From the
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
[
Insulin-like growth factor I (IGF I)
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.
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.
[
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
[
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
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,
[
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
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.
We thank Cynthia Curry for editorial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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) .
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
10
M), 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.
H]Thymidine (1 µCi/ml) was present during
the latter 24 h, and trichloroacetic acid-precipitable counts were
determined as described above.
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.
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).
).
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.
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.
/S phase of the cell cycle(7) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.