©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Vascular Smooth Muscle Cell Insulin-like Growth Factor I Receptors by Phosphorothioate Oligonucleotides
EFFECTS ON CELL GROWTH AND EVIDENCE THAT SENSE TARGETING AT THE ATG SITE INCREASES RECEPTOR EXPRESSION (*)

Patrick Delafontaine (1)(§), Xiao Ping Meng (1) (2), Li Ku (1), Jie Du (1)

From the (1)Division of Cardiology, Department of Medicine, Emory University Atlanta, Georgia 30322 and the (2)Second Clinic Hospital, Norman Bethune University of Medical Sciences, Changchun, China 130021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently shown that insulin-like growth factor I (IGF I) is a mediator of angiotensin II-induced mitogenesis in vascular smooth muscle cells (Delafontaine, P., and Lou H.(1993) J. Biol. Chem. 268, 16866-16870). To study the role of the IGF I receptor in vascular smooth muscle cell growth, phosphorothioate oligonucleotides were used to modulate IGF I receptors. An antisense oligonucleotide targeting the ATG site inhibited basal and serum-induced DNA synthesis in vascular smooth muscle cells. Mismatch oligonucleotide had no effect, while surprisingly sense oligonucleotide increased IGF I receptor number and basal and serum-induced DNA synthesis. A 51% reduction in IGF I receptor number following exposure to 5 µM antisense oligonucleotide markedly inhibited angiotensin II-induced mitogenesis. A 70% increase in IGF I receptor number following exposure to 5 µM sense oligonucleotide resulted in a 4-fold increase in basal [H]thymidine incorporation, and angiotensin II (1-1000 nM) had no additive stimulatory effect. An antisense oligonucleotide targeting a sequence starting at +109 base pairs (relative to ATG) also reduced IGF I receptor number, however, the corresponding sense oligonucleotide was without effect. These findings demonstrate that alterations in vascular smooth muscle cell IGF I receptor density play a critical role in the proliferative response of vascular smooth muscle cells to serum and to angiotensin II. In addition, the surprising observation that an ATG-directed sense oligonucleotide up-regulates IGF I receptors identifies a novel effect of oligonucleotides on gene expression.


INTRODUCTION

Insulin-like growth factor I (IGF I)()is a ubiquitous peptide that regulates growth and differentiation of multiple cell types, serving a crucial function in normal development (1, 2). Vascular smooth muscle cells (VSMC) synthesize and secrete IGF I, which acts as an autocrine/paracrine factor(3, 4, 5, 6) . The action of IGF I to stimulate cell progression through G to S phase places this factor at a privileged position within the cell cycle(7) . Thus anti-IGF I antiserum inhibits platelet-derived growth factor-mediated growth of VSMC(3) . Furthermore, we have recently reported that angiotensin II (ang II), a vasoactive and mitogenic peptide, transcriptionally regulates the IGF I gene in VSMC, and that neutralization of extracellular IGF I with an anti-IGF I antibody inhibits ang II-induced DNA synthesis in VSMC(8) .

The effects of IGF I are mediated by the IGF I receptor (IGF IR), a membrane tyrosine kinase(9, 10) . Several growth factors including platelet-derived growth factor, basic fibroblast growth factor, and ang II up-regulate VSMC IGF IR(11, 12) . Preincubation of VSMC with basic fibroblast growth factor leads to increased mitogenic responsiveness to IGF I(12) . Thus regulation of IGF IR density may play a critical role in the proliferative response of VSMC to agonists. In support of this hypothesis, it has been shown that antisense targeting of the IGF IR inhibits growth of SV40-transformed BALB/c3T3 fibroblasts(13) . Furthermore, overexpression of the IGF IR has been shown to induce transformation of NIH/3T3 cells(14) . To determine whether changes in IGF IR availability were important in the regulation of VSMC growth, we used oligonucleotides (ODNs) to modulate IGF IR expression in rat aortic VSMC. This approach has been used effectively to inhibit VSMC protooncogene expression(15, 16, 17) . Our findings demonstrate unequivocally that antisense (AS) ODNs specific for the IGF IR sequence markedly alter the proliferative response of VSMC to serum and to agonists such as ang II. In addition, our data demonstrate that a sense (S) ODN specific for the ATG site of the IGF IR up-regulates VSMC IGF IR, resulting in increased mitogenesis. These data provide strong evidence that alterations in IGF IR density have an important impact on the mitogenic response to serum and to ang II. Furthermore these data support the concept of cross-talk between various growth factor receptors systems on VSMC.


EXPERIMENTAL PROCEDURES

Oligonucleotide Synthesis

Phosphorothioate 20-mer ODNs (synthesized by the Microchemical Facility, Emory University) were high performance liquid chromatography purified, resuspended in 10 mM Tris- Cl, 1 mM EDTA, pH 7.4 (TE), and quantified by spectrophotometry. ATG-directed antisense, sense, and mismatch (M) ODNs targeting a sequence starting 2 bp 5` to the ATG site were as follows: AS-1, 5`-TCCGGAGCCAGACTTCATTC-3`; S-1, 5`-GAATGAAGTCTGGCTCCGGA-3`; M-1, 5`-AGCGGTCCCACTCTTGTTTG-3`. M-1 corresponds to AS-1 with 9 of 20 bp differences. A 2`-0-methyl phosphorothioate AS-1 ODN (Me-AS-1) was also synthesized. Non-ATG-directed ODNs targeting a sequence starting at bp +109 (relative to ATG) were AS-2, 5`-CAGCTGCTGATAGTCGTTGC-3` and S-2, 5`-GCAACGACTATCAGCAGCTG-3`. Oligonucleotides were filter-sterilized and used at a concentration of 0.1-10 µM.

Cell Culture

VSMC were isolated from rat thoracic aorta by enzymatic dissociation as described previously by Gunther et al.(18) . They were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (CS), 2 mM glutamine, antibiotics, and passaged twice a week at a 1:8 ratio in 75-cm flasks. For experiments, cells between passage levels 5 and 15 were seeded into 100-mm, 24- or 48-well cluster dishes. For certain experiments, cells were rendered quiescent by exposure to defined serum-free medium (SFM) containing DMEM and Ham's F-12 (1:1) supplemented with transferrin (5 µg/ml), insulin (5 10M), ascorbate (0.2 mM), glutamine, and antibiotics.

Measurement of DNA Synthesis

To measure effects of ODNs on the growth response to 10% serum, VSMC were grown to 80% confluence in 48-well plates, serum-deprived for 48 h without or with 0.1-10 µM ATG-directed or non-ATG-directed ODNs and then exposed to fresh SFM in the absence of ODNs, or to 10% CS, for 24 h. For some experiments, cells were exposed to 10% CS with a 1/200 dilution of normal rabbit serum or polyclonal anti-IGF I antiserum (kindly provided by Drs. L. Underwood and J. J. Van Wyk through the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases. 1 µCi/ml [H]thymidine was included during the last 24 h. Cells were then washed 3 times with ice-cold phosphate-buffered saline, incubated on ice for 15 min with 10% trichloroacetic acid, and, following two washes in ice-cold 95% ethanol, radioactivity was extracted with 0.4 N NaOH for assay by liquid scintillation spectrophotometry.

To measure the effects of ODNs on the mitogenic response to IGF I, VSMC were grown to 50% confluence and then incubated in DMEM with 10% CS alone or in the presence of 5 µM AS-1 or S-1 ODNs for 48 h. Cells were then washed in SFM and incubated in SFM with or without IGF I (1-50 ng/ml) for 24 h. [H]Thymidine (1 µCi/ml) was present during the latter 24 h, and trichloroacetic acid-precipitable counts were determined as described above.

To measure the effects of ODNs on the mitogenic response to ang II, VSMC were exposed to 5 µM AS-1, M-1, or S-1 ODNs for 48 h in the presence of 10% CS, washed in SFM, and exposed to SFM without or with 1-1000 nM ang II for 24 h. Cells were then incubated with [H]thymidine (1 µCi/ml) for 12 h in the continued presence of ang II. Trichloroacetic acid-precipitable counts were then determined.

Growth Assay

VSMC were grown to 50% confluence in 48-well plates and then exposed to SFM alone, 10% CS alone or 10% CS with 0.1-10 µM AS-1, M-1, or S-1 ODNs. Medium and ODNs were replaced at 48 h. At 96 h, cells were trypsinized and counted.

Binding Assays

To determine the effect of ODNs on IGF IR number and binding affinity, VSMC were grown to 80% confluence in 24-well plates and then exposed to SFM alone or with 5 µM AS-1, Me-AS-1, AS-2, M-1, S-1, or S-2 ODNs for 48 h prior to performing binding assays. For some experiments, cells were grown to 50% confluence and then exposed to 10% CS alone or with 5 µM AS-1, Me-AS-1, AS-2, S-1, or S-2 ODNs for 48 h, and binding assays were performed. To determine the effect of S-1 ODNs on ang II up-regulation of IGF IR, 50% confluent cells were preincubated in 10% CS alone or with 5 µM S-1 or M-1 ODNs for 48 h, and then exposed to SFM with or without 100 nM ang II for 24 h, prior to binding assays. Assays were performed by incubating cells with 0.1 nMI-IGF I and 0-0.1 µM unlabeled IGF I for 90 min at room temperature. Cells were washed in ice-cold binding buffer and solubilized in 2 N NaOH before counting. All assays were performed in duplicate for each experimental point. Data were analyzed using the LIGAND program. For measurement of ang II binding, cells were incubated for 48 h in 10% CS alone or with 5 µM AS-1, S-1, or M-1 ODNs. Binding studies were then performed essentially as described above, except 0.1 nM [I-Sar-Ile]ang II and 0-0.1 µM unlabeled ang II were used for displacement curves.

Solution Hybridization/RNase Protection Assay

For determination of IGF IR mRNA levels, a 203-bp EcoRI and KpnI cDNA fragment (containing 195 bp of the rat IGF IR cDNA sequence) was ligated into pGEM3(19) . The subclone p26K was linearized with EcoRI to allow generation of antisense RNA probe using SP6 RNA polymerase. The full-length antisense IGF IR probe (251 bp) includes a 56-bp flanking sequence. Solution hybridization assays were performed by hybridizing 30 µg of total RNA with 5 10 cpm [P]UTP-labeled AS IGF IR riboprobe and co-hybridizing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe as described previously(8) . Following RNase digestion, samples were analyzed by 6% polyacrylamide, 8 M urea denaturing gel electrophoresis. The IGF IR and glyceraldehyde-3-phosphate dehydrogenase-protected fragments are 195 and 156 bp, respectively. Autoradiographic bands were quantitated by two-dimensional laser densitometry.

Materials

Recombinant human IGF I was kindly provided by Dr. H.P. Guler, CIBA-GEIGY Corp., Summit, NJ. [H]thymidine (20 Ci/mmol), [-P]UTP (3000 Ci/mmol), I-IGF I (300 µCi/µg), and [I-Sar-Ile]ang II (2200 Ci/mmol) were obtained from DuPont NEN. Angiotensin II was purchased from Sigma.


RESULTS

Effect of ATG-directed-ODNs on Growth Response to Serum

As shown in Fig. 1, incubation of VSMC with increasing concentrations of AS-1 ODNs for 48 h in SFM decreased [H]thymidine incorporation (64% decrease with a concentration of 10 µM, compared with SFM alone, p < 0.01). In addition, the mitogenic response to 10% CS was inhibited (61% decrease with a concentration of 10 µM, compared with 10% CS alone, p < 0.025). There was no significant change in either basal or serum-induced DNA synthesis rates in cells exposed to M-1 ODNs. Incubation of VSMC in SFM for 48 h with S-1 ODNs significantly increased [H]thymidine incorporation, 118% increase above SFM alone, with a concentration of 10 µM S-1 ODNs (p < 0.05). Furthermore, the mitogenic response to 10% CS was maintained in cells preexposed to S-1 ODNs. Thus, the incorporation of [H]thymidine into cells exposed to 10 µM S-1 ODN for 48 h and then to 10% CS was 61% greater (p < 0.05) than that of cells exposed to SFM alone for 48 h and subsequently to 10% CS. In order to establish correlations between DNA labeling indices and cell proliferation, cell counts were performed. Treatment of VSMC with 10 µM AS-1 ODNs for 96 h in the presence of 10% CS reduced cell number by 64% compared with cells maintained in 10% CS alone (p < 0.01, Fig. 2). M-1 ODNs had no effect on cell proliferation, whereas S-1 ODNs increased cell number (28% increase with 10 µM S-1 ODN compared with 10% CS alone, p < 0.025). To demonstrate that the effect of S-1 ODNs to increase DNA synthesis was indeed related to an autocrine effect mediated by interaction of IGF I with its receptor, we measured DNA synthesis in S-1 ODN-exposed cells in the presence of anti-IGF I antibody. As shown in Fig. 3anti-IGF I antibody blocked the ability of S-1 ODNs to increase DNA synthesis.


Figure 1: Effect of ATG-directed IGF IR ODNs on DNA synthesis. VSMC were incubated in SFM alone or with 0.1-10 µM AS-1, M-1, or S-1 ODNs for 48 h. [H]Thymidine incorporation was then determined basally (in the continued presence of SFM) or in response to 10% CS. Results, expressed as percent of that in SFM, are the mean ± S.E. of values from three to 10 experiments for each condition.




Figure 2: Effect of ATG-directed IGF IR ODNs on proliferation of VSMC. 50% confluent VSMC were exposed to SFM, DMEM with 10% CS alone, or DMEM with 10% CS and 0.1-10 µM AS-1, M-1, or S-1 ODNs. Cell counts were determined at 96 h. Shown is the mean ± S.E. of duplicate measurements from four experiments.




Figure 3: Effect of anti-IGF I antiserum on S-1 ODN induced DNA synthesis. VSMC were incubated in SFM alone (Control) or with 5 µM S-1 ODNs for 48 h. [H]Thymidine incorporation was then determined in 10% CS alone or with a 1/200 dilution of normal rabbit serum (NRS) or polyclonal anti-IGF I antiserum (IGF I). Results are the mean ± S.E. of values from four experiments.



Mitogenic Response to IGF I

To assess the effects of ATG-directed-ODNs on IGF I-induced growth responses, cells were incubated in 10% CS with or without ODNs for 48 h and then exposed to SFM with or without increasing concentrations of IGF I (Fig. 4). IGF I caused a dose-dependent increase in [H]thymidine incorporation that was markedly blunted by pre-incubation of cells with AS-1 ODNs. Thus [H]thymidine incorporation stimulated by 20 ng/ml IGF I was reduced by 62% in AS-1 ODN-treated cells, compared with control (p < 0.025). Exposure of cells to 5 µM S-1 ODNs increased [H]thymidine incorporation basally (176% above control, p < 0.01) and in response to IGF I (100% increase above control following incubation with 20 ng/ml IGF I, p < 0.01).


Figure 4: Effect of ATG-directed IGF IR ODNs on mitogenic response to IGF I. VSMC were incubated for 48 h in DMEM containing 10% CS alone (Control) or with 5 µM AS-1 or S-1 ODNs. Cells were then exposed to fresh SFM with 0-50 ng/ml human recombinant IGF I, and [H]thymidine incorporation was determined. Results are the mean ± S.E. of duplicate determinations from four experiments.



Mitogenic Response to Ang II

We have previously shown that ang II increases IGF I secretion from (8) and up-regulates IGF I receptors on VSMC (12) and that a neutralizing antibody against IGF I inhibits ang II-induced DNA synthesis in VSMC(8) . These findings suggested that activation of the IGF IR was required for ang II-induced growth responses in VSMC. We therefore assessed the effects of ATG-directed ODNs on ang II-induced [H]thymidine incorporation. As shown in Fig. 5, ang II caused a dose-dependent increase in DNA synthesis that was unaltered by preexposure of cells to M-1 ODNs but markedly reduced following preexposure to AS-1 ODNs. Thus, there was a 81% reduction in [H]thymidine incorporation in response to 100 nM ang II in AS-1 ODN-treated cells compared with control (p < 0.05). As expected, S-1 ODN-treated cells had a marked increase in [H]thymidine incorporation basally (297% above control). However, ang II had no additive mitogenic effect on S-1 ODN-treated cells.


Figure 5: Effect of ATG-directed IGF IR ODNs on mitogenic response to ang II. VSMC were incubated for 48 h in DMEM containing 10% CS alone (Control) or with 5 µM AS-1, M-1, or S-1 ODNs. Cells were then exposed to fresh SFM containing 0-1000 nM ang II for 36 h, and [H]thymidine incorporation was measured. Results are the mean ± S.E. of duplicate measurements from three to seven experiments for each condition.



Effect of ATG-directed-ODNs on IGF IR Number

To establish correlations between the observed effects of ODNs on growth and IGF IR binding parameters, radioligand binding experiments were performed. Exposure of VSMC to 5 µM AS-1 ODNs for 48 h in SFM reduced IGF IR number by 52%, compared with control (Fig. 6A). M-1 ODNs had no effect on IGF I binding, whereas 5 µM S-1 ODNs increased IGF IR number by 42%. Exposure of VSMC to ATG-directed ODNs in the presence of 10% CS produced similar results (Fig. 6B). Thus 5 µM AS-1 ODNs reduced IGF IR by 51%, and 5 µM S-1 ODNs increased IGF IR by 70%, compared with 10% CS alone. There was no effect of IGF IR ODNs on IGF I binding affinity (K control, 2.7 ± 0.4 nM; K AS-1, 2.3 ± 0.4 nM; K M-1, 3.7 ± 1.4 nM; K S-1 3.1 ± 0.7 nM, mean of results from experiments performed in SFM and 10% calf serum, n = 5-8). To demonstrate the specificity of these findings [I-Sar-IIe]ang II binding studies were performed on cells exposed to IGF IR ODNs. AS-1, S-1, and M-1 ODNs had no effect on ang II receptor number (Fig. 5C) nor affinity (not shown).


Figure 6: Effect of ATG-directed IGF IR ODNs on IGF IR and ang II receptor number. VSMC were incubated in SFM (A) or in DMEM with 10% CS (B and C) alone (Control), or in the presence of 5 µM AS-1, M-1, or S-1 ODNs for 48 h. I-IGF I (A and B) and [I-Sar-Ile]ang II (C) displacement binding experiments were performed and data analyzed using the LIGAND program. Results shown are the mean ± S.E. of duplicate determinations from three to five separate experiments for each condition. *, p < 0.05, compared with control;**, p < 0.025, compared with control.



To determine whether the effect of AS-1 ODNs to down-regulate IGF IR was dependent on RNase H-mediated RNA cleavage, we performed binding assays using cells preincubated in SFM alone or with 5 µM Me-AS-1 ODNs. The RNase H-resistant ODN did not reduce IGF I receptors: control, 18.0 fmol/10 cells; Me-AS-1, 17.9 fmol/10 cells (mean of results from two independent experiments). To determine whether S-1 ODNs altered the ability of ang II to up-regulate IGF I receptors, we performed binding assays on cells exposed to SFM with or without 100 nM ang II, following a prior 48-h exposure to 10% CS with or without 5 µM S-1 or M-1 ODNs. Ang II up-regulated IGF I receptors by 48% in control cells (- ang II, 22 fmol/10 cells, + ang II, 32.6 fmol/10 cells), but not in cells exposed to S-1 ODNs (- ang II, 38.5 fmol/10 cells; + ang II, 34.7 fmol/10 cells) (mean of results from two independent experiments). The loss of the ability of ang II to up-regulate IGF IR was specific for the S-1 ODN because cells preincubated with M-1 ODNs showed the expected response to ang II: - ang II, 26.8 fmol/10 cells; + ang II, 35.6 fmol/10 cells, 33% increase (mean of results from two independent experiments).

Effects of Non-ATG-directed IGF IR ODNs

To determine whether the observed effects of S-1 ODNs on VSMC IGF IR number and growth responses were site-specific, experiments were conducted using AS-2 and S-2 oligomers targeting a sequence starting at bp +109 (relative to ATG). As shown in Fig. 7A AS-2 ODNs produced the expected reduction in [H]thymidine incorporation. Thus 10 µM AS-2 ODNs reduced DNA synthesis by 51% basally (p < 0.01) and by 63% in response to 10% CS (p < 0.05). However, in marked contrast to results obtained using S-1 ODNs specific for the ATG site, S-2 ODNs specific for a sequence 3` to the ATG site did not significantly alter DNA synthesis either basally or in response to serum. Furthermore radioligand binding studies established that these ODNs had the expected effects on IGF IR. Thus, exposure of VSMC to 5 µM AS-2 ODNs in the presence of 10% CS reduced IGF IR number by 37%, while there was no significant effect of S-2 ODNs on IGF IR number (Fig. 7B). Furthermore, exposure of VSMC to 5 µM AS-2 ODNs in SFM for 48 h reduced IGF IR number by 29% (mean of results from two independent experiments), whereas corresponding S-2 ODNs had no significant effect on IGF IR number.


Figure 7: Effect of non-ATG-directed ODNs on VSMC DNA synthesis and IGF IR number. A, VSMC were incubated in SFM alone or in the presence of AS-2 or S-2 ODNs (0.1-10 µM) for 48 h. [H]Thymidine incorporation was then determined basally (in the continued presence of SFM) or in response to 10% CS. Results are expressed as percent of SFM and are the mean ± SE of duplicate measurements from three to eight experiments for each condition. B, VSMC were incubated in 10% CS alone (Control), or in the presence of 5 µM AS-2 or S-2 ODNs for 48 h, and radiolabeled IGF I binding experiments were performed. Results are the mean ± S.E. of duplicate measurements from three separate experiments for each condition. *, p < 0.05, compared with control.



Effects of IGF IR ODNs on IGF IR mRNA Levels

To determine whether effects of ODNs on IGF IR number correlated with changes in steady-state levels of IGF IR mRNA, solution hybridization/RNase protection assays were performed (Fig. 8). Exposure of VSMC to 5 µM ATG-directed AS-1 ODNs in SFM caused a gradual decrease in IGF IR mRNA levels (p < 0.01 at 48 h). M-1 ODNs did not significantly alter IGF IR mRNA levels, whereas S-1 ODNs caused a transient increase in IGF IR mRNA, peaking at 12 h (p < 0.01). Non-ATG-directed AS-2 ODNs also decreased IGF IR mRNA levels (results not shown).


Figure 8: Effects of ATG-directed IGF IR ODNs on IGF IR mRNA Levels. A, representative solution hybridization assay. 30 µg of total RNA from VSMC incubated in SFM alone or with 5 µM S-1 ODNs for 12 h were co-hybridized using [P]UTP-labeled IGF IR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense riboprobes. B, time-course, densitometric analysis. VSMC were incubated for 6-48 h in SFM alone or with 5 µM AS-1, M-1, or S-1 ODNs. Total RNA was extracted and co-hybridized using [P]UTP-labeled IGF IR and glyceraldehyde-3-phosphate dehydrogenase antisense riboprobes. IGF IR mRNA levels (corrected for glyceraldehyde-3-phosphate dehydrogenase) are shown as percent change, compared with SFM (mean ± S.E., n = 3-6 for each condition).




DISCUSSION

Our findings clearly demonstrate that IGF IR density on VSMC is an important determinant of their growth responses to serum and to ang II. Thus, exposure of VSMC to ODNs complementary to a sequence of the IGF IR spanning the initiation codon as well as the sequence 109 bp downstream reduces IGF IR mRNA levels and IGF IR number without altering IGF IR binding-affinity (K). This results in a decrease in basal [H]thymidine incorporation as well as in the mitogenic response to the addition of 10% serum. The antiproliferative effect of the AS ODNs is reflected in the reduction of cell counts observed after 96 h of incubation of VSMC with increasing doses of AS-1 ODNs in the presence of 10% CS. Because serum contains a variety of growth factors, we measured DNA synthesis rates in response to IGF I and demonstrated that [H]thymidine incorporation in response to IGF I was inhibited dose-dependently by AS-1 ODNs. The mitogenic response to ang II was also inhibited by AS-1 ODNs, confirming the crucial role of the autocrine IGF I ligand-receptor system in ang II-induced growth of the VSMC. Thus, we have previously demonstrated that an anti-IGF I antibody inhibits ang II-induced mitogenesis in VSMC(8) . The effect of AS-1 IGF IR ODNs on ang II mitogenesis was specific, in that ang II binding was not altered.

In contrast to findings with AS-1 ODNs, a M-1 ODN (containing the antisense sequence with a 9 of 20 bp mismatch) had no effect on IGF IR number, [H]thymidine incorporation (basally or in response to serum), cell proliferation, and the mitogenic response to ang II. These data confirm the specificity of findings obtained with the AS-1 ODNs. However, unexpectedly, we found that a S-1 oligomer specific for the sequence spanning the initiation codon of the IGF IR resulted in an increase in IGF IR number, associated with an increase in [H]thymidine incorporation basally (in SFM) as well as in the presence of 10% CS. This was reflected in an increase in cell proliferation. The increase in IGF IR induced by S-1 ODNs was associated with an increase in the mitogenic response to IGF I. Interestingly, however, ang II had no additive stimulatory effect on [H]thymidine incorporation following preincubation of cells with S ODNs. This was not due to an inhibitory effect of the S-1 ODNs on ang II-induced IGF I secretion, because AS-1, MS-1, and S-1 ODNs do not alter either basal or ang II-stimulated IGF I secretion from VSMC.()Rather the lack of ang II-induced DNA synthesis in cells preexposed to the S-1 ODN is likely due to the inability of ang II to further up-regulate IGF IR in this condition. Our findings thus suggest that the mitogenic effect of ang II on VSMC is critically dependent on its ability to up-regulate IGF IR. It is important to note that the stimulatory effect of S-1 ODNs on DNA synthesis required interaction of IGF I with its receptor, since it was blocked by an anti-IGF I neutralizing antibody.

The lack of an effect of a 2`-0-methylated (RNase H-resistant) AS-1 ODN on IGF IR indicates that RNase-H mediated RNA cleavage, rather than translational inhibition, is the primary mechanism of action of the AS-1 ODN leading to down-regulation of IGF IR. Molecular mechanisms responsible for S-1 ODN induced increases in IGF IR number are not completely elucidated at present but could include effects on transcription, mRNA stability, and/or translation. The increase is associated with a transient increase in IGF IR mRNA levels and is site-specific. Thus, although the AS-2 ODNs targeting a sequence at bp + 109 (relative to ATG) reduced IGF IR number and inhibited VSMC growth, the S-2 ODNs targeting this site had no effect on IGF IR number nor VSMC growth. A potential explanation for S-1 ODN mediated up-regulation of IGF IR would be interaction with an endogenous IGF IR antisense mRNA species in VSMC. However, known antisense RNAs in eukaryotic systems are rare(20, 21, 22) , although an antisense transcript specific for basic fibroblast growth factor has been detected in Xenopus oocytes and human oocytes(23, 24) . Alternatively, the sense oligonucleotides could compete with IGF IR mRNA transcripts for binding to an mRNA-binding protein that acts as a translational repressor. One may also speculate that ATG-directed S ODNs could form triple helix structures or hybridize to the open loop created by RNA polymerase and alter transcription by facilitating DNA helix opening. Indeed, DNA helix openings have been shown to correlate with DNA template activity, and clearly epigenetic RNA molecules are capable of stabilizing these openings(25) . Sense ODNs could also potentially bind to transactivating factors. The increase in IGF IR mRNA levels in response to the S-1 ODNs is consistent with an increase in IGF IR transcription rates.

Our data indicating a key regulatory role of the IGR IR in VSMC growth are consistent with several recent observations in fibroblasts. Thus in BALB/c3T3 fibroblasts, epidermal growth factor up-regulates IGF I expression and secretion and down-regulation of the IGF IR through use of antisense oligonucleotides inhibits epidermal growth factor-induced growth(26) . Furthermore, in BALB/c3T3 cells overexpressing IGF I and IGF IR, IGF I-mediated growth occurs independent of the epidermal growth factor and platelet-derived growth factor receptors(27) . Constitutive expression of c-myb in 3T3 cells has been shown to up-regulate 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(28, 29) . Moreover, in SV40 T antigen-transformed BALB/c3T3 cells, use of antisense oligonucleotides to downregulate the IGF I receptor has been shown to inhibit growth (13). Our findings provide strong evidence that IGF IR density is an important factor in mediating ang II and serum-induced growth responses in VSMC. Even in the presence of high concentrations of IGF I, the mitogenic response of AS-1 ODN-treated cells is markedly blunted. The data suggest that IGF I, acting through its tyrosine-kinase receptor, may serve as an important co-factor or intermediary in the growth response to a variety of agonists. This is consistent with its known effects at the G/S phase of the cell cycle(7) .

In summary, our findings demonstrate that manipulation of IGF IR density on VSMC markedly alters the growth responses of these cells to IGF I, ang II, and serum. These findings establish that this ligand-receptor system is crucial for the control of VSMC growth in vitro and demonstrate that receptor availability is a critical determinant of growth responses of these cells. Furthermore, the surprising observation that a S ODN specific for the sequence spanning the initiation codon up-regulates IGF IR, identifies a novel effect of synthetic oligonucleotides on gene expression. Current studies are aimed at further characterizing molecular mechanisms involved in these effects and at defining the role of this growth factor in mediating vascular proliferative responses in vivo.


FOOTNOTES

*
This work is supported by National Institutes of Health Grants HL47035, HL45317, and DK 45215, and by a grant from the American Heart Association, Georgia Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: Div. of Cardiology, Dept. of Medicine, P. O. Drawer LL, Emory University, Atlanta, GA 30322. Tel.: 404-727-8119; Fax: 404-727-3330.

The abbreviations used are: IGF I, insulin-like growth factor I; ang II, angiotensin II; AS, antisense; IGF IR, IGF I receptor; M, mismatch; ODN(s), oligonucleotide(s); S, sense; SFM, serum-free medium; VSMC, vascular smooth muscle cell(s); CS, calf serum; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; Sar, sarcosine.

P. Delafontaine, unpublished results.


ACKNOWLEDGEMENTS

We thank Cynthia Curry for editorial assistance.


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