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(Circulation Research. 1995;76:963-972.)
© 1995 American Heart Association, Inc.

Articles

Inhibition of Vascular Smooth Muscle Cell Growth Through Antisense Transcription of a Rat Insulin-Like Growth Factor I Receptor cDNA

Jie Du, Patrick Delafontaine

From the Division of Cardiology, Department of Medicine, Emory University, Atlanta, Ga.

	Abstract

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Abstract Insulin-like growth factor I (IGF I) is an autocrine/paracrine growth factor that is produced in multiple tissues and is essential for normal developmental growth. Its effects are mediated by activation of a membrane-bound tyrosine kinase receptor, IGF IR. On the basis of the partial rat IGF IR -chain cDNA sequence previously reported, we cloned cDNA encoding the full-length rat IGF IR. The deduced amino acid sequence predicts a 1370–amino acid receptor precursor, which includes signal sequence, a 707–amino acid -chain, a 4-Arg cleavage site, and a 629–amino acid ß-chain. Overall, similarity to human IGF IR is 89% and 98% at the nucleotide and amino acid levels, respectively. Antisense IGF IR expression constructs in vectors incorporating Epstein-Barr virus replicative signals and the cytomegalovirus promoter/enhancer or the inducible human metallothionein IIa promoter/enhancer were assembled and stably transfected into cultured rat aortic smooth muscle cells. Clone CA9 (constitutively expressing abundant antisense IGF IR transcripts), clones MA5 and MA7 (expressing antisense IGF IR transcripts inducibly), and clones ME8 and ME10 (expressing vector alone) were characterized. There was a 57% reduction in IGF IR mRNA levels in clone CA9 after confluence compared with clone ME10. This resulted in a 51% decrease in IGF I binding sites in clone CA9, without a change in binding affinity (K_d), and a 55% and 57% reduction in DNA synthesis rates, basally and in response to 10 ng/mL IGF I, respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF IR number after confluence following exposure to 100 µmol/L ZnSO₄ and a 44% and 58% reduction in DNA synthesis, basally and in response to 10 ng/mL IGF I, respectively. Growth curves indicated that proliferation of clone CA9 in the presence of 10% serum was reduced by 60% compared with clone ME10. Thus, cloning of cDNA encoding the full-length rat IGF IR indicates that this receptor is highly conserved. Antisense targeting of this receptor in vascular smooth muscle cells (VSMCs) demonstrates that a decrease in IGF IR density results in marked inhibition of VSMC proliferation. These findings indicate an important role for this ligand-receptor system in regulating VSMC growth. Specifically, they suggest that modulation of VSMC IGF IR density may be an important mechanism whereby growth of these cells is controlled.

Key Words: insulin-like growth factor I • molecular cloning • antisense RNA • transfection • cellular proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin-like growth factor I (IGF I) is a ubiquitous peptide that circulates at high levels in serum and is expressed in multiple tissues. It has a broad spectrum of effects, including stimulation of embryonic and postnatal growth, cell growth and differentiation in vitro, various metabolic effects, and participation in tissue regeneration.^{1 2} The effects of IGF I are mediated via binding to a specific heterotetrameric membrane receptor (IGF IR) that consists of two extracellular -subunits and two membrane-spanning ß-subunits and is widely distributed in mammalian tissues.^{3 4 5 6} There is a high degree of similarity between the insulin and IGF I receptors,^{3 7 8} and both ligands may interact with each other's receptors, albeit with much lower affinity.⁶ Furthermore, hybrid receptors have recently been demonstrated.^{9 10}

Several studies have suggested that IGF I plays a unique role as an autocrine regulator of cellular function and that activation of the IGF I/IGF IR loop is required for the entry of cells into S phase in response to a variety of growth stimuli. Thus, in BALB/c3T3 fibroblasts, overexpression of the proto-oncogene c-myb stimulates the expression of IGF I and of its receptor and abrogates the requirement for exogenous IGF I as a growth stimulus.^{11 12} In these cells the transforming potential of simian virus 40 (SV40) T antigen is dependent on interaction of IGF I with its receptor and is inhibited by antisense targeting of IGF IR.¹³ Similar findings have been reported for epidermal growth factor (EGF)–stimulated mitogenesis.^{14 15} Overexpression of IGF IR has been shown to promote ligand-dependent neoplastic transformation.¹⁶ The potential participation of IGF IR in tumorigenesis has been studied for a variety of neoplasms^{17 18 19 20 21 22 23 24} and has clearly been demonstrated in Wilms' tumor.^{25 26}

The possible involvement of IGF I and IGF IR in cardiovascular pathology has recently raised interest. Thus, in vitro data have established that IGF I is a smooth muscle cell mitogen,^{27 28 29} and several reports have documented that vascular smooth muscle cells (VSMCs) in vitro and in vivo express IGF I and its receptor.^{30 31 32 33 34 35 36 37 38 39} In conditions in which there is VSMC proliferation, such as coarctation hypertension⁴⁰ or balloon angioplasty,⁴¹ there is an increase in IGF I mRNA expression. Recently, we have shown that angiotensin II (Ang II), a vasoactive peptide that is a VSMC mitogen,^{42 43 44 45} transcriptionally regulates the IGF I gene in VSMCs and that neutralization of extracellular IGF I abolishes Ang II–induced growth.⁴⁶ We and others have demonstrated that growth factors such as Ang II, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (FGF) increase IGF I receptors on VSMCs, suggesting that upregulation of IGF I receptors may play a role in growth factor–induced mitogenic responses.^{47 48}

To obtain insights into the function of IGF IR in the modulation of VSMC growth, we have cloned, sequenced, and characterized cDNAs encoding the entire rat IGF IR. Antisense IGF IR expression constructs were assembled, incorporating Epstein-Barr virus (EBV) replicative signals and a cytomegalovirus (CMV) promoter/enhancer or an inducible metallothionein promoter/enchancer, and stably transfected into primary cultures of rat aortic smooth muscle cells (RASMs). Functional analysis of these transfectants is consistent with an important role for IGF IR in VSMC proliferation in vitro. These findings provide a strong rationale for developing therapeutic strategies to target this ligand receptor system in the treatment of conditions in which VSMC proliferation and migration play a major role.

	Materials and Methods

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Cloning of IGF I Receptor cDNA
Total RNA was isolated from RASMs, and after first-strand cDNA synthesis, a 1.1-kb cDNA probe was generated by polymerase chain reaction (PCR). Primers D1 (5'-CCCCAAATAAAAGGAATG-3') and U1 (5'AGCAGATTGCCCTTC-3') were based on the published partial sequence of the rat IGF IR -chain.⁵ The PCR product was directly cloned into the pCRII vector (Invitrogen Inc) to obtain clone p118. This clone was sequenced on both strands, and results were in agreement with published data.⁵ Prior studies have indicated high-level IGF IR expression in adult rat brain.^{3 49} Accordingly, we screened a gt10 cDNA library generated from adult male Sprague-Dawley rat brain by using random and oligo(dT) primers (Clontech). Screening of 1.2x10⁶ clones identified 80 positive plaques, 30 of which were analyzed by use of PCR with universal gt10 arm primers and IGF IR internal primers to identify clones with longer inserts. After two additional cycles of screening, phage clones P611, P311, and P522 were purified and subcloned into pGEM3 vector, yielding plasmid clones p611, p311, and p522. Insert sizes were 2.6, 2.7, and 1.6 kb, respectively. The cDNA library was rescreened by using radiolabeled p611 cDNA, and phage clones P26 and P34, 1.2 and 1.3 kb in size, respectively, were purified. To obtain cDNAs encoding the 3' end of IGF IR, we synthesized a primer, based on sequence analysis of clone p26, encoding base pairs +2887 to +2906 (relative to ATG). p26 was linearized at an EcoRI site and used as a template to generate a 3'-specific probe by use of the primer and Klenow enzyme. This probe (from +2887 to +3065) was used to rescreen the library, and after PCR identification of clones with longer inserts, phage clones P384, P3121, P391, and P383 were purified, subcloned into pGEM3, and sequenced. In all, nine overlapping clones, which encompassed the full coding sequence of the rat IGF IR precursor, were obtained (Fig 1A). Sequencing was performed with the Sequenase version 2.0 kit (USB). Ambiguities were resolved by resequencing with a modified 40% formamide gel (J.T. Baker, Inc). DNA sequence data were assembled and analyzed by the Genetics Computer Group program. The deduced amino acid sequence data were analyzed PC GENE (IntelliGenetics, Inc). Sequencing strategy is depicted in Fig 1B. In addition to the protein coding sequence, 45 bp of 5' untranslated region and 538 bp of 3' untranslated region were sequenced.

View larger version (12K): [in this window] [in a new window]   Figure 1. Cloning of rat insulin-like growth factor I receptor (IGF IR) cDNAs. A, Overlapping cDNA clones were isolated from a rat brain gt10 cDNA library as described in "Materials and Methods." The 4113-bp open reading frame is depicted by the heavy bar and is flanked by untranslated 5' and 3' regions, which are depicted by thin lines. Selected restriction enzyme sites are indicated. B, All clones were sequenced using the dideoxy-chain termination method. Sequencing reactions are indicated by arrows.

Construction of Antisense Rat IGF IR Plasmids
A diagrammatic representation of vector assembly is shown in Fig 2. Briefly, the rat IGF IR cDNA clone p118 was digested by Xho I and Kpn I, and the 0.8-kb restriction fragment (from nucleotides +277 to +1086 relative to ATG) was ligated in an antisense orientation into pCEP4 or pMEP4 (Invitrogen), yielding plasmids pAnti–IGF IR and pAnti–IGF IR_i, respectively. Transcription of the antisense IGF IR cDNA is under control of the CMV immediate-early gene enhancer/promoter (pAnti–IGF IR) or of the human metallothionein IIa enhancer/promoter (pAnti–IGF IR_i) and yields a 1.0-kb antisense transcript (including linker and polyadenylated tail). The vectors contain two EBV-derived genes allowing episomal replication, namely, the EBV ori-P (origin of replication) and the EBV–nuclear antigen 1. The SV40 poly(A) tail provides a termination signal for transcription, and a hygromycin resistance gene allows selection of transfectants.

View larger version (19K): [in this window] [in a new window]   Figure 2. Construction of episomal vectors pAnti–IGF IR and pAnti–IGF IRi. The rat insulin-like growth factor receptor I (IGF IR) cDNA clone p118 was digested by Xho I and Kpn I, and the 0.8-kb restriction fragment was ligated in an antisense orientation into pCEP4 (shown), yielding pAnti–IGF IR, or into pMEP4, yielding pAnti–IGF IRi. CMV indicates cytomegalovirus immediate-early gene enhancer/promoter; SVpA, simian virus 40 poly(A); EBNA-1, Epstein-Barr virus (EBV)–encoded nuclear antigen 1; OriP, EBV origin of replication; Hyg, hygromycin resistance gene; Amp, ampicillin resistance gene; Km, kanamycin resistance gene; and IGFIR, IGF IR. pAnti–IGF IRi was constructed in a similar fashion by using pMEP4 (identical to pCEP4 but containing the human metallothionein IIa promoter).

Cell Transfection and Selection
VSMCs were isolated from rat thoracic aortas as described previously by Gunther et al.⁵⁰ Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged twice a week by harvesting with trypsin-versene and seeding at a 1:8 ratio in 75-cm² flasks. For transfections, 10% confluent passage-5 cells in 100-mm dishes were incubated in the presence of 10 µg of a calcium phosphate precipitate of carrier DNA (pRSV-CAT, Invitrogen) and the plasmids pAnti–IGF IR or pAnti–IGF IR_i in a 1:2 ratio for 6 hours. Cells were grown in hygromycin-free medium for 72 hours and then selected in the presence of hygromycin (Sigma) at a concentration of 100 µg/mL. This concentration of antibiotic was determined to effectively kill VSMCs. Selection medium was replaced every 3 days. After 6 weeks, hygromycin-resistant colonies were isolated, cloned into 24-well plates, and subsequently expanded into 12- and 6-well tissue culture plates. To confirm that hygromycin had no toxic effect on the selected clones, a growth curve of clones CA9 and ME10 was performed in the presence and absence of hygromycin (100 µg/mL), and results indicated no change in cell number with the antibiotic.

Northern Analysis
Transfected VSMCs were studied before confluence (80% confluent) and 2 days after confluence. Total RNA was prepared from transfected VSMCs by using the TRI-reagent method (Molecular Research Center, Inc), subjected to 1.2% agarose-formaldehyde gel electrophoresis, and transferred to a nylon membrane (Genescreen Plus, New England Nuclear). The filter was stained with methylene blue to monitor RNA loading and RNA integrity. A ³²P-labeled rat IGF IR sense RNA probe (120 bp, nucleotides +560 to +680) was generated by T7 polymerase transcription of the rat IGF IR cDNA clone p522, linearized with Pvu II. Hybridization conditions were as recommended by Promega, ie, 7% PEG 8000, 7% SDS, 50% formamide, 0.25 mol/L NaCl, 0.25 mol/L NaPO₄, pH 7.2, 100 µg/mL herring sperm DNA, and 100 µg/mL yeast tRNA for 24 hours at 60°C. Filters were washed at room temperature in 2x standard saline citrate (SSC) and then at high stringency (0.1x SSC, 30 minutes at 65°C) before autoradiography.

Solution Hybridization/RNase Protection Assay
For determination of IGF IR mRNA levels, a 203-bp EcoRI and Kpn I rat IGF IR cDNA fragment (from nucleotides +1839 to +2034) was ligated into pGEM3 vector. The subclone p26K was linearized with EcoRI to allow generation of antisense RNA probe using SP6 RNA polymerase. Solution hybridization/RNase protection assays were performed as previously described.^{37 38 46} In brief, 30 µg of total RNA was hybridized with 5x10⁵ cpm of [³²P]UTP-labeled antisense IGF IR riboprobe and cohybridized with a GAPDH riboprobe⁵¹ in a solution containing 80% deionized formamide, 40 mmol/L PIPES, pH 6.4, 0.4 mol/L NaCl, and 1 mmol/L EDTA. After hybridization at 42°C and RNase digestion using 40 µg/mL RNase A and 100 U/mL RNase T1, samples were proteinase K–treated, phenol-extracted, and analyzed by 6% polyacrylamide/8 mol/L urea denaturing gel electrophoresis. Full-length IGF IR probe is 251 bp and protected fragment is 195 bp. The GAPDH protected fragment is 156 bp. Autoradiography was performed for 1 to 3 days, and protected bands were quantified by two-dimensional laser densitometry.

Radioligand Binding Studies
Confluent and postconfluent (2 days) 24-well culture plates of VSMCs transfected with vector alone or with pAnti–IGF IR or pAnti–IGF IR_i were washed in PBS and incubated for 60 minutes at 37°C in binding buffer to allow dissociation of cell-bound IGF I. Cells were then rewashed and incubated for 90 minutes at 22°C in binding buffer (mmol/L: HEPES 20, NaCl 120, KCl 5, MgSO₄ 1.2, NaHCO₃ 10, CaCl₂ 1.3, and KH₂PO₄ 1.2, along with 0.25% bovine serum albumin, pH 7.4) in the presence of 10^-10 mol/L ¹²⁵I–IGF I and increasing concentrations of unlabeled IGF I (0 to 2x10^-7 mol/L). Cells were washed in ice-cold binding buffer and solubilized in 2N NaOH before counting by using an automated gamma counter with 80% efficiency. For some experiments (clones ME10, MA5, and MA7), radioligand binding studies were performed before confluence and between confluence and up to 2 days after confluence in the presence or absence of ZnSO₄ (100 µmol/L). For measurement of Ang II binding, confluent CA9 and ME10 cells were incubated with 10^-10 mol/L [¹²⁵I-Sar¹,Ile⁸]Ang II and 0 to 10^-6 mol/L unlabeled Ang II for 90 minutes at 22°C in binding buffer. Cells were washed, and bound counts were quantified as described above. All assays were performed in duplicate for each experimental point. Data were analyzed by using the LIGAND program.

Measurement of DNA Synthesis
Monolayers of confluent and up to 2 days postconfluent transfected cells in 24-well plates were exposed to fresh serum-free medium in the presence of 1 µCi/mL of [³H]thymidine for a 24-hour period. For some experiments, cells were incubated in the presence or absence of increasing doses of IGF I (0 to 100 ng/mL). Clones ME10, MA5, and MA7 were studied in the presence or absence of ZnSO₄ (100 µmol/L). Cells were washed three times with ice-cold PBS, incubated for 30 minutes with 10% trichloroacetic acid on ice, washed two times with ice-cold 95% ethanol, and allowed to air dry. Cellular radioactivity was extracted by incubation with 0.4N NaOH, and after neutralization, samples were counted by liquid scintillation spectrophotometry. All experiments were performed in triplicate for each experimental point.

Growth Curves
Transfected cells were split into 24-well culture plates at a density of 2000 cells per well and grown in the presence of 10% calf serum and 100 µg/mL hygromycin. Cells in duplicate wells were washed with PBS, harvested with PBS and 20 mmol/L EDTA, and counted manually. A growth curve with clones MA5 and ME10 was performed in the presence or absence of ZnSO₄ (100 µmol/L). Culture medium replacement and cell counts were performed every 2 days.

Materials
Recombinant human IGF I was kindly provided by Dr H.P. Guler, CIBA-GEIGY Corp. [³H]Thymidine (20 Ci/mmol), [-³²P]UTP (3000 Ci/mmol), [-³²P]dCTP (3000 Ci/mmol), and ¹²⁵I–IGF I (300 µCi/µg) were obtained from DuPont-New England Nuclear.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of cDNAs Encoding the Full-Length Rat IGF IR
Nucleotide sequence analysis of the full-length cDNA resulted in deduction of the primary protein sequence of the rat IGF IR precursor (Fig 3). The cDNA contains an opening reading frame of 4113 nt encoding a 1370–amino acid receptor precursor. Based on the structure of the human IGF IR,⁴ the first 30 amino acids likely represent signal peptide, with the putative start site of the -subunit being glutamine at position 31. Four arginine residues representing the putative cleavage site of the ß-proreceptor precede the putative start site of the ß-subunit at Asp 706. The 17–amino acid hydrophobic sequence between residues 938 and 954 is likely the transmembrane domain. Features consistent with tyrosine kinase activity are located between amino acids 1005 and 1260, including the tyrosine kinase class II signature pattern from Asp 1160 to Arg 1168 and a potential ATP-binding region from Leu 1006 to Val 1014.

View larger version (119K): [in this window] [in a new window]   Figure 3. Rat insulin-like growth factor I receptor (IGF IR) precursor nucleotide sequence and predicted amino acid sequence. Nucleotide residues are numbered beginning at the ATG initiation codon. The partial rat IGF IR -chain cDNA sequence reported by Werner et al5 corresponds to base pairs -45 to +1093 of our sequence (indicated by ). The putative start sites of the -subunit (after a 30–amino acid-signal peptide) and of the ß-subunit are indicated by arrows. The cysteine-rich domain is underlined. The putative 4-Arg receptor precursor cleavage site precedes the start site of the ß-subunit (boxed). The transmembrane domain and the tyrosine kinase domain are boxed. The protein kinase ATP binding signature (Leu 1006 to Val 1014), the tyrosine kinase–specific active-site signature (Phe 1132 to Val 1144), and the receptor tyrosine kinase class II signature (Asp 1160 to Arg 1168) are perfectly conserved between rat and human and are underlined within the tyrosine kinase domain. GenBank accession number is L29232.

Transfection of VSMCs With pAnti–IGF IR
To study the role of the IGF IR in VSMC growth, an antisense IGF IR cDNA under the control of a CMV promoter/enhancer was assembled as described in "Materials and Methods" (pAnti–IGF IR, Fig 2). A similar construct using the Zn²⁺-inducible metallothionein promoter/enhancer was also assembled (pAnti–IGR IR_i). Primary VSMCs were transfected using the calcium phosphate precipitation method,⁵² and 11 clones stably transfected with pAnti–IGF IR, 30 clones transfected with pAnti–IGF IR_i, and 25 clones transfected with vector alone were selected by hygromycin resistance and amplified. The presence of the vector sequence in transfected cells was confirmed by Southern blotting (not shown), and Northern analysis was used to screen for the presence of antisense IGF IR mRNA (not shown). Two clones (CA9 and CA11) transfected with pAnti–IGF IR exhibited high-level expression of the 1-kb antisense IGF IR mRNA, and of these, clone CA9 was selected for further study. Two clones (MA5 and MA7) transfected with pAnti–IGF IR_i showed inducible expression of antisense IGF IR mRNA transcripts and were chosen for characterization. Two clones transfected with vector alone (ME8 and ME10) were selected as controls.

Effect of Antisense IGF IR cDNA Transcription on IGF IR mRNA
To detect antisense IGF IR mRNA, Northern analysis was performed with a ³²P-labeled sense riboprobe generated by using T7 RNA polymerase transcription of a partial IGF IR sequence (nucleotides +560 to +680). As shown in Fig 4, left, CA9 cells both before and after confluence expressed abundant amounts of the 1.0-kb antisense transcript, whereas this transcript was undetectable in clone ME10. MA5 and MA7 cells likewise expressed the antisense IGF IR transcript after 48-hour exposure to ZnSO₄ (not shown). To determine the effect of antisense IGF IR RNA transcripts on endogenous IGF IR mRNA levels, solution hybridization/RNase protection assays were performed by using an antisense IGF IR riboprobe cohybridized with an antisense GAPDH riboprobe as an internal control. As shown in Fig 4, right, there was a small reduction in IGF IR mRNA levels in preconfluent CA9 cells and a marked reduction in postconfluent cells. Densitometric analysis of IGF IR mRNA levels (corrected for the GAPDH signal) indicated that CA9 cells, compared with ME10 cells, had a reduction in IGF IR mRNA of 18±4.5% (mean±SEM, n=2) before confluence and of 57±4.8% (mean±SEM, n=3) after confluence. Analysis of clones MA5 and MA7 yielded similar results. Thus, exposure of these cells to Zn²⁺ reduced IGF IR mRNA particularly after confluence, whereas exposure of control clone ME10 to Zn²⁺ had no effect on IGF IR mRNA levels (Fig 5). Analysis of densitometric data indicated that in preconfluent MA5/MA7 cells, exposure to Zn²⁺ reduced IGF IR mRNA levels by 17±4.8% (mean±SEM, n=3), whereas in postconfluent MA5/MA7 cells, exposure to Zn²⁺ reduced IGF IR mRNA levels by 39±6.2% (mean±SEM, n=3).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of antisense (AS) insulin-like growth factor receptor I (IGF IR) cDNA transcription on IGF IR mRNA levels. Left, Detection of AS IGF IR by Northern analysis. Total RNA (30 µg) from clone ME10 (transfected with vector alone [ME]) and from 80% preconfluent (Pre-) and 2-day postconfluent (Post-) clone CA9 (transfected with an AS IGF IR cDNA under control of a cytomegalovirus promoter/enhancer [CA]) were hybridized to a 32P-labeled sense IGF IR ribroprobe as described in "Materials and Methods." The 18S rRNA band (detected by methylene blue staining of membrane) is indicated. Right, Detection of IGF IR transcript by solution hybridization/RNase protection assay. Total RNA (30 µg) from preconfluent and postconfluent clones ME10 and CA9 were cohybridized to 32P-labeled AS IGF IR and GAPDH riboprobes, as described in "Materials and Methods."

View larger version (75K): [in this window] [in a new window]   Figure 5. Effect of inducible antisense insulin-like growth factor receptor I (IGF IR) cDNA transcription on IGF IR mRNA levels. ME10 cells (transfected with vector alone [ME]) and MA5 cells (transfected with an antisense IGF IR cDNA under control of a metallothionein promoter/enhancer [MA]) were grown in the absence or presence (+) of ZnSO4, and total RNA was harvested at 80% confluence (Pre) and 2 days after confluence (Post). Total RNA (30 µg) per lane was cohybridized to 32P-labeled antisense IGF IR and GAPDH riboprobes, as described in "Materials and Methods."

Effect of Antisense IGF IR cDNA Transcription on IGF IR Number and Binding Affinity
To assess the effect of antisense IGF IR cDNA transcription on IGF I binding, we performed ¹²⁵I–IGF I radioligand binding studies on CA9 cells and MA5/MA7 cells and on control ME8 and ME10 cells. Experiments were performed on cells before confluence and at confluence and for up to 2 days after confluence. Results from Scatchard analysis of displacement curves indicated that there was a 35% reduction in IGF IR number per cell in preconfluent CA9 cells compared with preconfluent control ME8/ME10 cells (CA9, 44±1.0 fmol/10⁵ cells; ME, 68±2 fmol/10⁵ cells; n=2). As shown in Fig 6A, there was a 51% reduction in IGF IR number in confluent CA9 cells compared with confluent ME8/ME10 cells. There was no significant difference in IGF IR binding affinity between CA9 cells (K_d=5.6±1.0 nmol/L, n=10) and ME8/ME10 cells (K_d=6.7±1.5 nmol/L, n=10) either before or after confluence. Radioligand binding studies of clones MA5/MA7 yielded similar results. Thus, in preconfluent MA5/MA7 cells, exposure to Zn²⁺ reduced IGF IR number by 18% (MA5/MA7, 51±1.9 fmol/10⁵ cells; MA5/MA7+Zn²⁺, 41.8±2.5 fmol/10⁵ cells; n=3). Exposure of preconfluent control ME10 cells to Zn²⁺ did not significantly alter the IGF IR number (ME10, 61±2.2 fmol/10⁵ cells; ME10+Zn²⁺, 58±3.5 fmol/10⁵ cells; n=3). As shown in Fig 7A, there was a 54% reduction in IGF IR in confluent MA5/MA7 cells after Zn²⁺ exposure, whereas Zn²⁺ treatment did not decrease IGF IR number in confluent ME10 cells.

View larger version (20K): [in this window] [in a new window]   Figure 6. Bar graphs showing the effect of antisense insulin-like growth factor receptor I (IGF IR) cDNA transcription on IGF IR number and on DNA synthesis. A, Confluent and postconfluent (2 days) monolayers of clone CA9 (transfected with an antisense IGF IR cDNA under control of a cytomegalovirus promoter/enhancer [CA]) and of clones ME8 and ME10 (transfected with vector alone [ME]) underwent 125I–IGF I radioligand binding studies as described in "Materials and Methods." Binding data were analyzed by using the LIGAND program. Shown is the mean±SEM of results from eight separate experiments. B, Confluent and postconfluent monolayers of clone CA9 and clones ME8/ME10 were incubated in serum-free medium in the presence of 1 µCi/mL [3H]thymidine for 24 hours, and incorporated counts were determined as described in "Materials and Methods." Shown is the mean±SEM of triplicate determinations from seven separate experiments.

View larger version (21K): [in this window] [in a new window]   Figure 7. Bar graphs showing the effect of inducible antisense insulin-like growth factor (IGF) I receptor (IGF IR) cDNA transcription on IGF IR number and on DNA synthesis. ME10 cells (transfected with vector alone [ME]) and MA5/MA7 cells (transfected with an antisense IGF IR cDNA under control of a metallothionein promoter/enhancer [MA]) were grown in the absence or presence (+) of ZnSO4 to confluence and up to 2 days after confluence. Cells were then used for radioligand binding studies as described in "Materials and Methods" (A) or incubated in serum-free medium in the presence of 1 µCi/mL [3H]thymidine for 24 hours, and incorporated counts were determined (B). Values are mean±SEM (n=3).

To further confirm the specificity of our results, we performed radioligand binding studies to quantify Ang II receptors on ME10 cells and CA9 cells. Analysis of two independent experiments indicated that there was no significant difference in Ang II receptor density between ME10 cells (97±4 fmol/10⁵ cells) and CA9 cells (90±3 fmol/10⁵ cells).

Effect of Antisense IGF IR cDNA Transcription on DNA Synthesis and Cellular Proliferation
To determine whether antisense RNA-mediated reductions in IGF IR density altered DNA synthesis, we measured [³H]thymidine incorporation in confluent CA9 cells and MA5 cells and in control ME10 cells, basally and in response to IGF I. As shown in Fig 6B, there was a 55% reduction in basal [³H]thymidine uptake in CA9 cells compared with ME10 cells. Likewise, exposure of MA5 cells to Zn²⁺ reduced [³H]thymidine uptake by 44%, whereas Zn²⁺ treatment of control ME10 cells did not alter DNA synthesis rates (Fig 7B). To determine whether the mitogenic response to IGF I was altered by reductions in IGF IR density, we measured DNA synthesis in confluent CA9 and ME10 cells exposed to IGF I (10 ng/mL, 24 hours). There was a 57% reduction in IGF I–stimulated [³H]thymidine incorporation in CA9 cells (441±135 dpm/10⁴ cells, n=5) compared with ME8/ME10 cells (1031±262 dpm/10⁴ cells, n=4). In addition, two dose-response experiments were performed with CA9 and ME10 cells. As shown in Fig 8, there was a marked blunting of the mitogenic response to IGF I in CA9 cells, even at very high doses of IGF I. Analysis of clone MA5 yielded a similar result. Thus, DNA synthesis in response to 10 ng/mL IGF I in MA5 cells was reduced by 58% in the presence of Zn²⁺ (MA5, 437±12 dpm/10⁴ cells; MA5+Zn²⁺, 182±9 dpm/10⁴ cells; n=3). IGF I–stimulated DNA synthesis in control ME10 cells was not altered in the presence of Zn²⁺ (ME10, 756±63 dpm/10⁴ cells; ME10+Zn²⁺, 654±21 dpm/10⁴ cells; n=3). To better characterize the effect of IGF IR antisense targeting on cell proliferation, growth curves were performed by using CA9 and ME10 cells maintained in the presence of 10% calf serum. As shown in a representative experiment (Fig 9), there was a marked inhibition of the proliferative response of CA9 cells. This antiproliferative effect became evident at day 7, as the cells reached confluence. The mean of results from three independent experiments indicated that there was a 60±3.4% reduction in cell number at day 7 after plating in CA9 cells compared with ME10 cells. Analysis of a growth curve of MA5 and ME10 cells in the presence and absence of Zn²⁺ yielded a similar result. Thus, there was a 49% reduction in cell number at 8 days in the presence of Zn²⁺ in MA5 cells (MA 5, 34.4x10⁴ cells per well; MA5+Zn²⁺, 17.5x10⁴ cells per well) but not in ME10 cells (ME10, 35.6x10⁴ cells per well; ME10+Zn²⁺, 33.8x10⁴ cells per well).

View larger version (24K): [in this window] [in a new window]   Figure 8. Bar graph showing the effect of antisense insulin-like growth factor (IGF) receptor I (IGF IR) cDNA transcription on IGF I–induced DNA synthesis. Confluent ME10 cells (transfected with vector alone [ME]) and CA9 cells (transfected with an antisense IGF IR cDNA under control of a cytomegalovirus promoter/enhancer [CA]) were incubated in the presence of increasing concentrations of human recombinant IGF I (0 to 100 ng/mL) for 24 hours, and [3H]thymidine incorporation was assessed as described in "Materials and Methods." Shown is the mean±SEM of triplicate determinations from two independent experiments.

View larger version (21K): [in this window] [in a new window]   Figure 9. Effect of antisense insulin-like growth factor receptor I (IGF IR) cDNA transcription on vascular smooth muscle cell proliferation. Bar graph shows growth of clone CA9 (transfected with an antisense IGF IR cDNA under control of a cytomegalovirus promoter/enhancer [CA]) and of clone ME10 (transfected with vector alone [ME]). Cells were seeded at a density of 2x103 cells per well and maintained in culture medium containing 10% calf serum. Shown is a representative experiment, which was repeated three times.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of full-length rat IGF IR cDNA revealed a high overall degree of similarity to the human sequence.⁴ Thus, within the protein coding sequence there is 89% similarity at the nucleotide level and 98% similarity at the amino acid level. The predicted rat -chain comprises 707 amino acids (compared with 706 for humans) with an 89% and 98% similarity to the human IGF IR -chain at the nucleotide and amino acid levels, respectively. There are 32 cysteine residues within the -chain, including 24 cysteine residues in a cysteine-rich region extending from Cys 178 to Cys 332. These are perfectly conserved between the rat and human receptors, consistent with the proposed important function of disulfide bridging in assembly of the ₂-ß₂ mature receptor. A putative cleavage site formed by four arginines (contrasting with the Arg-Lys-Arg-Arg sequence of the human receptor) after residue 737 precedes the putative start site of the ß-subunit at Asp 742. Like the -chain, the ß-chain is highly conserved between rat and human (90% and 98% similarity at nucleotide and amino acid levels, respectively). A 17–amino acid hydrophobic sequence (residues 938 to 954) likely represents the transmembrane domain and differs from the corresponding human sequence by the substitution of an isoleucine for valine at position 945. The highly conserved tyrosine kinase domain (88.9% similarity at the nucleotide level with humans) contains a protein kinase ATP binding signature (Leu 1006 to Val 1114), a tyrosine kinase specific active-site signature (Phe 1132 to Val 1144), and a receptor tyrosine kinase class II signature (Asp 1160 to Arg 1168). These sites are perfectly conserved between rats and humans, as are the positions of cysteines within the rat and human ß-chains. Like the human receptor, there are 17 potential N-glycosylation sites in the rat receptor. In contrast to the coding sequence, the 3'-untranslated region of the rat IGF IR has a lower (72%) degree of similarity to the human sequence.

IGF I has long been known to be an essential mediator of normal postnatal development, playing a central role in growth hormone–stimulated growth (reviewed in Reference 2² ). Recently, a critical role for this ligand-receptor system has been shown in embryonic growth, as evidenced by the severe growth deficiency and lethality that occurs in null mutants for these genes.^{53 54} IGF I and its receptor are widely expressed in tissues and cells and are thought to serve an important autocrine/paracrine function in the control of cellular growth and differentiation.^{1 2 3} The function of the IGF I/IGF IR pathway in regulating tumor growth has raised much interest,^{17 18 19 20 21 22 23 24} and this pathway clearly participates in the pathogenesis of Wilms' tumor.^{25 26} Evidence is also emerging for a role for this pathway in cardiovascular growth responses. Thus, VSMCs express and secrete IGF I,^{30 37} and anti–IGF I antiserum inhibits the growth of VSMCs in vitro.²⁹ In conditions in which VSMCs proliferate, such as after balloon angioplasty⁴¹ and the induction of coarctation hypertension,⁴⁰ there is an increase in vascular IGF I mRNA levels. The effects of IGF I are mediated after binding to its cell-surface high-affinity receptor rather than by interaction with intracellular receptors.⁵⁵ It has previously been shown that PDGF modulates IGF I receptor number on BALB/c3T3 fibroblasts⁵⁶ and that PDGF, FGF, and Ang II increase IGF I receptors on VSMCs.^{47 48} We have shown that FGF upregulation of IGF I receptors on VSMCs markedly increases the mitogenic response to IGF I.⁴⁸ These findings suggested that regulation of IGF IR on VSMCs could be an important determinant of their growth state.

To address the function of the IGF IR in VSMC growth, antisense IGF IR constructs were stably transfected into primary RASMs. Our results show that VSMCs constitutively expressing this antisense transcript have a decrease in endogenous IGF IR mRNA levels, particularly at confluence and after confluence. The reduction in IGF IR mRNA levels in postconfluent CA9 cells of 57% correlates well with the 51% decrease in surface IGF IR binding sites. DNA synthesis in antisense-expressing CA9 cells was decreased by 55%, indicating that a reduction in IGF IR number per cell inhibited VSMC growth. Furthermore, antisense-expressing cells had a 59% decrease in IGF I–stimulated DNA synthesis. Results obtained by using clones in which antisense IGF IR cDNA transcription was induced by exposure to Zn²⁺ indicated a reduction in IGF IR mRNA and protein levels particularly after confluence, correlating with a reduction in basal and IGF I–induced DNA synthesis. Control ME10 cells exposed to Zn²⁺ had no change in IGF IR number or basal or IGF I-induced DNA synthesis. To further confirm the specificity of our results, we determined that the Ang II receptor numbers for control and antisense-expressing cells were not different. Our findings were substantiated by growth curves that showed a marked reduction in the proliferation of CA9 cells maintained in 10% serum. Furthermore, a growth curve indicated that proliferation of MA5 cells in the presence of Zn²⁺ was inhibited by 49%, whereas Zn²⁺ exposure did not alter proliferation of control ME10 cells.

Interestingly, there was only a small decrease in IGF IR mRNA levels in antisense-expressing cells before confluence, correlating with an 18% to 35% reduction in IGF IR number. The lesser reduction in IGF IR mRNA in preconfluent cells may be related to differences in IGF IR mRNA stability in preconfluent cells or possibly to cell cycle–related changes in RNase H activity. The greater reduction in IGF IR at confluence in antisense cells correlates with a marked reduction in cellular growth rates in the presence of serum (Fig 9). Because serum contains a variety of growth factors, our results suggest that IGF IR participates in growth responses to multiple agonists. It is important to note, in this regard, that anti–IGF I antiserum has been shown to inhibit PDGF-mediated growth of VSMCs.²⁹ Recently, we have shown that Ang II transcriptionally regulates IGF I expression in VSMCs and that anti–IGF I antibody inhibits Ang II–induced DNA synthesis.⁴⁶ Furthermore, in neuroepithelial cells, FGF-mediated proliferation is dependent on IGF I.⁵⁷ It is unlikely that the antiproliferative effect of IGF IR downregulation is IGF I independent. Rather, the blunted growth response to serum probably results from inhibition of the response to exogenous IGF I (present in serum) and to autocrine/paracrine IGF I (released in response to growth factors present in serum). In this regard, we have previously demonstrated that serum increases IGF I mRNA levels in VSMCs.³⁷

IGF I has been shown to act at the G₁ phase of the cell cycle and to stimulate progression through S phase.⁵⁸ The concept that the IGF I–IGF IR interaction participates in the response to other growth factors is supported by several recent observations. Thus, in BALB/c3T3 fibroblasts, EGF has been shown to upregulate IGF I expression and secretion, and targeting of IGF IR through use of antisense oligonucleotides inhibits EGF-induced growth.¹⁴ Furthermore, in BALB/c3T3 cells overexpressing IGF I and IGF IR, IGF I–mediated growth occurs independently of the EGF and PDGF receptors.¹⁵ Constitutive expression of c-myb in 3T3 cells has been shown to upregulate IGF I and IGF IR expression, thereby abrogating the requirement of these cells for exogenous IGF I and suggesting that IGF IR activation may be important mechanistically in the effect of c-myb on cell proliferation.^{11 12} Recently, SV40 T antigen transformation of BALB/c3T3 cells has been shown to markedly increase the expression and secretion of IGF I.¹³ Antisense targeting of IGF IR inhibits the growth of SV40-transformed cells. Interestingly, these cells still require PDGF or 1% serum for growth; however, if IGF IR is overexpressed in these cells, they will grow in serum-free medium. This finding supports the concept that IGF IR number per cell is important in cellular growth responses. It is important to note that antisense targeting of the IGF I/IGF IR system has provided evidence for its role in normal organ growth⁵⁹ and in tumorigenesis.⁶⁰ Furthermore, overexpression of IGF IR has been shown to induce transformation of NIH/3T3 cells in vitro, allowing these cells to form tumors in nude mice.¹⁶

In summary, we have cloned cDNA encoding the full-length rat IGF I receptor. Comparison with the known sequence of the human receptor indicates a remarkable degree of similarity within the entire translated region. Constitutive or inducible antisense IGF IR cDNA transcription in stably transfected VSMCs decreases levels of IGF IR mRNA and IGF IR number and growth of these cells in 10% serum. These findings indicate that IGF IR plays a crucial role in VSMC proliferative responses and provides a rationale for targeting of this ligand-receptor system as a strategy to modulate vascular growth responses in vivo. Further study of antisense IGF IR VSMC clones will allow detailed analysis of the function of the IGF I/IGF IR pathway in the response of VSMCs to various agonists and, in particular, provide insights into crosstalk phenomena between various growth factor–receptor systems.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-47035, HL-45317, and DK45215 and by a grant from the American Heart Association, Georgia Affiliate, Inc (to Dr Delafontaine). Dr Delafontaine is an Established Investigator of the American Heart Association. We are grateful to Cynthia Curry for editorial assistance.

	Footnotes

 
Reprint requests to Patrick Delafontaine, MD, Division of Cardiology, Emory University, PO Drawer LL, Atlanta, GA 30322.

Received August 15, 1994; accepted February 13, 1995.

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				C. Duan and D. R. Clemmons Differential Expression and Biological Effects of Insulin-like Growth Factor-binding Protein-4 and -5 in Vascular Smooth Muscle Cells J. Biol. Chem., July 3, 1998; 273(27): 16836 - 16842. [Abstract] [Full Text] [PDF]

				R. N. N. Han, V. K. M. Han, S. Buch, B. A. Freeman, M. Post, and A. K. Tanswell Insulin-like growth factor binding proteins in air- and 85% oxygen-exposed adult rat lung Am J Physiol Lung Cell Mol Physiol, April 1, 1998; 274(4): L647 - L656. [Abstract] [Full Text] [PDF]

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				K. J. Scheidegger, B. Cenni, D. Picard, and P. Delafontaine Estradiol Decreases IGF-1 and IGF-1 Receptor Expression in Rat Aortic Smooth Muscle Cells. MECHANISMS FOR ITS ATHEROPROTECTIVE EFFECTS J. Biol. Chem., December 1, 2000; 275(49): 38921 - 38928. [Abstract] [Full Text] [PDF]

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