© 1999 American Heart Association, Inc.
Vascular Biology |
Angiotensin II Activation of Insulin-Like Growth Factor 1 Receptor Transcription Is Mediated by a Tyrosine Kinase–Dependent Redox-Sensitive Mechanism
From Emory University, Atlanta, Ga (J.D., T.P.), and the Division of Cardiology, University Hospital of Geneva, Switzerland (K.J.S., P.D.).
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Abstract—We have recently shown that angiotensin II activation of insulin-like growth factor 1 receptor (IGF-1R) transcription is a critical requirement for angiotensin-stimulated vascular smooth muscle cell growth; therefore, we examined the signaling pathway involved. In rat aortic smooth muscle cells, the antioxidants N-acetyl-L-cysteine (5 mmol/L) and pyrrolidine dithiocarbamate (100 µmol/L) completely inhibited angiotensin II–stimulated increases in IGF-1R mRNA and protein levels, suggesting the involvement of reactive oxygen species. Indeed, catalase abolished the Ang II–stimulated increase of IGF-1R protein expression, and accordingly, H2O2 (0.2 mmol/L) or the oxidized products of linoleic acid, hydroperoxyoctadecadienoic acids (10 µmol/L), increased IGF-1R mRNA levels at 3 hours by 74±20% and 107±22% and increased receptor number at 24 hours by 51±6.7% and 55±7.4%, respectively. The protein tyrosine kinase inhibitors genistein and tyrphostin A25 also blocked angiotensin II increases in IGF-1R mRNA and protein levels and blocked the ability of hydroperoxyoctadecadienoic acids and H2O2 to increase IGF-1R expression, suggesting that oxidative stress may be an early event in the angiotensin II signaling cascade. Furthermore, calcium chelation inhibited the angiotensin II effect. Transient transfection assays revealed that a -2350/+640 IGF-1R promoter/luciferase construct was fully responsive to angiotensin II stimulation (127±20% increase). Ten millimoles per liter hydroperoxyoctadecadienoic acids and 0.2 mmol/L H2O2 increased luciferase activity by 79±8.5% and 63±12%, respectively, and 5 mmol/L N-acetyl-L-cysteine blocked the angiotensin II–induced upregulation of luciferase activity by 70%. These data suggest that angiotensin II stimulates IGF-1R gene transcription via calcium-dependent activation of protein tyrosine kinase activity that lies downstream from an oxidant stimulus. These findings provide key insights into the signaling mechanisms whereby angiotensin II exerts its growth-promoting effects on the vasculature.
Key Words: insulin-like growth factor 1 receptor • reactive oxygen species • tyrosine kinases • gene regulation • signal transduction
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Introduction |
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Angiotensin II (Ang II) is the primary effector molecule of the renin-angiotensin system. Through its effects on the central nervous system, the kidney, the adrenal gland, and the vasculature, Ang II maintains normal volume and pressure homeostasis (reviewed in Reference 11 ). Interest in the potential role of Ang II in cardiovascular pathophysiology has been increasing. Ang II has growth-promoting effects both on the myocardium2 3 and on blood vessels,4 5 6 and inhibition of the effects of Ang II is beneficial in conditions such as congestive heart failure7 8 9 and certain nephropathies.10 11
The effects of Ang II are mediated on binding to its G protein–coupled
7–transmembrane domain receptor.12 The signaling cascade
initiated by Ang II binding includes activation of phospholipase C and
D,13 14 with resultant hydrolysis of membrane
phospholipids,15 increases in cytosolic
calcium,16 activation of the serine-threonine protein
kinase C (PKC), tyrosine phosphorylation of
multiple proteins,17 18 activation of the
mitogen-activated protein kinase (MAPK)
cascade,19 20 21 and increases in proto-oncogene
expression.22 23 24 Ang II has also been shown to stimulate
membrane oxidase systems, such as NADH/NADPH oxidase.25 26
Ang II induction of activating protein-1 activity has been shown to be
mediated by reactive oxygen species (ROS),27 and Ang II
has recently been shown to induce tyrosine kinase activity in vascular
smooth muscle, resulting in phospholipase C-
phosphorylation28 29 and stimulation of
the Jak/STAT pathway.30 Furthermore, Ang II stimulation of
MAPK has been shown to be dependent on
calcium/calmodulin-dependent tyrosine kinase activation of
p21 ras.31
Ang II stimulation of vascular smooth muscle cells modulates expression of other autocrine/paracrine growth factors and/or their receptors, such as platelet-derived growth factor,22 endothelin,32 transforming growth factor-ß,33 34 and insulin-like growth factor 1 (IGF-1).35 36 We have recently demonstrated that Ang II stimulation of the IGF-1 system plays an important role in the mitogenic effects of Ang II on vascular smooth muscle. Thus, Ang II upregulates vascular smooth muscle cell (VSMC) IGF-1 receptors,36 and inhibition of this effect by use of antisense phosphorothioate oligonucleotides inhibits Ang II–induced cellular growth.37 In addition, use of neutralizing anti–IGF-1 antibodies inhibits the mitogenic effects of Ang II.35 The signaling mechanisms that mediate the ability of Ang II to upregulate IGF-1 receptors (IGF-1R) are unknown; however, the effect is transcriptionally mediated and is not dependent on Ang II activation of PKC.38 This is in marked contrast to the effects of basic fibroblast growth factor on IGF-1R expression, which are PKC–dependent.38
In the present work, we explored signaling mechanisms that mediate the ability of Ang II to regulate IGF-1R. Our findings indicate that Ang II regulation of IGF-1R transcription is mediated via a tyrosine kinase–dependent redox-sensitive pathway. Our findings further suggest that cellular redox state is a proximal event in the regulation of IGF-1R expression and that it involves H2O2. These findings provide key insights into signaling mechanisms whereby Ang II transcriptionally regulates the IGF-1R and have important implications for understanding the mechanisms by which Ang II promotes cellular growth responses in vivo.
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Methods |
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Cell Culture
VSMCs were isolated from rat thoracic aorta as described previously.39 Cells were grown in DMEM supplemented with 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. They were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 and were passaged twice a week by harvesting with trypsin-versene and seeded at a 1:8 ratio in 75-cm2 flasks. For experiments, cells between passage levels 5 and 15 were seeded into 100-mm dishes, grown to 80% to 90% confluence, made quiescent by exposure for 48 hours to defined serum-free medium (SFM) containing DMEM and Ham's F-12 (1:1) supplemented with transferrin 5 µg/mL (Sigma Chemical Co), ascorbate 0.2 mmol/L (Sigma), glutamine, and antibiotics.40 Cells were then exposed to 100 nmol/L Ang II, 0.2 mmol/L H2O2, 10 µmol/L hydroperoxyoctadecadienoic acids (HPODEs), or 100 µmol/L linoleic acid for 3 hours in SFM. HPODEs were prepared by mixing 20 µL 100 mmol/L linoleic acid with 1000 U lipoxygenase in 1 mL PBS, incubating at 37°C for 1 hour, then adding to the cells hourly for 3 hours. Total RNA was collected for analysis of IGF-1R mRNA levels. Preliminary experiments showed a good response of Ang II on IGF-1R after 3 hours of stimulation. All of the following experiments therefore used this time point.
Depletion of Endogenous ROS by Antioxidants and Protein
Tyrosine Kinase Inhibition
To determine the effects of Ang II after depletion of ROS,
100 µmol/L pyrrolidine dithiocarbamate (PDTC) or 5 mmol/L
N-acetyl-L-cysteine (NAC) was applied
1 hour before cells were exposed to SFM with or without Ang II for 3
hours. Total RNA was then extracted for assay. To determine the effects
of Ang II after protein tyrosine kinase (PTK) inhibition, 60
µmol/L genistein or 20 µmol/L tyrphostin A25 was applied 1
hour before cells were exposed with or without Ang II for 3 hours.
Total RNA was then extracted for assay. Concentrations of PTK
inhibitors were at least 3 times the
IC50 values for these compounds.
Calcium Chelation by EGTA and BAPTA-AM
To determine the effects of Ang II after calcium chelation,
10 µmol/L BAPTA-AM (in DMSO) and 4 mmol/L EGTA were applied
1 hour before cells were exposed to SFM with or without Ang II for 3
hours. Total RNA was then extracted for assay.
IGF-1R mRNA Levels
Total RNA was extracted by the TRI-Reagent method (Molecular
Research Center), quantified by spectrophotometry, and was used
only if the 260/280 optical density ratios were between 1.8 and 2.0.
For determination of IGF-1R mRNA levels, a 203-bp EcoRI and
KpnI rat IGF-1R cDNA fragment in pGEM3Z was used to generate
a radiolabeled antisense probe, as previously
described.41 Solution hybridization/RNase protection
assays were performed as previously described.38 41
Briefly, 30 µg total RNA was hybridized overnight in a solution
containing 80% deionized formamide, 40 mmol/L PIPES (pH 6.4), 0.4
mol/L NaCl, 1 mmol/L EDTA, and 5x105 cpm of
32P-labeled riboprobe. After RNase digestion with
40 µg/mL ribonuclease A and 100 U/mL ribonuclease
T1, samples were treated with proteinase K,
extracted with phenol, precipitated with ethanol, and analyzed
on a 6% denaturing polyacrylamide urea (sequencing) gel. The
full-length probe is 251 bp, and the protected fragment is 195 bp in
length. As an additional control for RNA loading and to determine the
specificity of observed changes in IGF-1R mRNA levels, a GAPDH
riboprobe42 was included in the hybridization mixture.
This probe gives a 133-bp protected band after RNase digestion.
Autoradiograms were exposed for 1 to 3 days, and
protected bands were quantified by 2-dimensional laser densitometry.
The data were analyzed with NIH Image 1.51 software.
IGF-1R Binding Assays
To determine the role of ROS and PTK in Ang II regulation of
IGF-1R, quiescent rat aortic smooth muscle cells were incubated in SFM
with 100 nmol/L Ang II, with or without 60 µmol/L genistein or
5 mmol/L NAC. To define more precisely which ROS may be involved,
cells were also pretreated with or without 500 U/mL catalase, 500
µg/mL superoxide dismutase (SOD), 100 µmol/L
diphenyleneiodonium (a flavin inhibitor), or the
inhibitors of the mitochondrial respiratory chains
antimycin A 10 µmol/L or rotenone 10 µmol/L. Homologous
displacement binding assays were performed as previously
described.36 38 Briefly, cells in 24-well plates were
incubated with 0.1 nmol/L 125I-labeled IGF-1 and
0 to 0.1 µmol/L unlabeled IGF-1 for 90 minutes at room
temperature. Cells were washed in ice-cold binding buffer and
solubilized in 2N NaOH before counting. All assays were performed in
duplicate for each experimental point. Data were analyzed with
the Ligand program.
Transient Transfection Analysis
For transient cotransfection experiments, a genomic IGF-1R
fragment containing 2350 bp of sequence 5' to the transcription start
site and 640 bp of the 5'-untranslated region subcloned upstream of a
promoterless firefly luciferase reporter gene was used. Both the
full-length IGF-1R construct and the promoterless control vector pOLUC
were kindly provided by Dr H. Werner, NIH.43 Early-passage
(<10) confluent VSMCs were seeded into 100-mm dishes 24 hours before
transfection. IGF-1R/luciferase DNA (10 µg) and cytomegalovirus
(CMV)-74 Gal DNA (1 µg) were prepared in a final volume of 540 µL
in 1xPBS. Twenty-eight microliters of 10 mg/mL DEAE-dextran was added
and mixed by gentle tapping of the tube. For transfection, cells were
washed twice in PBS, and the DNA/DEAE-dextran mixture was added and
dispersed evenly over the cells. The final concentration of
DEAE-dextran in the salt solution was 
0.5 mg/mL. Plates were
incubated at 37°C for 30 minutes, 6 mL of DMEM with 10% FCS
was gently added, and after 3 hours of incubation, cells were exposed
to fresh DMEM with 10% FCS for 18 hours. SFM with or without Ang II,
HPODEs, or H2O2 was added.
Agonist was replaced every 3 hours for 12 hours, then every 6 hours for
72 hours.
Luciferase Assay
Cells were covered with 400 µL of reporter lysis buffer
(Promega) and incubated at room temperature for 15 minutes. The cell
lysate was transferred to a microcentrifuge tube, which was
vortexed for 10 to 15 seconds and then centrifuged for 15
seconds at room temperature. Cell extracts (20 µL) were mixed with
100 µL luciferase assay reagent (Promega), and luciferase activity
was measured in a Microlite ML3000 microtiter plate luminometer
(Dynatech). Cell extract (30 µL) was used for the measurement of
ß-galactosidase activity by use of a standard
protocol.44
Materials
Linoleic acid was purchased from Cayman Research lnc; Ang II,
lipoxygenase, EGTA, NAC, catalase, SOD, and antimycin A
from Sigma Chemical Co; genistein and tyrphostin A25 from LC
Laboratories (Alexis Corp); DEAE-dextran from Promega; PDTC from Fluka
Chemicals AG; and BAPTA-AM, diphenyleneiodonium, and rotenone from
Calbiochem.
Statistical Analysis
All experiments were performed at least 3 times. Data are
expressed as mean±SEM. Comparison between groups was performed with
Student's t test. A value of P<0.05 was
considered statistically significant.
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Results |
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Antioxidants NAC and PDTC Block Ang II Activation of IGF-1R Gene Transcription
We have previously shown that Ang II strongly induces IGF-1R transcription in VSMCs through a PKC-independent pathway.38 To define the potential signaling mechanisms involved, we determined the effect of the antioxidants NAC (1 to 5 mmol/L) and PDTC (100 µmol/L) on Ang II induction of IGF-1R mRNA. As shown in Figure 1
, 100 nmol/L
Ang II increased IGF-1R mRNA levels 2-fold at 3 hours (n=4,
P<0.02), and this increase was completely inhibited by NAC
and PDTC. To determine the effects of antioxidants on Ang
II–stimulated increases in IGF-1 receptors, radioligand
binding studies were performed. As shown in Figure 2, 100 nmol/L Ang II increased IGF-1R
numbers 2.5-fold at 24 hours (n=4, P<0.03), and this
increase was inhibited by NAC. Scatchard analysis indicated no
significant changes in IGF-1R binding affinity:
Kd control, 5.4±1.2 nmol/L;
Kd Ang II, 6.8±1.1 nmol/L;
Kd NAC 6.9±2.2 nmol/L; and
Kd NAC+Ang II, 6.1±1.5 nmol/L, mean±SEM,
n=4. These findings strongly suggest that an oxidant signal was
required for Ang II induction of IGF-1R transcription.
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Effects of H2O2 and of Linoleic Acid and
Its Oxidized Derivatives on IGF-1R Expression
To determine potential regulation of IGF-1R by ROS, we stimulated
RASM with 0.2 mmol/L
H2O2. As shown in Figure 3, H2O2 increased IGF-1R mRNA
levels at 3 hours. Recently, fatty acids such as linoleic acid and
their oxidized derivatives have been proposed to be important early
signaling molecules that can stimulate membrane oxidases,
proto-oncogene expression, and MAPK activation.25 45 46 We
therefore determined whether linoleic acid and its oxidized derivatives
regulated IGF-1R expression. As shown in Figure 3, linoleic acid
and HPODEs also increased IGF-1R mRNA levels at 3 hours. Results from 6
experiments indicate that
H2O2 and HPODEs increased
IGF-1R mRNA by 74±20% (P<0.05) and 107±22%
(P<0.01), respectively. To determine the effects of ROS on
IGF-1R, we performed radioligand binding studies.
H2O2 and HPODEs increased
IGF-1R numbers at 24 hours by 51±6.7% (n=4, P<0.01) and
55±7.4% (n=4, P<0.01), respectively (Figure 3B).
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PTKs Are Required for Ang II Activation of IGF-1R
Expression
Ang II activates several PTKs, which may be important in
its signaling cascade (reviewed in Reference 1818 ). To study the
role of PTKs in Ang II activation of IGF-1R transcription, rat aortic
smooth muscle cell(s) (RASMC) were treated with the PTK
inhibitors genistein (60 µmol/L) or tyrphostin A25
(20 µmol/L) in the presence or absence of Ang II for 3 hours,
and solution hybridization/RNase protection assays were performed. As
shown in Figure 4A and 4B, the induction
of IGF-1R mRNA by Ang II at 3 hours (2-fold increase, n=4,
P<0.05) was completely blocked by genistein and tyrphostin
A25. To further confirm the requirement of PTK in Ang II upregulation
of IGF-1R, we measured IGF-1R number in the Ang II–treated cells in
the presence or absence of genistein. As shown in Figure 4C, the
ability of Ang II to upregulate IGF-1R (69±10% increase at 24 hours,
n=3, P<0.01) was blocked by genistein. These data
demonstrated that PTKs are required for Ang II stimulation of IGF-1R
expression.
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Activation of IGF-1R Expression by H2O2 and
HPODEs Requires PTK Activity
Our findings indicated that both ROS and PTK were required for Ang
II modulation of IGF-1R expression. To determine whether an oxidant
stimulus also requires PTK to regulate IGF-1R, we treated RASMC with or
without the PTK inhibitor genistein, then exposed the cells
to H2O2 or HPODEs for 3
hours. As shown in Figure 5A and 5B, the
ability of H2O2 and of
HPODE to increase IGF-1R mRNA (1.75- and 2-fold increases,
respectively, n=6, P<0.05) was inhibited by genistein.
These data indicate that ROS activation of IGF-1R expression requires
PTK activity and suggest that an oxidant stimulus is an early event in
the Ang II–induced signaling pathway leading to transcriptional
activation of the IGF-1R gene. The experiments using various
inhibitors of either superoxide anions or
H2O2 formation or
inhibitors of the mitochondrial respiratory chain suggested
strongly that H2O2 was the
predominant oxidant stimulus involved, because catalase completely
abolished the increase in IGF-1R number induced by Ang II, whereas SOD
had no significant effect (Figure 5C). It is of note that
catalase inhibited basal IGF-1R numbers, quite in contrast to SOD.
Conversely, the flavin inhibitor diphenyleneiodonium and
also the inhibitor of the complex I of the mitochondrial
respiratory chain rotenone did not inhibit the Ang II response, and
antimycin A, an inhibitor of complex III, was toxic for the
cells (data not shown).
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Effect of Ang II, H2O2, and HPODEs on
IGF-1R Reporter Construct Activity
To gain insight into the promoter regions of the IGF-1R gene
responsive to Ang II activation, we transfected RASMC with an IGF-1R
promoter fragment including 2350 bp of sequence 5' to the transcription
start site and 640 bp 5'-untranslated region fused to the reporter
luciferase gene. This segment of the IGF-1R promoter contains consensus
sequences for a number of well-defined regulatory elements, including
several Sp-1, Ap-2, and wt1/Egr-1.43 A CMV ß-gal
construct was cotransfected to correct for transfection efficiency. As
shown in Figure 6, relative luciferase
activity corrected for ß-gal in Ang II–treated VSMCs was 127±20.3%
higher than in control VSMCs (n=3, P<0.05). Compared with
control, 10 µmol/L HPODEs and 0.2 mmol/L
H2O2 also caused increases
of 79±8.5% (n=6, P<0.01) and 63±12% (n=4,
P<0.01), respectively, in luciferase activity. NAC 5
mmol/L blocked the Ang II–mediated upregulation of luciferase activity
by 70% (n=3, P<0.05). These data indicate that the 2350-bp
IGF-1R promoter contains cis-acting elements responsive to
Ang II and to an oxidant signal.
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Calcium Is Required for Ang II Activation of IGF-1R
Expression
Ang II induces the release of calcium from intracellular stores,
and stimulation of calcium influx leads to elevations in cytosolic
calcium levels, an important component of the Ang II signaling
cascade.47 48 MAPK activation by Ang II has recently been
shown to be mediated by tyrosine kinase activity that is
calcium/calmodulin-dependent.31 To study the
role of calcium in Ang II activation of IGF-1R transcription, RASMs
were treated with the intracellular calcium chelator BAPTA-AM 10
µmol/L and the extracellular chelator EGTA 4 mmol/L in the
presence or absence of Ang II for 3 hours, and solution
hybridization/RNase protection assays were performed. As shown in
Figure 7A and 7B, the induction of
IGF-1R mRNA by Ang II at 3 hours (1.5-fold increase, n=4,
P<0.01) was completed blocked by BAPTA-AM/EGTA. Additional
evidence that Ca2+ is required comes from recent
findings that BAPTA-AM completely inhibited the increase induced by Ang
II on IGF-1R promoter transcription.49 These data
demonstrate that calcium is required for Ang II stimulation of IGF-1R
expression.
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Discussion |
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It has previously been demonstrated that the ability of Ang II to upregulate IGF-1R is a critical determinant of its mitogenic effects on VSMCs. Thus, IGF-1R gene–specific antisense oligonucleotides block Ang II–induced DNA synthesis.37 Furthermore, in VSMCs overexpressing IGF-1R, Ang II loses its ability to stimulate DNA synthesis, and this is related to the inability of Ang II in these conditions to further upregulate IGF-1R.37 The stimulatory effect of Ang II on IGF-1R transcription is independent of activation of the serine/threonine kinase, PKC, unlike that induced by basic fibroblastic growth factor.38 Identification of the signaling pathway involved in the Ang II activation of IGF-1R transcription is thus of major importance to understand the biological effects of Ang II. To define potential mechanisms involved in Ang II regulation of IGF-1R, we studied the effects of oxidant stress, of tyrosine kinase inhibitors, and of calcium chelators on Ang II regulation of IGF-1R expression.
Our findings clearly show that antioxidants suppress Ang II stimulation of IGF-1R. Furthermore, exposure of VSMCs to H2O2, which generates a variety of oxygen-derived free radicals,50 upregulates IGF-1R. To obtain further insights into potential regulation of IGF-1R by oxidant signals, we exposed VSMCs to linoleic acid and its oxidized derivative HPODEs. Our data indicate that these lipid peroxides rapidly increase IGF-1R mRNA levels, consistent with a model in which an oxidant signal is required for Ang II stimulation of IGF-1R transcription. Linoleic acid and HPODEs have been shown to stimulate proto-oncogene expression and MAPK activation in VSMCs.45 Furthermore, nordihydroguaiaretic acid, an inhibitor of the lipoxygenase system, reduced these effects markedly, consistent with HPODEs being the main mediator of the effects of linoleic acid.45 We did not find that arachidonic acid upregulated IGF-1R (data not shown). To the best of our knowledge, the relative degree of oxidation of arachidonic acid by VSMCs is not known, but one can speculate that it could be less than that of linoleic acid. In addition, one can speculate that Ang II–triggered release of linoleic acid by stimulation of phospholipase A2 activity51 or indirectly by stimulation of phospholipase D activity14 plays a role in early events leading to stimulation of IGF-1R transcription. It is important to note that changes in redox state have recently been shown to be involved in Ang II stimulation of c-jun and c-fos DNA binding activity and activating protein-1–driven transcription in myoblasts and myotubes.27
Further evidence that ROS, and more precisely H2O2, were required in the upregulation of IGF-1R protein expression was provided by the experiments using catalase or SOD. Indeed, the results of these experiments suggest that Ang II promotes the formation of H2O2, because catalase completely abolished the stimulatory effect of Ang II on IGF-1R numbers. In contrast, SOD, which catalyzes the detoxification of superoxide anions to water and oxygen, did not significantly reduce the Ang II effect, suggesting that superoxide anions were not absolutely required for the Ang II response. Rotenone, which inhibits complex I of the mitochondrial respiratory chain, and DPI, an inhibitor of flavin-containing enzymes and in particular NADH/NADPH oxidase, did not inhibit Ang II induction of IGF-1R. Also, we have shown that diphenyleneiodonium does not inhibit Ang II–induced IGF-1R promoter activity in CHO cells overexpressing Ang II AT1 receptors (K. Scheidegger et al, unpublished results).
To determine the potential participation of tyrosine kinases in the
signal leading to IGF-1R transcription, we determined the effects of
genistein and tyrphostin A25 on Ang II induction of IGF-1R mRNA and
protein. These tyrosine kinase inhibitors blocked the
ability of Ang II to increase IGF-1R mRNA and protein levels. Recent
studies have indicated that tyrosine phosphorylation is
an important early requirement in Ang II signaling.18
Thus, activation of the AT1 receptor by its
ligand leads to tyrosine phosphorylation of
phospholipase C-,28 most likely via activation of the
cytosolic tyrosine kinase c-Src,29 leading to phospholipid
hydrolysis, inositol 1,4,5-triphosphate generation, and calcium
mobilization. Furthermore, Ang II has been shown to induce tyrosine
phosphorylation of Jak2 kinase, leading to activation
of STAT 1 and STAT 2.30 Finally, p21 ras activation by Ang
II is blocked by tyrosine kinase inhibition31 and by
anti-Src antibodies.18 Our data indicate that tyrosine
phosphorylation is an essential signaling event
mediating Ang II induction of IGF-1R expression.
An important question is whether an oxidant signal requires
tyrosine kinase activity to induce IGF-1R transcription. To address
this question, we measured IGF-1R mRNA induction by
H2O2 and HPODEs in the
presence or absence of genistein or tyrphostin A25. Interestingly,
these inhibitors blocked agonist-stimulated increases in
IGF-1R mRNA, demonstrating that an oxidant signal requires tyrosine
kinase activity to stimulate IGF-1R transcription. This is
consistent with recent reports demonstrating redox modulation
of tyrosine kinases in human neutrophils.52 53
Furthermore, these data suggest that Ang II induction of IGF-1R
transcription utilizes an oxidant signaling event very early. To
further confirm these findings, we used an IGF receptor
promoter/luciferase construct. Note that the IGF-1R gene promoter lacks
TATA or CAAT motifs, with transcription starting from a unique
initiator sequence.43 The IGF-1R promoter contains
multiple SP-1, WT-1, and EGR motifs. Our data indicate that Ang II
significantly increased reporter gene activity and that this increase
was significantly blocked by an antioxidant. Additional evidence for
the involvement of an oxidant stimulus in the Ang II effect was
provided by the experiments using
H2O2 or HPODEs. Indeed,
both directly increased IGF-1R promoter activity 2-fold. These
findings support the model in which an oxidant signal is required by
Ang II–induced IGF-1R transcription (Figure 8).
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Early signaling events triggered by Ang II include release of calcium from intracellular stores and stimulation of calcium influx.16 47 48 Our data using calcium chelators indicate that Ang II–induced simulation of IGF-1R expression is calcium-dependent, although the exact components (intracellular versus extracellular calcium) of the calcium signal remain to be determined. In other experiments using promoter/reporter assays, however, we have shown that BAPTA-AM completely inhibits Ang II stimulation of IGF-1R transcription.49
In summary, we have shown that Ang II–induced IGF-1R expression is calcium-dependent and is mediated by a redox-sensitive, tyrosine kinase–dependent pathway. Oxidative stress, specifically H2O2 and lipid peroxides, regulates IGF-1R transcription via a tyrosine kinase–dependent mechanism. In view of the critical role played by the IGF-1R in mediating the growth effects of Ang II, these findings are particularly relevant to understanding the biochemical pathways required for Ang II stimulation of cellular proliferative responses.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-45317, HL-47035, and DK-45215 and by the Swiss National Science Foundation (FNSR3100-050799.97), the Swiss Cardiology Foundation, and the Gerbex-Bourget Foundation. Dr Delafontaine performed this project during the tenureship of an Established Investigator Award from the American Heart Association. We are grateful to Dr S. Parthasarathy for helpful discussions and to Kate W. Harris and Marjorie Burkhard for editorial assistance.
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Footnotes |
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Reprint requests to Patrick Delafontaine, MD, FACC, Cardiology Division, University Hospital, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland.
Received June 12, 1998; accepted February 5, 1999.
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