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J. Biol. Chem., Vol. 277, Issue 45, 42480-42487, November 8, 2002
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From the University of Cambridge, Department of Clinical
Biochemistry, Addenbrooke's Hospital, Hills Road,
Cambridge CB2 2QR, United Kingdom, the
Received for publication, June 21, 2002, and in revised form, August 27, 2002
Insulin and insulin-like growth factor-1 (IGF-1)
act through highly homologous receptors that engage similar
intracellular signaling pathways, yet these hormones serve largely
distinct physiological roles in the control of metabolism and growth,
respectively. In an attempt to uncover the molecular mechanisms
underlying their divergent functions, we compared insulin receptor (IR)
and IGF-1 receptor (IGF-1R) regulation of gene expression by microarray analysis, using 3T3-L1 cells expressing either TrkC/IR or TrkC/IGF-1R chimeric receptors to ensure the highly selective activation of each
receptor tyrosine kinase. Following stimulation of the chimeric receptors for 4 h, we detected 11 genes to be differentially
regulated, of which 10 were up-regulated to a greater extent by the
IGF-1R. These included genes involved in adhesion, transcription,
transport, and proliferation. The expression of mRNA encoding
heparin-binding epidermal growth factor-like growth factor (HB-EGF), a
potent mitogen, was markedly increased by IGF-1R but not IR activation. This effect was dependent on MAPK, but not phosphatidylinositol 3-kinase, and did not require an autocrine loop through the epidermal growth factor receptor. HB-EGF mitogenic activity was detectable in the
medium of 3T3-L1 preadipocytes expressing activated IGF-1R but not IR,
indicating that the transcriptional response is accompanied by a
parallel increase in mature HB-EGF protein. The differential abilities
of the IR and IGF-1R tyrosine kinases to stimulate the synthesis and
release of a growth factor may provide, at least in part, an
explanation for the greater role of the IGF-1R in the control of
cellular proliferation.
The insulin receptor
(IR)1 and the IGF-1 receptor
(IGF-1R) are highly homologous, both being class II receptor tyrosine
kinases with a disulfide-linked tetrameric structure. Both use similar signaling mechanisms, but in vivo the IGF-1R is associated
with a more "mitogenic" response, whereas the IR is associated with a more "metabolic" response. This is graphically illustrated by the
fact that mice in which the IR has been deleted transgenically are born
of normal size but develop severe diabetes and fatal ketoacidosis
shortly after birth (1, 2), whereas IGF-1R knockout mice have marked
intrauterine growth retardation (3).
Whereas tissue-specific expression could contribute to the differential
biological functions of these receptors, both receptors actually show
quite widespread expression, and thus it appears likely that at least
some of the in vivo specificity relates to differential
signaling properties of the receptors. Most previous studies examining
this issue have compared cells overexpressing the IR with those
expressing the IGF-1R or have used IR/IGF-1R chimeras. The majority of
these studies indicate that activation of the IGF-1R is more mitogenic
than the IR (4-6), although others (7) have failed to demonstrate such
differences. In addition, glycogen synthesis has been shown to be
coupled more strongly to the IR than the IGF-1R (6, 8). However, there
are several problems associated with the use of these systems,
including the formation of hybrid IR/IGF-1Rs and cross-reactivity
(ligand binding to non-cognate endogenous receptors, which is likely to
occur at the concentrations usually used in ex vivo
experiments), all of which may obscure any signaling specificity.
In an attempt to overcome these problems, we have generated TrkC
receptor chimeras, consisting of the extracellular domain of the TrkC
receptor fused to the intracellular domain of the IR or the IGF-1R (9).
Expression of these receptors in cells without endogenous TrkC
receptors allows specific stimulation of the receptors by
neurotrophin-3 (NT-3) without any background signaling, hybrid
formation, or non-cognate ligand binding. As the extracellular domains
of the two chimeras are identical, any signaling differences observed
are purely due to differences in the intracellular domains of the IR
versus IGF-1R. In 3T3-L1 preadipocytes stably expressing
these chimeras, we have reported previously (9) that the intracellular
domain of the IR couples more effectively to glycogen synthesis than
the intracellular domain of the IGF-1R, whereas both appear to couple
equally to DNA synthesis. The two receptors also appear to be equally
efficient in protecting fibroblasts and adipocytes against apoptosis
(10). In 3T3-L1 adipocytes, the IR chimera (TIR) stimulated GLUT4
translocation and glucose uptake to a greater extent than the IGF-1R
chimera (TIGR). Differences were also seen in signaling immediately
downstream of the receptors in adipocytes, with the IRS-1 signaling
pathway being more effectively stimulated by the TIR chimera, and the
Shc/MAPK signaling pathway being more stimulated by the TIGR chimera
(11). Thus, it seems that intrinsic signaling differences between the
two receptors do exist and are at least in part attributable to
sequence differences in the intracellular domains of the IR and
IGF-1R.
In order to elucidate further the reasons for the different effects of
insulin and IGF-1, we chose to compare the regulation of gene
transcription by the two chimeric receptors in 3T3-L1 preadipocytes,
using microarray technology to gain a global perspective on
transcriptional regulation. Overall, 3T3-L1 TIGR cells showed a greater
transcriptional response than TIR cells. Of the differentially regulated mRNAs, the potent mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) was shown to be highly selectively up-regulated by TIGR chimeras. The basis for this selective
effect on HB-EGF mRNA was explored further, because it was thought
to represent an excellent candidate for the preferential mitogenic
effects of the IGF-1R.
Materials--
Insulin (Actrapid) was supplied by Novo-Nordisk,
and IGF-1 was supplied by GroPep. NT-3 was a generous gift from
Regeneron Pharmaceuticals, and HB-EGF was supplied by Sigma. The
inhibitors PD153035, PD98059, and wortmannin were all from Calbiochem.
Cycloheximide and actinomycin-D were from Sigma. Anti-phosphotyrosine
antibody 4G10 was purchased from Upstate Biotechnology, Inc., and
anti-active MAPK antibody (specific for dual-phosphorylated MAPK) and
anti-Erk 1/2 antibody (which recognizes non-phosphorylated and
phosphorylated forms) were purchased from Promega. Unless indicated
otherwise, all cell culture and general purpose laboratory reagents
were supplied by Sigma.
Cell Culture--
3T3-L1 TIR/TIGR preadipocytes were maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
(v/v) newborn calf serum, 50 units/ml penicillin/streptomycin, and 2 mM glutamine. NIH-3T3 IR/IGF-1R cells and HaCat cells (12)
were maintained in DMEM supplemented with 10% fetal calf serum, 50 units/ml penicillin/streptomycin, and 2 mM glutamine.
Microarray Analysis--
Confluent 3T3-L1 TIR/TIGR cells were
serum-starved overnight and then stimulated with 4 nM NT-3
for 4 h. Total RNA from stimulated and unstimulated cells was
extracted using the RNeasy mini kit (Qiagen). This RNA was then
processed according to the protocol recommended by Affymetrix, using
the Superscript Choice kit (Invitrogen) for double-stranded cDNA
synthesis and the Enzo Bioarray kit for in vitro
transcription and labeling of cRNA. 15-µg samples of fragmented cRNA
were hybridized for 16 h at 45 °C to MG-U74A mouse arrays
(Affymetrix). Analysis of the scanned chips was carried out using
Affymetrix Microarray Suite version 4.0, using default settings and a
target intensity of 100 for each chip as a whole. Further analysis
(sorting into groups) was carried out using a relational database
program based on Filemaker Pro 5, developed for microarray analysis by
G. Denyer. The program allowed the formation of clusters based on both
data from the chips (mRNA intensity, fold change, etc.) and on
known functionality (e.g. pathways, etc.). The clusters were
combined in multiple comparison statements (AND/OR/NOT), and genes of
interest were followed up by break out to web databases
(e.g. SwissProt, BLAST, etc.) for the collection of sequence
and functional information.
In order to find transcripts expressed at a similar level in two chips,
we first eliminated all transcripts that were classified by the
Affymetrix software as increasing to or decreasing from a detectable
signal. We also eliminated all those sequences that were more than
1.5-fold different between the two chips, unless they were undetectable
in both. To find transcripts that were different between two chips, we
selected for mRNAs that were classified as increasing to or
decreasing from a detectable signal, with a difference of more than
2-fold. Transcripts were only included in the final lists if they
fulfilled these selection criteria in two independent microarray experiments.
Northern Blotting--
Confluent cells were serum-starved and
treated as indicated in the text. Total RNA was extracted using the
RNeasy mini-kit from Qiagen and quantified by GeneQuant (Amersham
Biosciences). Equal amounts of RNA were resolved by formaldehyde gel
electrophoresis in MOPS buffer before transfer and cross-linking to
Nytran N membrane (Schleicher & Schuell). Membranes were hybridized to
HB-EGF probe in Quickhyb solution (Stratagene) for 3-4 h at 65 °C.
Membranes were then washed in 2× SSC, 0.1% SDS (2 times for 15 min),
followed by a final wash in 0.1× SSC, 0.1% SDS for 30 min (all at
65 °C), and subsequent visualization using a PhosphorImager (Fuji).
HB-EGF probe was made by reverse transcriptase-PCR from a sample of
total RNA extracted from 3T3-L1 TIGR cells stimulated with NT-3 for
4 h. The PCR product was gel-purified (QIAquick, Qiagen) and
labeled with 32P using Ready-To-Go oligolabeling beads
(Amersham Biosciences). The labeled probe was purified using Nick
columns (Amersham Biosciences), and ~10,000 cpm were used for a
single hybridization. The probe was sequenced to verify its identity.
Western Blotting--
Confluent cells were serum-starved
overnight, before being treated as indicated in the text. They were
then rinsed twice with ice-cold phosphate-buffered saline (PBS) and
lysed in lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM
Na3VO4, 30 mM NaF, 10 mM Na4P2O7, 2.5 mM benzamidine, 1 µg/ml pepstatin A, 1 µg/ml leupeptin,
1 µg/ml antipain, 0.5 mM phenylmethylsulfonyl fluoride,
and 1% Triton X-100). Cleared lysates were assayed for total protein
concentration (Bio-Rad Dc kit), and equal amounts of total protein were
resolved by SDS-PAGE (using 8% minigels) and transferred by
electroblotting to polyvinylidene difluoride membrane (Millipore). The
membrane was then placed in blocking buffer (PBS, 0.05% Tween 20, 1%
bovine serum albumin) for 1 h at room temperature, followed by
incubation with primary antibody overnight at 4 °C. After extensive
washing in PBS, 0.05% Tween 20, detection was carried out with
horseradish peroxidase-conjugated anti-mouse/rabbit antibody (Dako) and
ECL Western blotting detection reagents (Amersham Biosciences).
Thymidine Incorporation Assay--
HaCat cells were seeded into
12-well plates at equal density and allowed to grow to ~80%
confluency. They were then serum-starved for 24 h, followed by
treatment for 16 h as described in the text. Culture medium was
then replaced with serum-free medium containing 1 µCi/well
[3H]thymidine for 4 h (37 °C), followed by two
washes in ice-cold PBS and precipitation at 4 °C for 30 min in 10%
trichloroacetic acid. Cells were then washed once in 5%
trichloroacetic acid and solubilized first in 0.2 M NaOH
and then in 0.4 M HCl. Lysates were combined for counting.
TIGR Chimera Signaling Stimulates Transcription to a Greater Extent
Than TIR Signaling in 3T3-L1 Preadipocytes--
We first compared
transcript levels in NT-3-stimulated TIR/TIGR cells with levels of the
same transcripts in untreated cells. Only genes/ESTs that were similar
at base line in untreated TIGR and TIR cells according to our selection
criteria (see "Experimental Procedures") were considered further.
From this set of transcripts with similar base-line levels, we selected
those that were increased or decreased from base line in either cell
line after chimeric receptor stimulation. This resulted in a list of
185 transcripts that were up-regulated and 38 that were down-regulated
in either cell line. Fig. 1 shows the
fold changes from base line for each of these genes/ESTs, represented
as a scatter plot with the fold change from base line after TIGR
stimulation along the horizontal axis, and that for TIR stimulation
along the vertical axis. Simple inspection of this plot demonstrates
clearly the greater effect of TIGR compared with TIR in terms of
numbers of genes whose expression level increased and the extent of
that change. Down-regulated genes, on the other hand, did not show such
a bias. The bias toward TIGR-mediated stimulation was confirmed by
linear regression analysis of the data that yielded a gradient of 0.66.
Similar and Differential Regulation of Transcripts by TIR and TIGR
Chimeras--
As would be expected from the similarity of their
receptors, insulin and IGF-1 do have overlapping effects, so we
searched for transcripts that were similarly regulated by the two
chimeric receptors after stimulation with NT-3, using stringent
selection criteria (see "Experimental Procedures"). From a list of
transcripts expressed at similar levels in TIR versus TIGR
comparisons at base line and after stimulation of the chimeric
receptors, we then selected only those genes that were increased or
decreased from base line in TIR cells. This approach revealed 23 transcripts that were up-regulated similarly by the TIR and TIGR (Table
I, part A). Some of these molecules have
known roles in various processes including proliferation, gene
expression from transcription to translation, protein modification and
degradation, and cellular adhesion. In addition, two secreted products
(protein related to DAN and cerberus, small inducible cytokine A2) were
up-regulated by both TIR and TIGR stimulation. These could be involved
in signaling to neighboring cells, thus adding another layer of
complexity to the downstream effects of TIR/TIGR signaling. Fewer
mRNAs (13) were equally down-regulated by the two receptors, in
agreement with the general expression patterns observed. These
transcripts encoded proteins involved in metabolism, apoptosis,
signaling, and vesicular transport (Table I, part B).
We defined differentially regulated transcripts as those that were
different in a comparison of the two stimulated cell lines but
expressed at similar levels in unstimulated cells, again using stringent selection criteria. Fewer mRNAs (11) were differentially regulated compared with those similarly regulated between the two cell
lines, consistent with the broadly overlapping effects of insulin and
IGF-1 (Table II). Of the 11 differentially regulated transcripts, 1 was an EST that was suppressed
to a greater extent by the TIR. The remaining 10 were all increased to
a greater extent by TIGR activation. The differentially regulated genes
with known functions had roles in transcription, transport, adhesion,
and proliferation.
Only a fraction of the 223 genes identified as being affected by
TIR/TIGR activation were found to be equally or differentially regulated. The remainder fell outside the boundaries of our selection criteria, for example by being between 1.5- and 2-fold different in
stimulated cells. The numbers of genes observed as being similarly or
differentially regulated were small because we designed our selection
criteria with high stringency to reduce the risk of false positive
results. Representative genes from each of the three groupings (equally
up-regulated, equally down-regulated, and differentially regulated)
were examined by Northern blotting, and in each case those results
confirmed the microarray data (data not shown).
The Mitogen HB-EGF Is Selectively Up-regulated by the IGF-1R
Intracellular Domain--
One of the genes selectively up-regulated by
TIGR was the mitogen HB-EGF. We hypothesized that TIGR-specific
up-regulation of HB-EGF could account for at least some of the more
mitogenic effects of IGF-1R signaling, and we therefore chose to study
the regulation of this gene in more depth.
In order to verify the microarray data and obtain a more detailed view
of HB-EGF gene regulation in response to TIR and TIGR signaling, a time
course experiment was carried out. Confluent 3T3-L1 TIR and TIGR cells
were serum-starved for at least 4 h, followed by stimulation with
4 nM NT-3 for 0, 1, 2, 4, 8, and 24 h. RNA extracts
were Northern-blotted for HB-EGF (Fig.
2A). Three independent
experiments confirmed the microarray result, showing a stimulation of
HB-EGF gene transcription in TIGR but not TIR cells. The differences
seen were not significant until the 4-h time point and continued up to
24 h, the last time point studied.
We proceeded to determine whether the differences in HB-EGF expression
observed in the cells expressing chimeric receptors would also be seen
when insulin and IGF-1 holoreceptors were activated. NIH-3T3 cells
overexpressing IR or IGF-1R were serum-starved for at least 4 h,
followed by 4 h of stimulation with varying concentrations of
insulin or IGF-1 (for NIH-3T3 IR or IGFR cells respectively). Equal
amounts of RNA extracts were then Northern-blotted for HB-EGF. Three
independent experiments showed that although insulin did cause an
increase in HB-EGF transcription in this system, IGF-1 clearly
stimulated HB-EGF transcription to a greater extent than insulin, at
both 5 and 10 nM concentrations (Fig. 2B).
Therefore, the selective regulation of HB-EGF by chimeric receptors is
also a property of whole IR/IGF-1Rs.
In order to discern whether TIGR signaling was acting on transcription
of HB-EGF or on the stability of its mRNA, we stimulated 3T3-L1
TIGR cells with NT-3 for 4 h and then incubated with actinomycin D
(to block further RNA synthesis) with or without NT-3. HB-EGF mRNA
levels at various time points after actinomycin D treatment were
studied by Northern blotting to observe any effect of TIGR signaling on
mRNA degradation. TIGR signaling did not have any effect on
mRNA stability, with both NT-3-treated and -untreated samples
degrading to ~35% of their initial levels by 4 h (Fig. 2C). Thus it appears likely that TIGR signaling stimulates
transcription of the HB-EGF gene.
Finally, we determined whether the up-regulation of HB-EGF was a
primary transcriptional response or a secondary effect. 3T3-L1 TIGR
cells were stimulated with NT-3 for 4 h in the presence or absence
of cycloheximide. Cycloheximide did not inhibit HB-EGF mRNA
induction, and in fact caused a slight superinduction relative to NT-3
alone (Fig. 2D). Therefore, TIGR-mediated up-regulation of
HB-EGF is a primary transcriptional response that does not require
de novo protein synthesis.
The Activation of the IGF-1R but Not the IR Also Increases the
Release of Bioactive EGF Receptor Ligands from 3T3-L1
Preadipocytes--
To determine whether the selective increase in
HB-EGF mRNA was accompanied by a similarly selective release of
HB-EGF bioactivity, we examined conditioned medium from cells
expressing activated TIGR or TIR. Briefly, 3T3-L1 TIR or TIGR cells
were stimulated with NT-3 for 24 h. Conditioned medium from these
cells was used to treat HaCat cells (12), a human keratinocyte cell
line that expresses abundant EGF receptors (EGFR). TIGR-conditioned
medium caused a significant increase in MAPK phosphorylation in HaCat cells, whereas TIR-conditioned medium had no significant effect (Fig.
3, A and B). This
pattern was mirrored almost exactly at the level of proliferation in
HaCat cells, with TIGR-conditioned medium stimulating thymidine uptake
to the same extent as seen for MAPK phosphorylation, whereas
TIR-conditioned medium had no effect (Fig. 3C). In the case
of both MAPK phosphorylation and thymidine incorporation, this
stimulatory effect was completely abrogated by concomitant treatment
with the EGFR kinase inhibitor PD153035, indicating that the mitogenic
activity in the medium is due to an EGFR ligand. It is, of course,
possible that other EGFR ligands are contributing to this effect. Of
the other members of the EGF family, only epiregulin was detectable in
the microarray experiments, with EGF, transforming growth factor- TIGR Signaling to HB-EGF Transcription Requires Activation of
MAPK--
Selective inhibitors were used to elucidate the signaling
pathways leading from the TIGR to HB-EGF gene transcription.
Serum-starved 3T3-L1 TIR and TIGR cells were treated with NT-3 for
4 h, alone or in combination with wortmannin (an inhibitor of
phosphatidylinositol 3-kinase (PI3K)) or PD98059 (an inhibitor of MAPK
pathway signaling). Northern blots from three independent experiments
demonstrated that wortmannin had no effect on the TIGR-mediated
stimulation of HB-EGF transcription, whereas the MAPK pathway inhibitor
PD98059 completely abrogated the response (Fig.
4A). Therefore, TIGR signaling to HB-EGF gene transcription appears to depend on MAPK but does not
require PI3K activity.
Differences in HB-EGF mRNA Regulation Are Not Explained by
Differences in MAPK Phosphorylation--
The chimeric receptors have
been shown to signal differentially to MAPK in 3T3-L1 adipocytes (11),
so we studied MAPK phosphorylation in 3T3-L1 TIR/TIGR preadipocytes to
test the hypothesis that differential MAPK phosphorylation could be
responsible for the differential regulation of HB-EGF. We initially
verified that there were no significant differences in chimeric
receptor phosphorylation between the two cell lines (Fig.
4B). Chimeric receptor tyrosine phosphorylation was
sustained over 24 h, whereas MAPK phosphorylation was transient, decreasing almost back to basal levels by 15 min (Fig. 4C).
Activated TIR and TIGR were both capable of causing robust stimulation
of MAPK phosphorylation. Thus, although the activation of MAPK is essential for the up-regulation of HB-EGF expression, differences in
the extent or time course of MAPK activation cannot explain the
selective effect of the TIGR versus the TIR on HB-EGF gene transcription.
TIGR-mediated Up-regulation of HB-EGF Gene Transcription Does Not
Involve the EGFR--
It has been suggested previously (13) that the
activation of MAPK by the IGF-1R is completely dependent on the
cleavage and release of membrane-bound, cell surface HB-EGF with
subsequent activation of EGFRs. Additionally, HB-EGF has been shown to
up-regulate the transcription of its own gene (14). Thus it was
possible that the IGF-1R-mediated increase in HB-EGF mRNA levels
was in fact occurring via cleavage of HB-EGF already present at the
cell surface and subsequent activation of the EGFR. To examine whether the EGFR itself was critically involved in the induction of HB-EGF expression by the TIGR, we used the specific EGFR kinase inhibitor PD153035. 3T3-L1 TIR/TIGR cells were incubated for 4 h with or without NT-3, in conjunction with PD153035 or in conjunction with a
combination of PD153035 and the MAPK inhibitor PD98059. Northern blots
from three independent experiments showed that the EGFR kinase
inhibitor PD153035 had no effect on HB-EGF gene transcription, whereas
the MAPK pathway inhibitor PD98059 suppressed the response of HB-EGF to
TIGR signaling as expected (Fig. 5). This
indicates that TIGR-mediated regulation of HB-EGF gene transcription,
although requiring MAPK activation, does not require a functional
EGFR.
We chose to compare the effects of signaling from IR and IGF-1R
intracellular domains to gene transcription using a chimeric receptor
system previously developed in our group (9). The use of TIR/TIGR
chimeras avoids many of the problems associated with the comparative
study of holoreceptors, including background expression of endogenous
receptors, cross-reactivity of ligand with non-cognate receptors, and
the formation of hybrid receptors. As both chimeras are identical in
their extracellular regions and bind the same ligand, any differences
observed between the two will be entirely due to differences in the
sequence and function of their intracellular domains. If, as has been
suggested, some of the differential signaling properties of the IR and
IGF-1R relate to the residence time of the ligand on the receptor, with IGF-1R having more sustained activation (15, 16), this real biological
difference will not be observed in our system. However, we feel that
our findings of consistent and repeatable differential biological
responses using these chimeras, both herein and in previous reports
(9-11), adequately justifies their continued use for this purpose.
We observed a generally greater stimulatory effect of TIGR than TIR on
transcription using our selection criteria, and almost all of the
differentially regulated genes were up-regulated to a greater extent in
TIGR cells than in TIR cells. This is in agreement with the general
patterns observed in previous microarray work (17). Dupont et
al. (17) analyzed gene expression in NIH-3T3 cells overexpressing
IR or IGF-1R using spotted cDNA arrays, and they found 30 genes
were up-regulated more in IGF-1R cells, whereas 9 genes were
up-regulated more in IR cells. The greater response to IGF-1R kinase
signaling could be because both 3T3-L1 preadipocytes and NIH-3T3
fibroblasts are more IGF-1-responsive cell lines, in which case IR
kinase signaling would be expected to cause a greater response in a
more classically insulin-responsive system, such as adipocytes.
Alternatively, the IGF-1R kinase may intrinsically couple more to
transcriptional responses regardless of the cell system.
The set of genes revealed to be differentially regulated in our system
did not overlap at all with the results of Dupont et al.
(17). This may be due, in part, to differences in our selection criteria. We eliminated transcripts that were differentially expressed in unstimulated cells, whereas this was not the case in the study of
Dupont et al. (17). Certain genes in our experiments
followed a similar pattern to that of Dupont et al. (17) but
were just outside the borders of our selection criteria
(e.g. Glvr-1, Jun). In addition, there were differences in
the cell system and receptor constructs employed, microarrays used, and
time points studied (4 h in our study compared with 90 min chosen by
Dupont et al. (17)).
Our microarray analysis revealed more similarly regulated transcripts
than differentially regulated ones. This is in contrast to the work by
Dupont et al. (17), who found more differentially regulated
genes. However, insulin and IGF-1 do have broadly overlapping effects
in cell culture, with no all-or-nothing differences being described
thus far, so one might expect more similarities than differences
between the two in terms of transcriptional regulation. Three genes
with roles in proliferation were found to be up-regulated in a similar
manner by the TIR and TIGR. Similar induction of these genes by TIR and
TIGR is consistent with both receptors being capable of signaling to
cellular proliferation. Several factors involved in various stages of
gene expression from transcription through RNA processing and
translation to protein modification and finally protein degradation
were up-regulated, and these processes would also be important for the
correct regulation of cell growth.
Fewer genes were down-regulated equally by TIR and TIGR signaling.
Pyruvate dehydrogenase kinase (PDK4) was down-regulated by more than
4-fold. Decreasing PDK4 levels would be expected to relieve the
inhibition of pyruvate dehydrogenase, thus encouraging glucose rather
than fatty acid oxidation (18). It therefore seems logical that
insulin, which would normally indicate abundant availability of
carbohydrate in vivo, would down-regulate this gene. The
purpose of IGF-1-mediated PDK4 down-regulation, however, is unclear.
Turning now to genes preferentially stimulated by activation of
the TIGR, our data revealed two up-regulated transcripts involved in
cell adhesion. A selective regulation of the The microarray data revealed one growth factor, HB-EGF, which was
up-regulated by TIGR much more than TIR. This gene was of particular
interest to us, because selective regulation of a soluble growth factor
by the IGF-1R but not the IR could contribute toward the differential
effects the two receptors have on cell proliferation. HB-EGF was first
discovered as a 22-kDa heparin-binding growth factor secreted by
macrophage-like U-937 cells. It was identified as a new member of the
EGF family and was shown to be mitogenic for fibroblasts and smooth
muscle cells. HB-EGF binding to EGF receptors was demonstrated, and in
smooth muscle cells it appeared to be a more potent mitogen than EGF
(21).
Since its discovery, the mitogenic properties of HB-EGF have been
implicated in several processes, including wound healing, liver
regeneration, and cancer (14, 22-24). It is synthesized as a
transmembrane precursor (also known as the diphtheria toxin receptor
(25)) which is cleaved to release mature soluble HB-EGF. However, both
the transmembrane and soluble forms of HB-EGF are thought to be active
signaling molecules. Soluble HB-EGF has been proposed to act in a
paracrine fashion, whereas the transmembrane form acts in a juxtacrine
manner on neighboring cells (26).
We verified the differential regulation of HB-EGF by Northern blot in
both the original 3T3-L1 TIR/TIGR system and in NIH-3T3 cells
overexpressing whole IR/IGF-1Rs. Replication of the data in NIH-3T3
cells demonstrates that the specific regulation of HB-EGF in 3T3-L1
TIR/TIGR cells is not simply due to chimera-specific or clone-specific
effects. Some increase in HB-EGF transcription was observed in NIH-3T3
IR cells, in contrast with 3T3-L1 TIR cells which showed no significant
change. This could be due in part to insulin acting through endogenous
IGF-1Rs in the NIH-3T3 IR cells, thus demonstrating the advantages of
using the chimeric system for detection of IR/IGF-1R specificity.
We found TIGR regulation of HB-EGF to be a primary transcriptional
response. This agrees with previous work showing that HB-EGF is an
immediate early gene up-regulated following EGFR stimulation (27). We
further demonstrated that MAPK is needed for TIGR signaling to HB-EGF,
a requirement also observed in the IGF-1R-selective stimulation of
Twist and VEGF transcription (28, 29). In our study, MAPK was capable
of being activated by both chimeric receptors, whereas other studies
(11)2 showed greater
phosphorylation of MAPK in response to TIGR signaling. However, the
relative differences in MAPK phosphorylation seen in those studies are
unlikely to be sufficient to account for the all-or-nothing response we
have observed in terms of HB-EGF transcription. In our system, MAPK
phosphorylation was transient as has been shown previously (30) in
other cell types, despite the long term chimeric receptor
phosphorylation. Thus, although MAPK activation is essential for the
effects of the IGF-1R on HB-EGF expression, the extent and duration of
MAPK activation are unlikely to underlie the major differences between
the IR and IGF-1R in their respective abilities to increase HB-EGF
mRNA levels. We hypothesize that specificity is achieved by
activation of a pathway in addition to MAPK phosphorylation, for
instance a stimulatory signal involving the IGF-1R-specific residues
Tyr-1250 to Tyr-1251 and/or Ser-1280 to Ser-1283 already implicated in mitogenesis and transformation (31, 32).
The secretion of active HB-EGF into culture medium by NT-3-stimulated
3T3-L1 TIGRs, but not TIRs, was detected by HaCat cells (chosen for
their abundance of EGFRs). We observed mitogenic activity in the
culture medium in terms of both MAPK phosphorylation and thymidine
incorporation in HaCat cells, with almost exact correlation between the
two. Inhibition of this activity by the EGFR kinase inhibitor PD153035
demonstrated that the mitogenic activity in the medium was an EGFR
ligand. Our microarray data indicate that only one other EGF family
member, namely epiregulin, was detectable in 3T3-L1 TIR/TIGR cells.
However, HB-EGF expression correlates far better with both MAPK
activation and proliferation selectively induced by conditioned medium
taken from NT-3-stimulated TIGR versus TIR cells. Therefore,
the mitogenic effects observed are likely to result mainly from the
specific production of HB-EGF in response to TIGR stimulation, although
at this stage we cannot rule out a minor contribution from epiregulin.
If selective induction of HB-EGF mRNA is indeed able to cause
differential cell growth, why did previous work in the same cell system
show no specificity in terms of proliferative signaling from the two
chimeric receptors (9)? Western blotting indicates that these cells
have few EGFRs, with MAPK being poorly phosphorylated in response to
HB-EGF, whereas NIH-3T3 cells showed much stronger HB-EGF-stimulated
MAPK phosphorylation (data not shown), and do show a differential
effect of insulin and IGF-1 on proliferation (4). Therefore, HB-EGF may
act in an autocrine or juxtacrine/paracrine fashion, depending on its
cellular origin. A juxtacrine role for HB-EGF has already been
demonstrated in the process of liver regeneration (24).
Roudabush et al. (13) reported that IGF-1 receptor signaling
to Shc, but not IRS-1, depends on IGF-1-induced cleavage of pro-HB-EGF
and subsequent phosphorylation of EGFRs. Rather than considering HB-EGF
as an end point of the IGF-1 signaling pathway, as suggested by our
work, Roudabush et al. (13) assigned a role to HB-EGF as an
integral part of the IGF-1 signaling pathway, with EGFR signaling
accounting for IGF-1 stimulation of the MAPK cascade. However, in our
system TIGR regulation of HB-EGF mRNA was
MAPK-dependent but EGFR-independent, suggesting that, in
this cell line at least, the cleavage of cell surface HB-EGF and
activation of the EGFR are not involved in IGF-1R kinase signaling to
MAPK and HB-EGF gene transcription. The two possible roles of HB-EGF in
IGF-1 signaling are summarized in Fig.
6.
In summary, we have used microarray technology to study the regulation
of transcription by insulin and IGF-1. The global patterns of
expression found reflected the broadly overlapping effects of the IR
and IGF-1R kinase on cell function, with more genes being similarly
regulated than differentially by the two. However, of the genes that
were differentially regulated, the great majority showed a stronger
response to TIGR activation. HB-EGF, a mitogenic protein already
implicated in the IGF-1R signaling cascade, is also selectively induced
by IGF-1 at the level of transcription. Selective regulation of this
gene could be responsible, at least in part, for the greater effects of
IGF-1 on cell proliferation.
We are grateful to Regeneron Pharmaceuticals
for recombinant NT-3 and to N. Fusenig (DKFZ, Heidelberg, Germany) for
the generous gift of HaCat cells. We are also grateful to J. Sethi for
assistance in importing and formatting the microarray data.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 3, 2002, DOI 10.1074/jbc.M206206200
2
A. Parmar and K. Siddle, unpublished data.
The abbreviations used are:
IR, insulin
receptor;
IGF-1R, insulin-like growth receptor;
HB-EGF, heparin-binding
epidermal growth factor-like growth factor;
MAPK, mitogen-activated
protein kinase;
NT-3, neurotrophin-3;
DMEM, Dulbecco's modified
Eagle's medium;
MOPS, 4-morpholinepropanesulfonic acid;
PBS, phosphate-buffered saline;
EGFR, EGF receptor;
PI3K, phosphatidylinositol 3-kinase.
Microarray Analysis of Insulin and Insulin-like Growth
Factor-1 (IGF-1) Receptor Signaling Reveals the Selective Up-regulation
of the Mitogen Heparin-binding EGF-like Growth Factor by IGF-1*
,
Department of
Biochemistry, the University of Sydney,
Sydney, New South Wales 2006, Australia, § MRC Human
Genome Mapping Programme-RC, Wellcome Trust Genome Campus,
Hinxton CB10 1SB and ¶ University of Cambridge, Cambridge
Institute for Medical Research, Addenbrooke's Hospital,
Cambridge CB2 2XY, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 1.
Changes in gene expression from basal
after a 4-h stimulation of TIR and TIGR chimeras. 3T3-L1 TIR/TIGR
cells were serum-starved overnight and then treated with 4 nM NT-3 for 4 h before RNA extraction. Untreated
samples were extracted at the start of the stimulation period to
provide a base line, and RNA expression was studied by microarray
analysis. The scatter plot shows all genes that were up-regulated or
down-regulated from similar base lines by either receptor (see
"Experimental Procedures" for selection criteria). The dotted
line indicates a 1:1 ratio, and data from a representative of two
independent experiments are shown.
Genes regulated in a similar fashion by TIR and TIGR after 4 h
stimulation
Genes differentially regulated by TIR/TIGR signaling after
4 h stimulation
View larger version (35K):
[in a new window]
Fig. 2.
IGF-1 receptor kinase regulation of HB-EGF
gene transcription. A, time course of HB-EGF mRNA
regulation by chimeric receptors. Serum-starved 3T3-L1 TIR/TIGR cells
were incubated with 4 nM NT-3 for the times indicated
before total RNA was extracted. RNA extracts were Northern-blotted for
HB-EGF mRNA, and a representative blot (with ribosomal bands shown
below) and means and S.E. relative to TIGR 24 h are shown
(n = 3). Open circles, TIR; filled
circles, TIGR. I, TIR; G, TIGR.
B, dose-response curve for whole IR/IGF-1R regulation of
HB-EGF mRNA. Serum-starved NIH-3T3 IR/IGFR cells were stimulated
with the indicated concentrations of insulin (NIH-3T3 IR cells) or
IGF-1 (NIH-3T3 IGFR cells) for 4 h. Equal amounts of RNA were then
Northern-blotted for HB-EGF mRNA (n = 3, representative blot and means and S.E. shown, ribosomal bands shown
below blot). I, IR cells plus insulin;
G, IGFR cells plus IGFI. Open circles, IR;
filled circles, IGF-1R. C, TIGR signaling to
HB-EGF mRNA stability. Serum-starved 3T3-L1 TIGR cells were
stimulated for 4 h with 4 nM NT-3. Cells were then
washed and placed in serum-free medium containing 2.5 µg/ml
actinomycin-D ± 4 nM NT-3 for the indicated times
before RNA extraction. Extracts were then Northern-blotted for HB-EGF
(n = 4, means and S.E. shown). Open circles,
actinomycin-D alone; filled circles, actinomycin-D + NT-3.
D, role of de novo protein synthesis in HB-EGF
transcription. Serum-starved 3T3-L1 TIGR cells were stimulated
with 4 nM NT-3 (N) for 4 h in the presence
or absence of 10 µg/ml cycloheximide (C). RNA extracts
were Northern-blotted for HB-EGF (n = 3, representative
blot shown with ribosomal RNA bands below).
,
amphiregulin, neuregulin, and betacellulin all being classed as absent
by the Affymetrix software. Interestingly, the mRNA levels of
epiregulin itself were also induced somewhat more by activated TIGR
than TIR (3.8-fold), and epiregulin was only excluded from our list of
differentially regulated genes by a narrow margin. However, TIR
stimulation also induced expression of epiregulin mRNA, in contrast
to the all-or-nothing response seen in the case of HB-EGF. Because the
EGFR ligand detected correlates more with the expression of HB-EGF
mRNA than epiregulin, it is highly likely to be HB-EGF, although a
minor contributory role for epiregulin cannot be entirely ruled
out.
View larger version (23K):
[in a new window]
Fig. 3.
Detection of active HB-EGF in 3T3-L1
TIR/TIGR-conditioned medium using human keratinocyte (HaCat)
cells. Serum-starved 3T3-L1 TIR/TIGR cells were stimulated for
24 h with 4 nM NT-3. This conditioned medium was then
removed and tested for mitogenic activity in HaCat cells, which express
abundant EGF receptors. A and B, stimulation of
MAPK phosphorylation in HaCat cells. Serum-starved HaCat cells were
treated as indicated for 5 min, and then equal amounts of total protein
were Western-blotted with antibodies specific for dual-phosphorylated
MAPK and for Erk-1/2 protein as a control. A, representative
Western blots of dual-phosphorylated MAPK (ppMAPK) and total
Erk-1/2 (MAPK) are shown. B, band densities from
three independent experiments were quantified and expressed relative to
MAPK phosphorylation in cells treated with 20 ng/ml HB-EGF. Means and
S.E. are shown. C, stimulation of DNA synthesis in HaCat
cells. ~80% confluent HaCat cells were serum-starved for 24 h
and then treated as indicated for 16 h and assayed for thymidine
incorporation. Results are expressed relative to cells treated with 20 ng/ml HB-EGF, and the means and S.E. of duplicate samples from
four independent experiments are shown. SFM, serum-free
medium; HB-EGF, 20 ng/ml HB-EGF; TIR24/TIGR24,
conditioned medium from 3T3-L1 TIR/TIGR cells, respectively, diluted
2:5 in SFM; PD, 1 µM PD153035 (EGFR kinase
inhibitor).
View larger version (23K):
[in a new window]
Fig. 4.
Signaling pathways to HB-EGF gene expression
in 3T3-L1 TIR/TIGRs. A, involvement of PI3K and MAPK in
signaling to HB-EGF transcription. Serum-starved 3T3-L1 TIR/TIGR
cells were incubated for 4 h in the presence/absence of 4 nM NT-3, along with 50 nM wortmannin or 50 µM PD98059. Total RNA was extracted and HB-EGF expression
was detected by Northern blot of equal amounts of total RNA
(n = 3, representative blot shown with ribosomal bands
below). I, TIR; G, TIGR; O,
serum-free medium only; N, 4 nM NT-3.
B, chimeric receptor phosphorylation levels. Serum-starved
3T3-L1 TIR/TIGR cells were incubated with 4 nM NT-3 for the
times indicated above, and then protein extracts were
Western-blotted for phosphotyrosine to study chimeric receptor
activation (n = 3, means and S.E. shown). Open
columns, TIR; filled columns, TIGR. C, MAPK
activation by chimeric receptors. Serum-starved 3T3-L1 TIR/TIGR cells
were stimulated with 4 nM NT-3 for the indicated times, and
then protein extracts were Western-blotted for dual-phosphorylated MAPK
(n = 3, representative blot and means and S.E. shown).
Open columns, TIR; filled columns, TIGR.
View larger version (39K):
[in a new window]
Fig. 5.
Role of the EGF receptor in TIGR regulation
of HB-EGF transcription. Serum-starved 3T3-L1 TIR/TIGR
cells were stimulated for 4 h with 4 nM NT-3 along
with the EGFR inhibitor PD153035 (1 µM) either alone or
in conjunction with PD98059 (50 µM). Total RNA extracts
were then Northern-blotted for HB-EGF mRNA (n = 3, representative blot shown with ribosomal bands below).
I, TIR; G, TIGR; O, serum-free medium
only; N, 4 nM NT-3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6
integrin subunit by IGF-1 rather than insulin has been previously
demonstrated, and differentiation of neuroepithelial cells into retinal
neurons was found to be dependent on expression of 6
integrin (19). Interaction of 6 integrin with laminin
also promotes cell survival and proliferation (20). The selective
regulation of 6 integrin is therefore consistent with
the more growth- and differentiation-regulatory functions of IGF-1.
View larger version (19K):
[in a new window]
Fig. 6.
HB-EGF involvement in IGF-1 receptor
signaling. The diagram combines the findings of Roudabush et
al. (13) with ours to show the two ways in which HB-EGF is
involved in IGF-1 signaling. The pathway described by Roudabush
et al. (13) is shown on the left in
gray. Briefly, IGF-1 stimulates cleavage of HB-EGF already
present at the plasma membrane, and this then acts through EGFRs to
phosphorylate Shc and activate the MAPK pathway. In their model, the
majority of IGF-1 signaling to MAPK activation was carried out via this
pathway. We have discovered an involvement of HB-EGF further
downstream, as a longer term transcriptional response selectively
induced by the IGF-1R but not the IR. The pathway we have hypothesized
(shown on the right in white) implicates MAPK in
IGF-1R-mediated stimulation of HB-EGF gene transcription, and the
resulting HB-EGF protein is capable of stimulating MAPK phosphorylation
and proliferation in an EGFR-expressing cell line. We saw no dependence
of HB-EGF gene induction on EGFR signaling in our system.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
44-1223-336855; Fax: 44-1223-330598; E-mail:
sorahill@hgmp.mrc.ac.uk.
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