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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2119-2126.)
© 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

Jie Du; Tao Peng; Kathrin J. Scheidegger; Patrick Delafontaine

From Emory University, Atlanta, Ga (J.D., T.P.), and the Division of Cardiology, University Hospital of Geneva, Switzerland (K.J.S., P.D.).

	Abstract

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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

	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 myocardium^{2 3} and on blood vessels,^{4 5 6} and inhibition of the effects of Ang II is beneficial in conditions such as congestive heart failure^{7 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- phosphorylation^{28 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.

	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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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: K_d control, 5.4±1.2 nmol/L; K_d Ang II, 6.8±1.1 nmol/L; K_d NAC 6.9±2.2 nmol/L; and K_d 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.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of NAC and PDTC on Ang II stimulation of IGF-1R expression. A, Representative solution hybridization/RNase protection assay. Quiescent VSMCs were incubated without (SFM) or with 100 nmol/L Ang II and/or 5 mmol/L NAC and/or 100 µmol/L PDTC. Total RNA (30 µg/lane) was cohybridized to 32P-labeled IGF-1R and GAPDH antisense riboprobes. After RNase digestion, products were analyzed by sequencing gel electrophoresis. The first 2 lanes show probes alone and probes hybridized to 30 µg tRNA. B, Densitometric analysis of solution hybridization/RNase protection assays. Shown is the percent increase in IGF-1R mRNA induced by Ang II in control cells (Ctrl) or in cells coexposed to NAC or PDTC (n=4).

View larger version (19K): [in this window] [in a new window]   Figure 2. NAC inhibits Ang II–induced upregulation of IGF-1R. Quiescent VSMCs were exposed without (SFM) or with 100 nmol/L Ang II and/or 5 mmol/L NAC for 24 hours. Radioligand binding studies were performed, and Scatchard analysis was done with the Ligand program. Shown is the mean±SEM of results from 4 separate experiments.

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).

View larger version (28K): [in this window] [in a new window]   Figure 3. H2O2 and HPODEs regulate IGF-1R expression. A, Representative solution hybridization/RNase protection assay. Quiescent VSMCs were treated without (SFM) or with 0.2 mmol/L H2O2, 10 µmol/L HPODEs, or 100 µmol/L linoleic acid (LA). Total RNA (30 µg/lane) was cohybridized to 32P-labeled IGF-1R and GAPDH antisense riboprobes. After RNase digestion, products were analyzed by sequencing gel electrophoresis. B, Radioligand binding studies. Quiescent VSMCs were incubated alone (SFM) or in the presence of 0.2 mmol/L H2O2 or 10 µmol/L HPODEs. Radioligand binding studies were performed and data analyzed with the Ligand program. Results are the mean±SEM of determinations from 6 separate experiments.

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.

View larger version (29K): [in this window] [in a new window]   Figure 4. PTK inhibitors block Ang II–induced upregulation of IGF-1R expression. A, Representative solution hybridization/RNase protection assay. Quiescent VSMCs were incubated without (SFM) or with 100 nmol/L Ang II and/or 60 µmol/L genistein (Gen) and/or 20 µmol/L tyrphostin A25 (TA25) for 3 hours. Total RNA (30 µg/lane) was cohybridized with 32P-labeled IGF-1R and GAPDH antisense riboprobes. After RNase digestion, products were analyzed by sequencing gel electrophoresis. B, Densitometric analysis of solution hybridization/RNase protection assays. Cells were treated as described in A. Shown is the percentage increase in IGF-1R mRNA levels induced by Ang II in control cells and in cells coincubated with tyrphostin A25 or genistein. Results are the mean±SEM of determinations from 4 separate experiments. C, Radioligand binding studies. Quiescent VSMCs were incubated alone (SFM) or with 100 nmol/L Ang II and/or genistein for 24 hours. Radioligand binding studies were performed and data analyzed with the Ligand program. Shown is the mean±SEM of results from 3 separate experiments.

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).

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of PTK inhibitor on H2O2 and HPODE induction of IGF-1R mRNA and IGF-1R numbers. A, Representative solution hybridization/RNase protection assay. Quiescent VSMCs were incubated alone (SFM) or with 0.2 mmol/L H2O2 or 10 µmol/L HPODEs and/or 60 nmol/L genistein (Gen) for 3 hours, and solution hybridization/RNase protection assays were performed as described in experimental procedures. B, Densitometric analysis of solution hybridization/RNase protection assays. Shown is the mean±SEM of results from 6 separate experiments. C, Radioligand binding experiments. Quiescent VSMCs were treated with or without Ang II 100 nmol/L in the presence or absence of catalase 500 U/mL or SOD 500 µmol/L for 24 hours. Data are mean±SEM of 4 to 9 separate experiments.

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.

View larger version (30K): [in this window] [in a new window]   Figure 6. Transfection assays. A, Quiescent VSMCs were cotransfected with a -2350/+640 IGF-1R promoter/luciferase reporter construct and a CMV ß-gal DNA construct. Cells were incubated alone (SFM) or with 100 nmol/L Ang II, 10 µmol/L HPODEs, or 0.2 mmol/L H2O2 for 72 hours, and reporter gene activity was measured as described in the Methods section. Results, corrected for ß-gal activity, are the mean±SEM of measurements from 3 to 6 experiments. B, Quiescent VSMCs were transfected as described above and stimulated with 100 nmol/L Ang II and/or the antioxidant NAC for 72 hours, and reporter gene activity was determined. Results are corrected for ß-gal activity and are the mean±SEM from 4 separate experiments.

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.

View larger version (37K): [in this window] [in a new window]   Figure 7. Effect of calcium chelators on Ang II induction of IGF-1R mRNA expression. A, Representative solution hybridization/RNase protection assays. Quiescent VSMCs were exposed alone (SFM) or in the presence of 100 nmol/L Ang II and/or 10 µmol/L BAPTA-AM and 4 mmol/L EGTA for 3 hours, and solution hybridization/RNase protection assays were performed as described in experimental procedures. B, Densitometric analysis of solution hybridization/RNase protection assays. Shown is the mean±SEM of results from 4 separate experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (16K): [in this window] [in a new window]   Figure 8. Schematic of the signaling pathways proposed to mediate Ang II and growth factor regulation of IGF-1R. RTK indicates receptor tyrosine kinase; FGF, fibroblast growth factor.

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.

	Acknowledgments

 
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.

	Footnotes

 
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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