In contrast to
c-fos, insulin increased the egr-1 expression levels
to the same level as serum. This difference also suggests the
regulation of c-fos and egr-1 expression by insulin
is using different mechanisms. This hypothesis was supported further by
the finding that PI 3-kinase was activated by insulin only in
CHO
cells, consistent with IR and IRS-1 phosphorylation.
One might argue that only a small, even undetectable, amount of IR
and IRS-1 phosphorylation is enough to cause a downstream signaling
cascade and account for the dose-dependent and submaximal to maximal
stimulation of egr-1 expression in CHO or
CHO
cells. Even if that is true, one must conclude
that the overexpression of the insulin receptor in the CHO
cells and the increase in insulin receptor
-subunit and
IRS-1 phosphorylation did not change egr-1 transcription
compared with the endogenous levels of insulin receptor in the
CHO
cells but had a marked effect on PI 3-kinase activity
and c-fos transcription. In addition, kinase-negative insulin
receptors in CHO
cells form hybrid heterotetrameric
receptors between endogenous insulin receptors and the mutant receptor
that may inhibit phosphorylation of endogenous receptors (dominant
negative inhibition, reviewed in (18) ). Therefore, there is
virtually no receptor tyrosine phosphorylation in CHO
cells. Some investigators(21, 22) reported that
insulin failed to activate IRS-1, Shc, Ras, and mitogen-activated
protein kinase in CHO cells expressing ATP binding site mutant insulin
receptor (CHO
cells). Regulation of c-fos expression followed this activation pattern, suggesting that
c-fos is downstream of these molecules. However, we believe
the virtually identical and insulin concentration-dependent egr-1 mRNA response in the three cell types demonstrates that
insulin's stimulation of egr-1 gene transcription in CHO
cells is independent of the level of insulin receptor and IRS-1
phosphorylation. We do not believe that insulin binding to IGF-1
receptors in the CHO clones explains the similar effects of insulin on egr-1 expression. Insulin-induced phosphorylation of IGF-1
receptors was only observed in CHO
cells, and even then
no IRS-1 phosphorylation was detected. It might be possible that high
insulin concentrations could maximally stimulate egr-1 expression in CHO
cells by binding to endogenous
IGF-1 receptors, thus masking the effects of the transfected insulin
receptor. However, the dose response curve shown in Fig. 3demonstrates that at insulin concentrations resulting in
submaximal stimulation of egr-1 expression the wild type
insulin receptor had no effect on insulin-induced egr-1.
Whether or not insulin occupancy of IGF-1 receptors activates egr-1 transcription, the data in Fig. 3indicate that wild type
insulin receptors did not increase the insulin sensitivity of the
CHO
cells.
Our results are different from those of Stumpo et al.(23) and Jhun et al.(24) using Rat 1 fibroblasts expressing high levels of normal or mutated human insulin receptors. They found that insulin did not increase c-fos and egr-1 mRNA accumulation in Rat 1 fibroblasts expressing tyrosine kinase-defective insulin receptors. The reasons for these differences are not clear. However, it is possible that different cell types have different signaling pathways and that the response may not be always the same. Wong et al.(25) demonstrated that final insulin responsiveness was strongly dependent on the stage of cell growth, and Rat 1 fibroblast cells with kinase-deficient insulin receptors (A1018K) have similar biological responsiveness to insulin if growth and incubation conditions are optimized. The difference in these conditions may also account for the different results we observed.
The first step of the
signaling pathway of various growth factors, including insulin, is
believed to be the binding of growth factors to its specific cell
surface receptor and receptor autophosphorylation. In the case of
insulin, tyrosine phosphorylation of the insulin receptor causes
sequential phosphorylation of intracellular substrates, such as IRS-1,
IRS-2, or Shc (reviewed in (1) ). These substrates bind and
activate several cytoplasmic signal proteins, such as the 85-kDa
subunit of PI 3-kinase or GRB-2 through their Src homology 2 binding
sites. Activation of these molecules and the following activation of
intracellular molecules, such as p21, raf-1,
mitogenactivated protein kinase, or S6 kinase are believed to be
responsible for many, if not all, of insulin's biological
responses. However, the requirement of growth factor receptor
phosphorylation for nuclear events, such as DNA synthesis or gene
transcription, is still controversial. Recent observations demonstrated
that platelet-derived growth factor-induced egr-1 expression (7) or EGF-induced c-fos expression (8) was
independent of its receptor tyrosine phosphorylation. These results
suggest the existence of another signaling mechanism capable of gene
induction that operates independently of platelet-derived growth factor
or EGF receptor tyrosine kinase activity.
The mechanisms involved in
these tyrosine kinase-independent pathways are not yet clear. One
possibility is that the cells with mutant insulin receptors could
utilize several compensatory mechanisms to overcome the lack of
autophosphorylation. Insulin receptors, even without tyrosine
phosphorylation, could interact with intracellular proteins and cause a
signal transduction cascade to induce egr-1 expression. This
hypothesis is supported by the data showing that
autophosphorylationdefective EGF receptors can tyrosine-phosphorylate
Shc, which then serves as a binding protein site for GRB-2/Sos, leading
to activation of the Ras signaling pathway and
mitogenesis(26) . We observed that tyrosine phosphorylation of
a 120-kDa protein (pp120) was increased by insulin more in CHO and CHO
cells than in CHO
cells.
The basal phosphorylation level was lower as well in the CHO
cells. The increase of tyrosine phosphorylation of this protein
by insulin suggests that an insulin-sensitive tyrosine phosphorylation
pathway exists in CHO
and CHO
cells,
which is independent of IRS-1 phosphorylation. This band at 120-kDa
seems to be made up of heterogeneous proteins such as focal adhesion
kinase(27) , ecto-ATPase (28) , Syk- or phospholipase
C
-associated pp120(29) , or RasGAP(30) . Recent
observations showed that insulin increased tyrosine phosphorylation of
protooncogene cbl (120 kDa) (31) or Syp (tyrosine
phosphatase)-associated protein pp115(32) . Their role in
insulin signaling is unclear. However, our observations raise the
possibility that phosphorylation of the pp120 in CHO
and
CHO
cells might be involved in a compensatory signal
transduction pathway.
Another possible explanation for insulin-stimulated egr-1 expression in the CHO cells is the involvement of internalized insulin. Some studies suggested that the translocation of growth factors or hormones to the nucleus is essential for mitogenesis. It has been reported that various hormones and growth factors, e.g. EGF (33) , aFGF(34) , bFGF(35) , interleukin-1(36) , prolactin(37) , nerve growth factor(38) , IGF-1(39) , or growth hormone (40) internalize and translocate to the nucleus (reviewed in (41, 42, 43) ). The studies with nuclear localization sequence mutants of aFGF (44) or prolactin (37) showed that the nuclear translocation of these hormones or growth factors was important for DNA synthesis or cell proliferation. A recent study on aFGF (34) demonstrated two, i.e. receptor and nuclear, mechanisms of signal transduction. Prolactin signaling in T lymphocytes appears to utilize a classical receptor-mediated kinase cascade and a novel peptide hormone activation pathway involving nuclear translocation(45) . Insulin's signaling mechanisms may be similar. We have demonstrated translocation of insulin to the nucleus in several rapidly proliferating cell types(46, 47, 48, 49) . Microinjection of insulin into the cytoplasm of Xenopus oocytes increased RNA and protein synthesis(50) . Insulin failed to stimulate growth in the PG19 mouse melanoma cells, which had internalization-defective, but kinase-competent insulin receptors(51) . When added to isolated nuclei, insulin affected various nuclear processes, such as nucleo-cytoplasmic transport of macromolecules(52) , protein phosphorylation(53) , enzymatic activities(54) , and mRNA release from nuclei(55) . Recently, we demonstrated that trypsin treatment, which resulted in undetectable insulin binding to the insulin receptor and phosphorylation of insulin receptor and IRS-1(56) , did not change insulin's ability to stimulate immediate early gene transcription in H35 rat hepatoma cells(57) . Taken together, these studies suggest that the internalization of insulin, and possibly its direct interaction with the cell nucleus, may be an important mechanism by which insulin regulates nuclear events.
In summary, we
have demonstrated that insulin-induced egr-1 mRNA expression
is independent of the tyrosine phosphorylation of insulin receptor and
IRS-1 in CHO cells overexpressing wild type or kinase-defective human
insulin receptor as well as in CHO cells. This result is
in stark contrast to insulin-induction of c-fos mRNA
expression and activation of PI 3-kinase, which require insulin binding
to its receptors and the tyrosine phosphorylation of insulin receptor
and IRS-1. These findings suggest that different mechanisms are
involved in regulating expression of some immediate early genes.
Further observations are necessary to characterize insulin receptor and
IRS-1 tyrosine phosphorylation-independent pathways. Differences in
pp120 phosphorylation between CHO
cells and the other
two CHO cell types may indicate that compensatory signaling pathways
exist in cells expressing low numbers of, or kinase-defective, insulin
receptor.