Published online before print May 10, 2001, doi: 10.1161/hh1001.090840
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© 2001 American Heart Association, Inc.
Cellular Biology |
Thrombin Regulates Insulin-Like Growth Factor-1 Receptor Transcription in Vascular Smooth Muscle
Characterization of the Signaling Pathway
From the Division of Nephrology (J.D.) and Department of Pathology (T.P.), Emory University, Atlanta, Ga; the Division of Cardiology (M.B., B.M.), University Hospital of Geneva, Geneva, Switzerland; and the Division of Cardiovascular Diseases (P.D.), University of Kansas, Kansas City, Kans.
Correspondence to Patrick Delafontaine, MD, Division of Cardiovascular Diseases, 1001 Eaton Building, Kansas University Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160. E-mail pdelafontaine{at}kumc.edu
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Abstract |
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Abstract—We have previously demonstrated that thrombin upregulation of insulin-like growth factor-1 receptor (IGF-1R) is essential for thrombin-induced mitogenic signaling. To characterize the mechanisms involved, we studied transcription of the IGF-1R gene in rat aortic smooth muscle cells. Thrombin markedly increased IGF-1R mRNA levels, peaking at 3 hours (112±7% above control). This effect was mimicked by the hexapeptide SFFLRN (that functions as a tethered ligand) and was blocked by the thrombin inhibitor hirudin. Nuclear run-on assays indicated that thrombin stimulated IGF-1R gene transcription by 2.1-fold, and this was confirmed with the use of actinomycin D. Thrombin-mediated upregulation of IGF-1R mRNA and protein levels was protein kinase C independent but was completely inhibited by the protein tyrosine kinase inhibitor genistein and by the antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate, suggesting the involvement of reactive oxygen species. The thrombin-induced increase in IGF-1R mRNA was inhibitable by diphenyleneiodonium chloride but not by other inhibitors of cellular oxidase systems, suggesting that NAD(P)H oxidase was necessary for the increase. Furthermore, inhibitors of the epidermal growth factor receptor kinase, Janus kinase-2 kinase, and Src kinase did not block the effect. Thus, thrombin transcriptionally regulates the IGF-1R gene via a redox-sensitive protein tyrosine kinase–dependent pathway that does not require protein kinase C activation. In view of our prior data indicating that IGF-1R density is a critical determinant of vascular smooth muscle cell growth, our findings have particular relevance to understanding mechanisms whereby growth factors such as thrombin regulate vascular proliferation in vivo.
Key Words: growth factors • signal transduction • gene regulation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The serine proteinase thrombin has multiple physiological effects.1 It is a procoagulant proinflammatory molecule2 3 4 5 and is a mitogen for vascular smooth muscle cells (VSMCs).6 7 8 Thrombin activates its receptor by a unique mechanism involving cleavage of the N-terminus of the receptor, generating a new N-terminus, which, in turn, acts as a tethered ligand.9 Cellular responses induced by thrombin are, in most cases, due to activation of protease-activated receptor (PAR)-1.1 PAR-1 is a proteolytically activated 7-transmembrane–spanning receptor that has been linked to a variety of cellular pathways, including hydrolysis of phosphoinositides, calcium mobilization, and activation of heterotrimeric G proteins, protein tyrosine kinases (PTKs), and monomeric G proteins.10 G-protein–coupled receptors, including the thrombin receptor, do not possess intrinsic tyrosine kinase activity. Using thrombin receptor–activating peptides and PTK inhibitors, some investigators have demonstrated that along with activation of its G-protein–coupled receptor, thrombin also requires PTK activity, possibly of a receptor type, for its mitogenic effect.11 12 13 Thus, thrombin has been shown to activate Src and c-Jun N-terminal kinase in VSMCs.14 15 16 Recently, evidence has been obtained indicating that thrombin exerts some of its effects by transactivating the epidermal growth factor (EGF) receptor.17
Insulin-like growth factor-1 (IGF-1) is a critical regulator of cell growth and differentiation.18 Its effects are mediated by the IGF-1 receptor (IGF-1R), a membrane tyrosine kinase. Evidence from a variety of studies using different cell types has shown that the IGF-1R is at a convergence point in the control of cell growth. Thus, a functional IGF-1/IGF-1R autocrine loop is required for the mitogenic effects of growth factors, such as platelet-derived growth factor,19 20 EGF,21 22 and angiotensin II (Ang II),23 and for cell transformation.24 Furthermore, the IGF-1R has been implicated in tumorigenesis.25 26
We have demonstrated that a functional IGF-1/IGF-1R pathway is essential for thrombin stimulation of VSMC mitogenesis.27 Thrombin upregulates VSMC IGF-1R, and inhibition of this effect using antisense phosphorothioate oligonucleotides inhibits thrombin-induced cellular proliferation. In addition, use of neutralizing anti–IGF-1 antibodies inhibits the mitogenic effects of thrombin. In the present study, we explored mechanisms and signaling pathways mediating the ability of thrombin to upregulate IGF-1R. Our findings indicate that thrombin regulates IGF-1R gene transcription via stimulation of its classic receptor, PAR-1, and that the signaling pathway is redox sensitive, requiring NAD(P)H oxidase and tyrosine kinase activity. These findings have important implications for understanding the mechanisms by which thrombin promotes cellular growth responses in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
VSMCs were isolated from rat thoracic aorta and grown as described.27 For experiments, cells from passages 5 to 16 were seeded into 100-mm dishes, grown to 80% to 90% confluence, and reached quiescence by exposure to defined serum-free medium (SFM) for 48 hours. Cells were then exposed to human thrombin (1 U/mL, 1000 U/mg) or to the rat thrombin receptor–specific peptide SFFLRN (100 µmol/L). The peptide was synthesized by the Microchemical Facility, Emory University, and purified by high-performance liquid chromatography. Some experiments were performed in the presence of the thrombin inhibitor hirudin (1 U/mL) or the transcriptional inhibitor actinomycin D (10 µg/mL).
Inhibition of ROS and PTKs
To determine the effects of reactive oxygen species
(ROS) depletion, pyrrolidinedithiocarbamate (PDTC, 100 µmol/L) or
N-acetyl-L-cysteine
(NAC, 5 mmol/L) was applied 1 hour before exposing the cells to
thrombin. In other experiments, cells were pretreated with
diphenyleneiodonium chloride (DPI, 10 or 100 µmol/L), allopurinol
(100 µmol/L), catalase (CAT, 500 U/mL), superoxide dismutase (SOD,
200 U/mL), or a combination of CAT and SOD for 1 hour before growth
factor stimulation. To determine the effects of PTK inhibition,
genistein (60 µmol/L) was applied 1 hour before exposing the cells to
thrombin. To analyze the specific kinases involved, cells were
pretreated with AG490 (10 to 50 µmol/L), AG1478 (10 to 50 µmol/L),
PP1 (10 µmol/L), or PP2 (1 µmol/L) (all from
Calbiochem).
Inhibition of PKC
To determine the effects of protein kinase C (PKC)
downregulation, phorbol 12,13-dibutyrate (PDBu, 200 nmol/L) was added
during the second 24-hour period of serum deprivation and
maintained during the period of exposure to the agonist. A PKC
inhibitor, bisindolylmaleimide (50 nmol/L) or chelerythrine
(3 mmol/L), was applied at time 0 after 48-hour serum
deprivation, immediately before the cells were exposed to
the agonist. Concentrations of PKC inhibitors were at least
3 times the IC50 values for these
compounds.28
IGF-1R mRNA Levels
Control and inhibitor-treated cells were
exposed to SFM without or with thrombin or SFFLRN for 3 hours, total
RNA was extracted with the use of TRI-reagent (Molecular Research
Center), and IGF-1R mRNA levels were determined by solution
hybridization/RNase protection assays with a radiolabeled antisense
probe transcribed from a rat IGF-1R
cDNA.29 The full-length
probe is 251 bp, and the protected fragment is 195 bp. GAPDH or 18S
riboprobes were included as a control, giving 133- and 158-bp protected
bands, respectively. Autoradiograms of protected bands
were quantified by densitometry.
Radioligand Binding Assays,
Immunoblotting, and Measurement of DNA
Synthesis
To determine the effect of thrombin on IGF-1R number
and binding affinity, quiescent VSMCs were exposed to SFM without or
with thrombin or SFFLRN for 24 hours. The dependence of thrombin
upregulation of IGF-1R on PKC and ROS was determined by coincubating
the cells with thrombin and inhibitors for 24 hours before
the binding assays. Binding assays were performed and analyzed
as described
previously.29
To analyze the effect of kinase inhibitors on IGF-1R protein expression and DNA synthesis, quiescent VSMCs were incubated with an inhibitor for 1 hour before the administration of growth factors. After 16 hours, proteins were collected for analysis of IGF-1R by Western blotting.30 DNA synthesis was measured at 16 hours by adding [3H]thymidine (1 µCi/mL) during the last 4 hours of incubation. Samples were processed as described previously.29
Nuclear Run-On Transcriptional
Analysis
These assays were performed as previously
described,31 with
modifications. Briefly, quiescent VSMCs were exposed to SFM in the
presence or absence of thrombin for 3 hours. Nuclei were isolated with
the use of a sucrose gradient, and transcription was carried out with
the use of 2 to 5x107 nuclei in a 300-µL
reaction mixture containing 37 mmol/L Tris-HCl, pH 8.0, 26%
glycerol, 5.5 mmol/L MgCl2, 0.066
mmol/L EDTA, 140 mmol/L KCl, 0.1 mmol/L each ATP, GTP, and
CTP, and 20 µL [
-32P]UTP (3000
Ci/mmol, Du Pont–New England Nuclear). RNA was extracted, and
unincorporated nucleotides were separated by using Bio-Gel
P-30 columns. Plasmids containing cDNAs for the IGF-1R (-610 to +200
bp relative to ATG), GAPDH (1.2-kb fragment), Ang II type 1 receptor
(full-length rat sequence), and plasmid without insert (pGEM3) were
linearized, denatured, and applied to a nylon membrane with use of a
Bio-Rad slot-blot apparatus. After prehybridization in
100 mmol/L TES-HCl, pH 7.4, 0.2% SDS, 10 mmol/L EDTA, 0.3
mol/L NaCl, 1x Denhardt’s solution, and 100 µg/mL yeast tRNA, the
membrane was hybridized in the same solution containing 1 to
5x106 cpm/mL of 0.2 mol/L NaOH treated
32P-labeled nuclear RNA for 40 hours at
65°C. Membranes were washed twice in 2x SSC and 0.1% SDS at room
temperature and then in 0.1x SSC and 0.1% SDS for 2 hours at 65°C.
Autoradiographic signals were quantified by
densitometry.
Statistical Analysis
All experiments were performed at least 3 times. Data
are expressed as mean±SE. Analysis of repeated measures was
performed by ANOVA. Comparison between groups was performed by using a
protected Tukey t test or
Student t test when
appropriate.
Materials
Thrombin, PDBu, CAT, allopurinol, and NAC were
purchased from Sigma Chemical Co. Bisindolylmaleimide, chelerythrine,
and genistein were from LC Laboratories, Alexis Corp. PDTC was from
Fluka Chemical Corp. DPI, SOD, PP1, PP2, AG490, and AG1478 were from
Calbiochem. TRI-reagent was from Molecular Research Center. The 18S
riboprobe was from Ambion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of Thrombin on IGF-1R mRNA Levels
Exposure of VSMCs to thrombin caused a marked increase in IGF-1R mRNA levels (Figure 1A
). To assess the time course of induction in IGF-1R
expression, IGF-1R and GAPDH mRNAs were measured after exposure to an
agonist for 0 to 24 hours. Thrombin induction of IGF-1R mRNA peaked at
3 hours (2.1-fold increase,
P<0.0001; n=8), and levels
were still elevated at 24 hours
(P<0.01,
Figure 1B). Thrombin caused a more delayed increase in GAPDH
mRNA (P<0.01 at 24 hours). To
determine whether the effect was mediated via proteolytic activation of
the thrombin receptor, we measured IGF-1R mRNA after exposure to the
hexapeptide SFFLRN, which acts as a tethered ligand after cleavage of
the amino terminus of the thrombin receptor. SFFLRN increased IGF-1R
mRNA by 47% (P<0.001, n=6),
mimicking the effect obtained with the parent agonist thrombin
(Figure 1C). To further demonstrate that the effect was
mediated by a receptor-dependent mechanism, we blocked the binding of
thrombin to its receptor with hirudin, which resulted in complete
inhibition of the induction of IGF-1R mRNA
(P<0.01,
Figure 1D).
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Effects of Thrombin on Newly Transcribed IGF-1R
mRNA
To determine whether the increase in IGF-1R mRNA was
transcriptionally mediated, we performed in vitro transcription with
the use of nuclei from VSMCs treated without or with thrombin for 3
hours. Hybridization of radiolabeled nuclear RNA probes to IGF-1R cDNA
demonstrated a 2.1-fold increase in IGF-1R signal from
thrombin-stimulated cells (n=4,
P<0.05,
Figure 2). There was a small (1.25-fold) increase between
nuclear transcripts from control or agonist-exposed cells after
hybridization to GAPDH cDNA (n=5,
P=0.13). Hybridization to the
Ang II type 1A receptor cDNA gave a significant 2-fold decrease in
signal intensity (n=4,
P<0.025), consistent
with a prior study indicating that thrombin decreases Ang II receptor
expression.32 Vector alone
gave a minimal signal. To confirm that thrombin induces IGF-1R gene
transcription, we inhibited RNA synthesis with actinomycin D, resulting
in complete loss of the upregulation of IGF-1R mRNA
(Figures 3A and 3B) and protein
(Figure 3C) levels by thrombin.
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Effect of PKC Downregulation on
Thrombin-Induced IGF-1R Expression
PKC activation is an important signaling pathway for
the cellular effects of multiple
mitogens.33 34 To
define the PKC dependence of thrombin regulation of IGF-1R, we measured
IGF-1R mRNA levels after 24 hours of preincubation with the phorbol
ester PDBu. Chronic exposure to PDBu has been shown to downregulate PKC
activity in VSMCs by 80% to
90%.35 After preexposure to
PDBu, thrombin still increased IGF-1R mRNA (1.7-fold increase,
P<0.025; n=6) and binding
(1.5-fold increase, P<0.05;
n=4) levels
(Figure 4A). To further define the PKC dependence of the
effect, we used 2 structurally unrelated inhibitors of PKC
(Figure 4B). Thrombin still increased IGF-1R mRNA by 2.0-fold
in the presence of bisindolylmaleimide (n=5,
P<0.05) or chelerythrine (n=5,
P<0.05). A small increase in
IGF-1R mRNA was observed with the inhibitors alone
(P=NS). Furthermore, thrombin
upregulated the IGF-1R number by 1.8-fold in the presence of
bisindolylmaleimide (n=4,
P<0.05) and by 1.5-fold in the
presence of chelerythrine (n=4,
P<0.025), without altering
IGF-1R binding affinity
(Kd)
(not shown).
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Effect of Inhibition of ROS on Thrombin-Induced
IGF-1R Expression
We have previously shown that Ang II induces IGF-1R
transcription in VSMCs through a redox-sensitive
pathway.36 To test whether
the action of thrombin to increase the IGF-1R is mediated through ROS,
we determined the inhibitory effect of the antioxidant PDTC
(Figure 5A) or NAC
(Figure 5B). Both compounds completely inhibited the
induction of IGF-1R mRNA. The findings for NAC were confirmed in
radioligand binding assays. Thrombin increased the IGF-1R
number 1.8-fold at 24 hours (n=6,
P<0.01), and this increase was
inhibited by NAC
(Figure 5B). Scatchard analysis indicated no
significant changes in IGF-1R binding affinity:
Kd
control, 4.7±0.1 nmol/L;
Kd
thrombin, 5.5±0.4 nmol/L;
Kd NAC,
4.8±0.3 nmol/L; and
Kd
NAC+thrombin, 7.1±1.0 nmol/L (n=5,
P=NS). These findings strongly
suggest that an oxidant signal was required for thrombin induction of
IGF-1R transcription.
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ROS-producing systems include various NADPH and NADH
oxidases, xanthine oxidase, and NO synthase. We examined the effects of
inhibitors of different oxidase systems on the
thrombin-induced increase in IGF-1R. This increase was completely
blocked by DPI at 100 µmol/L (n=5,
P<0.05), an
inhibitor of flavoprotein-containing enzymes, such as
NAD(P)H oxidase
(Figure 6). DPI alone had negligible effects on IGF-1R mRNA
levels in the absence of thrombin. No inhibition was observed with
allopurinol, which is an inhibitor of xanthine oxidase
(data not shown), with CAT, which blocks the production of the
reactive oxygen intermediate
H2O2, with superoxide
dismutase, which blocks the reactive oxygen intermediate
O2 · -, or with a
combination of CAT and SOD
(Figure 6). Taken together, these results indicate that the
NAD(P)H oxidase system is involved in the response to
thrombin.
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Effect of PTK Inhibition on Thrombin-Induced
IGF-1R Expression
Thrombin activates several PTKs, which may be
important in its signaling cascade, including nonreceptor tyrosine
kinases, such as Src14 and
focal adhesion kinase,37 and
receptor tyrosine kinases, such as the IGF-1
receptor38 and the EGF
receptor.39 To study the
role of PTK in thrombin-induced IGF-1R transcription, VSMCs were
treated with genistein. The induction of IGF-1R mRNA by thrombin
(2.8-fold increase, P<0.00001;
n=5) was completely blocked by genistein
(Figures 7A and 7B). Furthermore, the ability of SFFLRN to
increase IGF-1R mRNA (1.5-fold increase,
P<0.001; n=6) was inhibited by
genistein
(Figure 7C). We further confirmed the requirement of PTK in
thrombin upregulation of IGF-1R by measuring IGF-1R numbers. In these
experiments, thrombin upregulated the IGF-1R number by 1.8-fold (n=4,
P<0.01), and genistein
completely inhibited this effect
(Figure 7D). These data demonstrate that PTKs are required
for thrombin stimulation of IGF-1R expression.
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To obtain insight into the specific PTKs involved, we
studied the effects of inhibitors of receptor and
nonreceptor kinases. Thrombin has been shown to transactivate
the EGF receptor.17 However,
the selective EGF receptor kinase inhibitor AG1478 did not
abolish the induction of IGF-1R mRNA or protein by thrombin at 10, 20,
or 50 µmol/L
(Figure 8), concentrations that have been shown effective in
inhibiting Ang II–induced extracellular signal–regulated kinase
activation in rat VSMCs.40
As expected, the induction of IGF-1R mRNA and protein by EGF was
inhibited by AG1478
(Figures 8A and 8B). Furthermore, the EGF-induced increase in
[3H]thymidine incorporation was
significantly inhibited by 50 µmol/L AG1478
(Figure 8C), confirming activity of the drug.
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The Janus kinase (JAK)-2 inhibitor AG490 had no
effect on thrombin-induced increases of IGF-1R mRNA, protein, or DNA
synthesis at doses of 10, 20, and 50 µmol/L. This
inhibitor also failed to block induction of the IGF-1R by
basic fibroblast growth factor (bFGF) (not shown) or EGF
(Figures 8A
and 8B). In a
[3H]thymidine incorporation assay, AG490
significantly reduced the thrombin- and EGF-induced increases in DNA
synthesis (P<0.01),
demonstrating that the compound was active.
Furthermore, we studied the effects of Src kinase
inhibitors. In these experiments, the increase in IGF-1R
mRNA levels was slightly enhanced after incubation with PP1 before
thrombin treatment
(Figure 8A). The same results were obtained with PP2 (not
shown). bFGF significantly increased IGF-1R mRNA and protein, and this
increase was not inhibited by PP1
(Figures 8A and 8B); however, PP1 significantly reduced
bFGF-induced increases in DNA synthesis
(Figure 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been demonstrated that the ability of thrombin to upregulate IGF-1R is a critical determinant of its mitogenic effects on VSMCs.27 Thus, use of IGF-1R gene–specific antisense oligonucleotides or of an anti–IGF-1 antibody blocks thrombin-induced DNA synthesis. In the present study, we demonstrate that the ability of thrombin to upregulate IGF-1R on VSMCs is mediated by transcriptional activation of the IGF-1R gene, resulting in a rapid increase in IGF-1R mRNA levels, with a peak at 3 hours that was maintained for at least 24 hours. The effect is transduced by the classic PAR-1, inasmuch as it is mimicked by the PAR-1–specific peptide SFFLRN. We further examined pathways whereby the G-protein–coupled receptor agonist thrombin increases expression of the tyrosine kinase IGF-1R.
A variety of signaling events are induced by thrombin. These
include stimulation of PTK and PKC and activation of multiple
intracellular kinases and
phosphatases.1 10 12
Because PKC is postulated to be an important mediator of the effects of
multiple growth factors, we assessed its role in thrombin regulation of
IGF-1R mRNA. Chronic stimulation of PKC by incubation with PDBu results
in marked downregulation of PKC
activity.35 In this
condition, thrombin caused a further significant 1.7-fold increase in
IGF-1R mRNA and 1.5-fold increase in IGF-1R protein levels, suggesting
that thrombin induces IGF-1R transcription via a PKC-independent
pathway. To confirm this, we used 2 structurally unrelated PKC
inhibitors, but in all cases, the stimulatory effect of
thrombin on IGF-1R was retained. It is of note that atypical PKC
isoforms (eg, PKC
) lack a phorbol ester binding site and are not
downregulated by PDBu
treatment.33 34
However, the failure of 2 different PKC inhibitors (and in
particular, chelerythrine, which blocks the catalytic
domain28 ) to block
thrombin-induced increases in IGF-1R mRNA and protein argues against an
involvement of PKC. Thus, the present findings are
consistent with a PKC-independent pathway whereby thrombin
stimulates IGF-1R expression.
To further define the potential mechanisms involved in thrombin regulation of IGF-1R, we studied the role of oxidant stress. Our findings show that the antioxidants PDTC and NAC suppress thrombin stimulation of IGF-1R expression. Patterson et al41 have recently shown that thrombin induces the generation of ROS in VSMCs at least in part through activation of NAD(P)H oxidase and that blocking this enzyme with DPI inhibits thrombin-induced proliferation. Consistently, we have demonstrated in the present study that DPI blocks the induction of IGF-1R by thrombin. Thus, unlike Ang II,36 thrombin activation of NAD(P)H oxidase provides a mechanism whereby activation of the thrombin receptor could lead to IGF-1R transcription. Again, unlike Ang II, the ability of thrombin to upregulate IGF-1R was not blocked by CAT. SOD also did not block the ability of thrombin to regulate IGF receptors. This lack of effect may be due to ineffective scavenging of intracellular reactive oxygen intermediates, or other intermediates may play a role.42
Recent studies have shown that PAR-1 couples functionally to multiple heterotrimeric G proteins that activate the mitogen-activated protein kinase (MAPK) pathway via the small GTP-binding protein p21ras.37 43 44 45 46 In various cell types, thrombin activation of the Ras/MAPK cascade requires non–receptor-dependent tyrosine kinase activity, such as Src44 46 or focal adhesion kinase,45 or receptor-dependent tyrosine kinase activity, eg, through the EGF receptor, which is transactivated by thrombin in rat 1 fibroblasts45 and in VSMCs.17 Although PKC activation provides a potential Ras-independent pathway whereby G-protein–coupled receptors may activate the MAPK pathway, our data using PDBu and PKC inhibitors argue strongly against the possibility that thrombin upregulation of IGF-1R uses PKC. To examine the potential role of PTK in the signal leading to IGF-1R transcription, we determined the effects of genistein. This tyrosine kinase inhibitor blocked the ability of thrombin to increase IGF-1R mRNA and protein levels. To determine which specific tyrosine kinases are involved, we determined the effect of Src, EGF receptor kinase, and JAK-2 kinase inhibitors. None of the inhibitors tested blocked IGF-1R induction by thrombin. JAK-2 is also not involved in IGF-1R induction by bFGF or EGF, indicating that JAK-2 is not required for IGF-1R regulation. However, growth factor–induced DNA synthesis was blocked by AG490, suggesting that JAK-2 is involved downstream from the IGF-1R. Taken together with our data using genistein, our results indicate that a kinase other than Src, EGF receptor, or JAK-2 must be involved in thrombin regulation of the IGF-1R.
Significant data have established that the IGF-1/IGF-1R
autocrine loop is a critical regulator of the cell cycle. Thus,
fibroblasts from mice expressing a null mutation for the IGF-1R gene
have a marked reduction in their growth rate in
serum.24 Normal growth
cannot be restored by exposure to other growth factors but can be
corrected by expression of the IGF-1R. A functional IGF-1/IGF-1R
pathway is essential for the growth response to other growth factors,
such as EGF,21 22
platelet-derived growth
factor,19 20 and
thrombin.27 Furthermore,
simian virus 40 T-antigen transformation of cells requires a functional
IGF-1R.47 It is of note that
the effects of growth factors on IGF-1R expression may be critical in
allowing cells to subsequently respond to endogenously
synthesized IGF-1. Thus, in VSMCs, Ang II–induced and thrombin-induced
mitogenesis is blocked by antisense phosphorothioate
oligonucleotides targeting the IGF-1R
mRNA.48 49
Furthermore, in VSMCs transfected with an IGF-1R antisense expression
plasmid, growth responses are markedly
blunted.29 In these cells,
as in cells exposed to antisense oligonucleotides
targeting the IGF-1R mRNA,49
there is a reduction in IGF-1R density of 
50%. Therefore,
relatively small changes in IGF-1R density appear to be critical for
the regulation of VSMC growth. We have previously shown that thrombin
doubles IGF-1R density on VSMCs and that this is a critical requirement
for thrombin-induced DNA synthesis. Our present study has defined
the signaling cascade involved. It is important to emphasize that the
IGF-1R gene promoter lacks TATA or CAAT motifs and that transcription
starts from a unique "initiator"
sequence.50 It will be of
paramount importance to determine how transcription factors interact
with this receptor promoter and, specifically, to determine whether
thrombin regulates transcription via a common
cis-acting
sequence.
In summary, we have demonstrated that thrombin upregulates IGF-1R via PAR-1–mediated transcriptional activation of the IGF-1R gene. Thrombin-dependent regulation of IGF-1R is mediated via a PKC-independent pathway that is redox sensitive and involves the activation of NAD(P)H oxidase. It requires a tyrosine kinase activity other than Src, EGF receptor, or JAK-2. In view of the critical role played by the IGF-1R in mediating the growth effects of thrombin, these findings are particularly relevant to understanding biochemical pathways required for thrombin stimulation of cellular proliferative responses.
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Acknowledgments |
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This study was supported by National Institutes of Health Grants HL-47035, HL-45317, and DK-45215; an American Heart Association Scientist Development Award (J.D.); the Swiss Cardiology Foundation; Swiss National Science Foundation Grant FNSR 3100-050799.97; and the Gerbex-Bourget Foundation. We are grateful to M. Burkhard for her excellent editorial assistance.
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Footnotes |
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Original received December 29, 1999; resubmission received March 27, 2001; accepted March 29, 2001.
1 Both authors contributed equally to this study. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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