Insulin, Insulin-Like Growth Factor I
and Platelet-Derived Growth Factor
Interact Additively in the Induction of
the Protooncogene c-myc and
Cellular Proliferation in Cultured
Bovine Aortic Smooth Muscle Cells
Nirmal K. Banskota*, Rebecca Taub, Karen Zellner, and G. L. King
Research Division
Joslin Diabetes Center
Department of Medicine
Brigham and Women's Hospital
and Harvard Medical School
Boston, Massachusetts 02215
Department of Human Genetics (R.T.)
Howard Hughes Medical Institute
University of Pennsylvania
Philadelphia, Pennsylvania 19104
Vascular smooth muscle cell (SMC) growth is under
the influence of various growth factors. We demonstrate that platelet-derived growth factor (PDGF)
stimulates DNA synthesis of cultured bovine aortic
SMCs by 2.5- to 3.5-fold. PDGF also exhibits additivity with insulin and insulin-like growth factor I (IGFI) for DNA synthesis and cellular proliferation. Insulin
(2 x 1<r6 M), IGF-I (1 x 10"8 M), and PDGF (1 x 10"9
M) cause a 60-80% increase in cell numbers over
basal, but PDGF with insulin or IGF causes a 40150% increase over basal. No additivity between
insulin and IGF-I is evident. PDGF also induces commitment to DNA synthesis earlier than insulin or IGFI. After exposure to PDGF for 4 h, SMCs incorporate
3
H-thymidine to 60% of maximum (with PDGF alone)
levels (achieved after exposure of 12 h or longer).
Insulin and IGF-I exposure for 4 h, on the other hand,
achieves 3H-thymidine incorporation that is only a
20-30% of maximum (with insulin or IGF-I alone).
Insulin, IGF-I, and PDGF increase mRNA levels of
the protooncogene c-myc. This induction begins
within 30 min of exposure to these growth factors
which causes a 4- to 6-fold increase in c-myc mRNA
levels. Additivity is also observed between PDGF
with insulin or IGF-I, but not between insulin or IGFI, in c-myc induction. C-myc mRNA levels remain
elevated as long as the hormones are present, although there's a tendency for the mRNA levels to
fall off with insulin and IGF-I. These data suggest
that c-myc induction is an early marker for growth in
response to these growth factors. Furthermore,
these growth factors interact in the regulation of
SMC growth, but do so via discrete pathways as
evidenced by additivity in growth and c-myc induction. Characterization of insulin, IGF-I, and PDGFs
growth effect on cultured vascular SMCs and on
specific genes such as c-myc which may be associated with growth control, may provide an understanding of the abnormal growth of SMCs in atherosclerosis. (Molecular Endocrinology 3: 1183-1190,
1989)
INTRODUCTION
Vascular smooth muscle cell (SMC) proliferation is the
dominant cellular lesion in atherosclerotic plaques (1).
The extent of this proliferation is an important determinant of the clinical consequences of atherosclerosis (2).
Although the exact nature of growth regulation of SMCs
in physiology or in disease is incompletely understood
currently, growth factors are thought to play a central
role (3). Platelet-derived growth factor (PDGF), a cationic glycoprotein (4), is a potent mitogen (5), as well as
a chemotactic agent for SMCs (6). Insulin-like growth
factor I (IGF-I), a 7500 dalton polypeptide with structural
similarities with proinsulin (7), and insulin, both stimulate
mitosis in smooth muscle cells (8, 9) in vitro. PDGF and
IGFs also interact to regulate growth in these cells (10).
The mechanism of action of these agents in smooth
0888-8809/89/1183-1190S02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
1183
Vol 3 No. 8
MOL ENDO-1989
1184
muscle cell growth is not established. Induction of the
protooncogene c-myc is recognized, however, to be an
early event in the mitotic effects of growth factors,
including PDGF, in lymphocytes and fibroblasts (11).
To further define the characteristics of SMC growth
regulation, we have used cultured bovine aortic SMCs
in response to PDGF, insulin, and IGF-I. We demonstrate that each of these agents are mitogens, and they
also interact synergistically to enhance growth. As an
early marker in their growth effect, each of these growth
factors increases levels of c-myc RNA levels (11). PDGF
demonstrates additivity with insulin and IGF-I in both cmyc induction and mitosis. No additivity is observed
between insulin and IGF-I in any growth parameter,
suggesting a common mechanism of action (8). Differences are observed between PDGF and IGF-I (and
insulin) in the temporal pattern of commitment to DNA
synthesis after exposure to these growth factors.
These data indicate to us that PDGF and IGF-I (and
insulin) interact to stimulate growth in cultured bovine
aortic SMCs via separate pathways.
RESULTS
DNA Synthesis and Cellular Proliferation
The effects of insulin and IGF-I on DNA synthesis and
cellular proliferation on bovine aortic smooth muscle
cells has been previously characterized by us (8). The
effects of PDGF are illustrated in Fig. 1. 3H-Thymidine
incorporation (top panel) is stimulated 2.5-fold by 1.5 x
10~10 M PDGF (5 ng/ml). A maximal stimulation of 3.5fold increase over basal is caused by concentrations of
3 x 10"10 M (10 ng/ml) and higher. Cell number changes,
shown in the bottom panel, follow similar dose responses. At 1.5 x 10~10 M, a 75% increase over basal
is observed while a 90% increase in cell number is seen
at 3 x 1O~10 M (ng/ml) and higher concentrations.
To examine whether PDGF, IGF-I, and insulin stimulated DNA synthesis and cell growth occur through
common or discrete pathways, additive dose responses
were done. As illustrated in Fig. 2, insulin (1 x 10~6 M)
causes a 60% increase, IGF-I (1 x 10~8 M) a 70%
increase and PDGF (1 x 1O~9 M) a 90% increase over
basal in cell numbers. Insulin and IGF-I, each at optimal
concentrations when added simultaneously cause a
75% increase over basal. Thus, insulin and IGF-I do not
exhibit additivity. This is in agreement with previous
data on human fibroblasts (12) and supports our observation on human smooth muscle cells that insulin
and IGF-I act through the IGF-I receptor to cause
growth (13); PDGF, on the other hand, causes a 150%
increase over basal with insulin and 140% with IGF-I,
thus clearly demonstrating additivity with insulin and
IGF-I. Similar additive effects between PDGF and insulin
and IGF-I (but not between insulin and IGF-I) occurs for
DNA synthesis as well (data not shown).
Figure 3 illustrates the time course of exposure to
various growth factors that is required before vascular
PDGF
CONCENTRATION
Fig. 1. Growth Effects of PDGF on Aortic Smooth Muscles
Bovine aortic smooth muscle were cultured to subconfluence, and then serum deprived for 18 h. The cells were then
stimulated with PDGF in varying concentrations, and 3H-thymidine incorporation measured (top panel), as described in
Materials and Methods. Cellular proliferation, in response to
PDGF is shown in the bottom panel. Cells were plated at a
density of 1 x 103/well, stimulated by different concentrations
of PDGF and cell number changes quantified. These data
illustrate the mean of triplicate cultures from a typical experiment with the SE bars plotted as well.
smooth muscle cells commit themselves to DNA synthesis. A 2-h exposure to PDGF (1 x 10~9 M), followed
by PDGF removal, causes an increase in DNA synthesis
which is 50% of the maximum (achieved when exposure to PDGF is for 12 h or longer). A 2-h exposure to
insulin or IGF-I elicits a response which is only 20% of
maximum. At least 4-6 h of exposure to insulin and
IGF-I is required before greater than 50% of the maximum effect on DNA synthesis is achieved. After 4-6 h
of exposure, IGF-I causes a greater increase in DNA
synthesis than insulin, but lags behind PDGF between
2-8 h of exposure. Beyond 8 h of exposure, most cells
exposed to PDGF, insulin, or IGF-I seem committed to
DNA synthesis and removal of these growth factors still
causes maximal increases in 3H-thymidine incorporation. Thus, shorter exposure time is required for PDGF
to commit SMCs to DNA synthesis than for insulin and
IGF-I.
1185
Protooncogene Expression and Growth in Vascular Muscle Cells
x
II
O
INS
IGF-I
INS
IGF-I
Fig. 2. Additivity of PDGF with Insulin and IGF-I in Bovine
Aortic SMC Proliferation
Cells were plated at a density of 1 x 103 cells per well and
stimulated with insulin (1 x 10~7 M), IGF-I (1 x 1O~8 M) and
PDGF (1x10~ 9 M) and in combination (insulin and IGF-I, insulin
and PDGF, and IGF-I and PDGF, each at the above concentrations). Cell number changes were quantified, as described in
Materials and Methods. The mean ± SEM of triplicate cultures
from a sample experiment is illustrated here, and has been
reproduced in four different experiments. The differences between any single growth factor and combination with PDGF
are significant with P < 0.01. Interexperimental differences
were not more than 15%.
nonmuscle /?- and 7-actins in these cells (2,14,15) (Fig.
4). The predominant actin mRNA detected in our bovine
aortic culture system is a 2.1 kilobase (kb) species,
which migrates just above the 18S ribosomal RNA (1.9
kb). This species in rat myoblasts has been shown to
be a mixture of mRNAs coding for both the /?- and 7actins (16). The muscle a-actin mRNA which is 1.5 kb
long (16) was not detected in our assay. IGF-I ( 1 x 1 0 " 8
M) and PDGF (1 x 10~9 M) (stimulate this 2.1 kb species
of mRNA 8- to 10-fold over basal. Furthermore, this
induction is relatively rapid because the dose-response
was done for 2 h only. IGF-I and PDGF also demonstrate additivity, causing a 15-fold increase over basal.
Insulin also stimulates this species of actin and demonstrates additivity with PDGF, but not IGF-I (data not
shown). Insulin, however, is only half as potent as IGFI or PDGF.
c-myc mRNA Levels
The dose response, time course of induction and additivity of insulin, IGF-I and PDGF on c-myc mRNA levels
in bovine aortic SMCs was assessed by Northern analysis of total RNA obtained from these cells after stimulation with the hormones. Figure 5 shows the dose
response of insulin (5A), IGF-I (5B), and PDGF (5C).
Although the predominant band is the 2.3 kb c-myc
mRNA band, the c-myc unspliced precursor band at
5.1 kb is also visualized, but it is also possible that this
may represent adherence of the probe to 28S ribosomal
RNA. Insulin causes a 2- to 3-fold increase in c-myc
mRNA levels at 1 x 10~9 M and 5- to 6-fold increase at
1 x 10~7 M. IGF-I causes a 3-fold increase at 1 x 10~9
M and a near 7-fold increase at 1 x 1O~7 M. Although
the maximal effects on c-myc induction achieved at
IGF-I PDGF
6
8
IO
TIME (HOURS)
Fig. 3. Time Course of Preincubation Periods Required for
DNA Synthesis on Stimulation of Bovine Aortic SMCs by
Insulin, IGF-I, and PDGF
Subconfluent cultures were serum deprived for 18 h. Then
insulin (1 x 10"7 M), IGF-I (1 x 10"8 M), or PDGF (1 x 10~9 M)
was added for various durations, then washed off. Serum-free
medium was replaced and at the end of 24 h, the cells were
pulsed with 3H-thymidine and incorporation was measured (as
described in Materials and Methods). 3H-Thymidine incorporation is expressed as a percentage of the incorporation
obtained after exposure to hormone for 24 h.
Messenger RNA Studies
We have measured the expression of a and /3-actin in
cultured SMCs since the previous reports have shown
the loss of normal muscle a-actin and an increase of
- 28 s
/3- ACTIN
18s
Fig. 4. Actin mRNA in Response to IGF-I and PDGF Stimulation
Confluent cultures of bovine aortic SMCs were serumdeprived for 72 h, then stimulated with IGF-I, PDGF, and IGFI and PDGF-I in combination for 2 h. The Northern blot was
probed with a Pst-Pst fragment of the 0-actin cDNA (as described in Materials and Methods).
MOL ENDO-1989
1186
Vol 3 No. 8
INSULIN
IGF-I
5
_, o
i
2 9X
<t
CD
5
co
<j>
i
h-
5
_, o
i
X
X
0)
1
S 9 o
<
X
X
9 9 9
X
CQ
—
—
28s
28s
28s
c-MYC
18s
(A)
PDGF(h)
-
.
c-MYC
18s
(B)
«
m c-MYC
18s
(C)
Fig. 5. Dose Response of Insulin, IGF-I, and PDGF on c-myc mRNA Levels
Confluent cultures of bovine aortic SMCs were serum-deprived for 72 h, followed by stimulation with insulin, IGF-I, and PDGF
for 2 h. The Northern blots were hybridized with a c-myc probe. Insulin {panel A) causes a 5- to 6-fold increase over basal in cmyc mRNA levels at optimal concentrations. A similar 5- to 6-fold increase is also seen with IGF-I (panel B) and PDGF (panel C).
The quantification stated in the text and the legend are derived from densitometry scanning.
high doses of insulin and IGF-I are similar, IGF-I consistently achieves higher induction at lower doses, suggesting that insulin may be inducing c-myc by acting
via the IGF-I receptor. However, insulin effect on c-myc
mRNA levels are seen at low concentrations of insulin
as well, suggesting that it can induce c-myc through its
own receptor, but optimal induction occurs via IGF-I
receptor (at high insulin concentrations). PDGF also
potently stimulates c-myc mRNA levels, as shown in
Fig. 5C. At 1 x 10~10 M, a 5-fold induction is achieved
while a 8-fold increase is seen at 1 x 10~9 M. Bovine
aortic SMCs as has been reported in the literature (17),
expresses relatively high levels of c-myc in the basal
state. Thus, prolonged serum deprivation (>72 h) is
required to lower the levels of c-myc expression for
determining dose-response studies.
The time course of c-myc induction in response to
insulin, IGF-I, and PDGF is illustrated in Fig. 6. These
growth factors induce a rapid increase in c-myc mRNA
levels, seen within 30 min. c-myc mRNA levels tend to
remain elevated as long as the stimuli exist for PDGF.
However, with insulin and IGF-I, levels of c-myc mRNA
tend to fall after 2-4 h of exposure, despite continued
presence of growth factors. These levels however are
still above basal.
PDGF demonstrates an additive effect on c-myc
mRNA levels with insulin and IGF-I shown in Fig. 7.
Insulin at 1 x 10~7 M and PDGF at 1 x 10" 9 M, and IGFI each at 1 x 10r8 M causes a 5- to 7-fold increase in
c-myc mRNA levels. Insulin and PDGF act synergistically to cause a 12-fold increase, while IGF-I and PDGF
cause a 15-fold increase. Thus, PDGF along with insulin
or IGF-I cause c-myc induction which is substantially
higher than that achieved by the optimal dose of any of
these hormones singly. Insulin and IGF-I, however, do
not demonstrate additivity (data not shown).
DISCUSSION
The study of regulation of vascular SMC growth is of
great interest because of the central role smooth muscle cell hyperplasia plays in atherosclerotic plaques (1,
2). We have used cultured bovine aortic SMCs to
characterize some of the growth effects of insulin, IGFI, and PDGF. Aortic SMCs in culture exhibit morphological traits that are similar to smooth muscle cells in
atherosclerotic plaques (2,14). In cultured murine aortic
SMCs, as well as in cells from human atherosclerotic
plaques, biochemical evidence of a less differentiated
state is evidenced by the change in the expression of
muscle actin (a-actin) to nonmuscle actins (jS- and 7actins) (15). We provide evidence here that in bovine
cultured SMCs the dominant species of actin mRNA
seen basally and in response to hormonal stimuli is the
2.1 kb species (which has been characterized in murine
cells to be coding for /3- and 7-actins (16). a-Actin mRNA
(1.5 kb species) was not detected in our assays. Thus,
cultured bovine aortic smooth muscles may be a useful
model to study growth regulation by insulin, IGF-I, and
PDGF because of some morphologically and biochemical similarities to SMCs from atherosclerotic plaques.
The roles of various growth factors in the control of
smooth muscle cell growth is becoming apparent (3,
18, 19), but a precise understanding is lacking. PDGF
is a well recognized smooth muscle mitogen (5). Our
data using purified porcine PDGF confirms this property
of PDGF in bovine aortic SMCs. PDGF has been documented to be produced from sources other than platelets. Endothelial cells in culture (20,21), activated monocytes (22,23), and most interestingly SMCs themselves
(24-26) have been shown to produce PDGF. Thus,
multiple sources of PDGF exist which may affect
smooth muscle growth under different conditions. A
recent observation has also demonstrated increased
Protooncogene Expression and Growth in Vascular Muscle Cells
INSULIN
I x IO"7M
f~ ~~^~. ~ ~
1187
IGF-I
lxlO" 8 M
PDGF
I x IO"9M
S2 ~ co c/j co co
_i £: £ o : 0:0:0:
<
5 5
x x x x
< O OOJ V CM ^
QD ro cn
— CM
28s
c-MYC
18s
28s
« # * » # •
(A)
(B)
c-MYC
18 s
28s
! * • • *
c-MYC
' 18s
(C)
Fig. 6. Time Course of c-myc mRNA Stimulation in Response to Insulin, IGF-I, and PDGF
Quiescent confluent cultures of bovine aortic SMCs were exposed to insulin ( 1 x 1 0 " 9 M), IGF-I (1 x 10' M), or PDGF (1 x 10M) for various durations. The Northern blots were hybridized with a c-myc probe.
BASAL
INS lxlO"7M
f
PDGF lxlO*9M
I G F - I lxlO'8M
INS/PDGF
*
g
— o
0 IGF-I /PDGF
00
CO
Fig. 7. Additivity between PDGF and Insulin and IGF-I in cmyc Induction in Aortic SMCs
Quiescent confluent cultures of bovine aortic SMCs were
stimulated with insulin (1 x 10~7 M), IGF-I (1 x 10" 8 M), or
PDGF (1 x 10~9 M) or in combination (insulin and PDGF and
IGF-I and PDGF). Ten micrograms of total RNA were electrophoresed and processed. The blot was hybridized with a cmyc probe.
levels of c-sis mRNA (coding for the 0 chain of PDGF)
in human atherosclerotic lesions (27).
IGF-I and insulin are also well established mitogens
for SMCs (8, 9). The role of insulin as a growth factor
is well known (28) and its putative role in atherogenesis
among diabetics and nondiabetics is of great interest
(29). It is important to define further the role of insulin
in SMC growth, because of the current tendency to
achieve strict glycemic control in diabetics who often
are hyperinsulinemic in the systemic circulation (30).
Insulin and IGF-I act through a common pathway to
produce growth in some cells (12), and more recently
we have demonstrated that in human SMCs, the IGF-I
receptor is the common pathway for growth effects of
insulin and IGF-I (13), although in liver cells, insulin acts
through its own receptor to promote growth (31).
The interaction between PDGF and IGF-I (and insulin)
in smooth muscle cell growth regulation is becoming
evident. PDGF modulates SMC growth in response to
IGF-I (10, 32). Furthermore, PDGF has been demonstrated to induce production of IGF-I like peptides by
smooth muscle cells (33) and that both PDGF and IGFI regulate receptors homologously and heterologously
(34). It has also been reported that in porcine vascular
SMCs, PDGF induces SMC growth largely by the endogenous production of IGF-I from porcine vascular
smooth muscles (33). Our data presented here show
that PDGF and IGF-I or PDGF and insulin interact
additively to induce SMC growth. Additivity at optimal
doses of PDGF with insulin or IGF-I suggests to us that
PDGF is acting through a different pathway than insulin
and IGF-I. If IGF-I production with subsequent autocrine
stimulation of SMCs was the mode of growth effect by
PDGF, additivity at optimal doses of PDGF and IGF-I
would be unlikely. Different pathways are also suggested by our data on the time course of preincubation
required for each of these hormones before commitment to DNA synthesis occurs. PDGF causes commitment earlier, and precedes insulin or IGF-I induced
commitment by at least 2-4 h. Thus, this data again
suggests to us that induced commitment to DNA synthesis would follow IGF-I induced commitment if PDGF
was acting predominantly via IGF-I production.
The mechanism of action for the growth effects of
these growth factors on SMCs is not known. It is well
established that the receptors for insulin (25), IGF-I (36),
and PDGF (37) are tyrosine kinases, but the sequence
of events leading from hormone receptor interaction
and the tyrosine kinase to biological effect is essentially
unknown. It is recognized, however, that induction of
the protooncogene c-myc is an early event in the action
of many mitogens (38) among them insulin (31) and
Vol 3 No. 8
MOL ENDO-1989
1188
PDGF (11). The translated product of c-myc, a DNA
binding protein (39), is thought to be associated with
cellular growth (40). Recent studies indicate a role of cmyc in regulating entry of cells into the S-phase of the
cell cycle (40). c-myc induction in smooth muscle cells
is largely unexplored. Kindy et al. (17) have demonstrated c-myc induction in response to serum to be a
complex phenomenon occurring at multiple levels (17).
The complexity of the nature of serum, however, necessitates further characterization of c-myc responses
to individual growth factors in vascular SMCs. Here we
demonstrate that PDGF, IGF-I, and insulin potently
stimulate c-myc mRNA levels individually. Furthermore,
PDGF is additive with insulin and IGF-I in c-myc induction, whereas insulin and IGF-I are not. This finding
supports our suggestion that PDGF is acting through a
separate pathway from insulin and IGF-I. c-myc induction by these growth factors is an early event, occurring
within 30 min. The early induction of c-myc, and its
associated role in regulating DNA synthesis in other cell
systems (41), suggest c-myc may be involved in regulating SMC growth in response to insulin, IGF-I, and
PDGF.
In summary, we demonstrate that PDGF, IGF-I, and
insulin potently stimulate c-myc and actin mRNA levels,
DNA synthesis and cellular proliferation in cultured bovine aortic SMCs. The pathways taken by PDGF appear
to be different from that of insulin and IGF-I. By defining
further cellular and genetic responses to these and
other growth factors in cultured bovine aortic SMCs,
we may gain more insight into understanding the SMC
hyperplasia characteristic of atherosclerotic plaques.
additive studies optimal concentrations for insulin (1 x 10~6
M), IGF-I (1 x 1(T8 M ), and PDGF (1 x 10" 9 M) were added in
combinations. Studies to illustrate the time course of commitment to DNA synthesis were initiated as above. After exposure
of the cells to optimal concentrations of the various growth
factors for 2-10 h, the growth factors were removed, the cells
were washed, and the medium was replaced with DMEM and
0.25% BSA. Incubation was continued for up to 24 h after the
initial exposure to growth factors. At the end of 24 h (dose
response studies, additivity, and time course) the cells were
pulsed with 2.0 fiC'\/m\ 3H-thymidine (New England Nuclear,
Boston, MA) for 1 h, and the samples processed as previously
described (42).
Labeling with 3H-thymidine is linear from 30-90 min and the
basal 3H-thymidine incorporation for the SMCs are stable for
24 h. In addition, no significant increase in 3H-thymidine incorporation was noted for 10% serum or any growth factors until
8-10 h, and maintained a steady state maximum from 16-24
h. After 24 h, stimulated 3H-thymidine will start to decay rapidly.
2) Cellular proliferation was assessed by changes in cell
number. Approximately 10 x 103 cells per well were plated on
to 12-well cluster dishes. One milliliter of DMEM with 0.25%
calf serum was added to each well. Six hours later, cells were
dispersed with 0.5% trypsin (Cooper Biomedical, Malvern, PA),
collected in 20 ml ISOTON II (azide-free balanced electrolyte
solution, Coulter Diagnostics, Hialeah, FL), and counted (Coulter Electronics, Inc., Hialeah, FL) to assess plating efficiency.
To the other cells, insulin, IGF-I, or PDGF were added in
various concentration. For additive studies insulin (1 x 1O~6
M), IGF-I (1 x 10~7 M), PDGF (1 x 10~9 M) were added in
combination. Forty-eight hours later the cells were trypsinized,
collected, and counted. Some cells were grown throughout in
DMEM with 0.25% BSA and served as control specimens.
All protein assays were done by the methods of Lowry et
al. (43) using albumin as standards. IGF-I was purchased from
Amgen (Thousand Oaks, CA), PDGF, from PDGF, Inc. (Boston,
MA) and Bioprocessing Ltd. (Durham, UK), and porcine insulin
from Elanco Products Co. (Indianapolis, IN).
Analysis of Actin and c-myc mRNA Levels
MATERIALS AND METHODS
Cell Isolation and Culture
Vascular SMCs were established in culture from explants of
bovine calf aortas as previously described (8). Briefly, the
bovine aorta was splayed open and rinsed several times with
chilled PBS. The internal elastic lamina was removed, exposing
the underlying muscularis media. Small pieces of the media
(1 - 2 mm) were cut and placed on 35-mm plastic petri dishes
(NUNC, Copenhagen, Denmark). Dulbecco's modified essential medium (DMEM), (GIBCO, Grand Island, NY) with 15%
fetal bovine serum (Hyclone, Logan, UT) was added. They
were allowed to explant undisturbed for 1-2 weeks, after
which the media was changed on alternate days until confluency was achieved. The cells were then passaged into
experimental dishes. Cells between passages 3-8 only were
used for experiments. The SMCs were identified by positive
staining to smooth muscle actin and myosin using specific
antibodies (Sigma, St. Louis, MO).
Growth Studies
The effects of various growth factors on vascular SMC growth
were studied by two methods:
1) DNA synthesis was assessed by 3H-thymidine incorporation. Cells were plated on to 12-well cluster dishes (Costar,
Cambridge, MA) and allowed to reach confluency. The medium
was replaced with DMEM with 0.25% BSA for 12 h. Insulin,
IGF-I, or PDGF was added in various concentrations. For
Confluent cells grown on 100-mm plastic petri dishes (NUNC)
were serum deprived for 72 h by replacing the growth medium
with DMEM with 0.25% BSA. They were then stimulated with
insulin, IGF-I, or PDGF in varying concentrations for doseresponses studies, and with 1 x 10~6 M insulin, 1 x 10~8 M
IGF-I, and 1 x 10~9 M PDGF in combination for additive studies,
and for variable durations for time course experiments.
The cells were scraped into a 4 M quanidine thiocyanite
solution and RNA extracted by the method of Chirgwin (44),
and quantified very carefully by spectrophotometry. Ten micrograms of total RNA were separated on a 1 % agarose-0.6
M formaldehyde denaturing gel, followed by transfer to a nylon
membrane (BioTrace, Gelman Sciences Inc., Ann Arbor, Ml)
(45). Ethidium bromide stains were done to check the quantity
of RNA in each lane. The blot was hybridized in a solution
containing 47% formamide and 10% dextran sulfate at 42 C
for 12-18 h with actin and c-myc probes. The actin probe was
a Pst\/Pst\ fragment of mouse /3-actin cDNA (46), and the cmyc probe was a £CORI/C/al fragment encoding the human
c-myc third exon (47). These probes were radiolabeled by the
technique of Feinberg and Vogelstein (48) with a commercially
available kit Prime Time, International Biotechnologies, Inc.
(IBI, New Haven, CT). After overnight hybridization, the blots
were washed under stringent conditions in 0.1 x SSC, 0.1%
sodium dodecyl sulfate (15 mM NaCI, 1.5 ITIM NaCitrate, 0.1%
at 50 C (46). They were then exposed to photographic film
(Kodak) for 48-72 h, and developed in an automatic processor
(X-Omat M20 Processor Kodak, Rochester, NY). The autoradiographic bands were quantified by laser scanning densitometry (G5300 Scanner, Hoefer Scientific Instruments, San Francisco, CA).
As an internal control of the amount of RNA loaded per
Protooncogene Expression and Growth in Vascular Muscle Cells
lane, the gels were also probed with /32 microglobulin (mouse),
but no hybridization occurred. Thus, an internal control like /3actin (which in our system is hormonally regulated) was not
available for these experimental situations, but the careful
spectrophotometric quantification and ethidium stains showed
approximately equivalent amounts of RNA in each lane. In
addition, 3H-uridine incorporation was performed to determine
total RNA which approximated ethidium stain very well. Insulin
and IGF-I did not increase uridine incorporation by more than
15% within the 2-hour period.
Acknowledgments
The authors wish to express their thanks to Dr. C. Ronald
Kahn for providing invaluable suggestions throughout the project; to Terri-Lynn Bellman, Wanda Mutter, and Leslie Balmat
for their excellent secretarial assistance; and to Kirstie Saltsman for her technical help.
Received June 7, 1988. Revision received April 24, 1989.
Accepted May 1,1989.
Address requests for reprints to: George L. King, M.D.,
Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215.
This work was supported by NIH Training Grant AM-07260
and NIH Grant EY-05110.
REFERENCES
1. Ross R 1986 The pathogenesis of atherosclerosis-an
update. N Engl J Med 314:488-500
2. Schwartz SM, Campbell GR, Campbell JH 1986 Replication of smooth muscle cell in vascular disease. Circ Res
58:427-444
3. Weinstein R, Stemerman MB, Maciag T. 1981 Hormonal
requirements for growth of arterial smooth muscle cells
in vitro: an endocrine approach to atherosclerosis. Science 212:818-820
4. Deuel TF, Huang JS 1984 Platelet-derived growth factor.
Structure, function and roles in normal and transformed
cells. J Clin Invest 74:669-676
5. Ross R, Raines EW, Bowen-Pope DF 1986 The biology
of platelet-derived growth factor. Cell 46:155-169
6. Grotendorst GF, Chang T, Seppa HEJ, Kleinman HK,
Martin GR 1982 Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J Cell Physiol
113:261-266
7. Rinderknecht E, Humbel RE 1978 The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 253:27692776
8. King GL, Goodman DA, Buzney S, Moses A, Kahn C
1985 Receptors and growth-promoting effects of insulin
and insulin-like growth factors on cells from bovine retinal
capillaries and aorta. J Clin Invest 75:1028-1036
9. Pfeifle B, Ditschuneit H 1981 Effect of insulin on growth
of cultured human arterial smooth muscle cells. Diabetologia 20:155-158
10. Clemmons DR 1985 Exposure to platelet-derived growth
factor modulates the porcine aortic smooth muscle cell
response to somatomedin-C. Endocrinology 117:77-83
11. Kelly K, Cochran BH, Stiles CD, Leder P1983 Cell-specific
regulation of the c-myc gene by lymphocyte mitogens and
platelet-derived growth factor. Cell 35:603-610
12. King GL, Kahn CR, Rechler MM, Nissley SP 1980 Direct
demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating
activity (an insulin-like growth factor) using antibodies to
the insulin receptor. J Clin Invest 66:130-140
1189
13. Banskota NK, Taub R, Zellner K, Olsen P, King GL 1987
Insulin and IGF-I rapidly induce the proto-oncogene cmyc, and stimulate DNA synthesis and cellular proliferation in cultured human vascular smooth muscle cells via
the IGF-I receptor. Diabetes, in press
14. Chamley-Campbell JH, Campbell GR, Ross R 1981 Phenotype-dependent response of cultured aortic smooth
muscle to serum mitogens. J Cell Biol 89:379-383
15. Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J,
Weber K 1984 Actin expression in smooth muscle cells
of rat aortic intimal thickening, human atheromatous
plaque, and cultured rat aortic media. J Clin Invest
73:148-152
16. Strauch AR, Offord JD, Chalkley R, Rubenstein PA 1986
Characterization of actin mRNA levels during BC3H1 cell
differentiation. J Biol Chem 261:849-855
17. Kindy MS, Sonenshein GE 1986 Regulation of oncogene
expression in cultured aortic smooth muscle cells. J Biol
Chem 261:12865-12868
18. Chamley-Campbell J, Campbell GR, Ross R 1979 The
smooth muscle cell in culture. Physiol Rev 59:1-61
19. Gospodarowicz D, Hirabayashi K, Giguere L, Tauber JP
1981 Factors controlling the proliferative rate, final cell
density, and life span of bovine vascular smooth muscle
cells in culture. J Cell Biol 89:568-578
20. DiCorleto PE, Bowen-Pope DF 1983 Cultured endothelial
cells produce a platelet-derived growth factor-like protein.
Proc Natl Acad Sci USA 80:1919-1923
21. Collins T, Ginsburg D, Boss JM, Orkin SH, Pober JS 1985
Cultured human endothelial cells express platelet-derived
growth factor /3-chain: cDNA cloning and structural analysis. Nature 316:748-750
22. Martinet Y, Bitterman PB, Mornex JF, Grotendorst GR,
Martin GR, Crystal RG 1986 Activated human monocytes
express the c-sis proto-oncogene and release a mediator
showing PDGF-like activity. Nature 319:156-160
23. Shimokado K, Raines EW, Madtes DK, Barrett TB, Benditt
EP, Ross R1985 A significant part of macrophage-derived
growth factor consists of at least two forms of PDGF. Cell
43:277-286
24. Seifert RA, Schwartz SM, Bowen-Pope DF 1984 Developmentally regulated production of platelet-derived
growth factor-like molecules. Nature 311:669-671
25. Sejersen T, Betsholtz C, Sjolund M, Heldin CH, Westermark B, Thyberg J 1986 Rat skeletal myoblasts and
arterial smooth muscle cells express the gene for the A
chain but not the B chain (c-sis) of platelet-derived growth
factor (PDGF) and produce a PDGF-like protein. Proc Natl
Acad Sci USA 83:6844-6848
26. Walker LN, Bowen-Pope DF, Ross R, Reidy MA 1986
Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci
USA 83:7311-7315
27. Barrett TB, Benditt EP 1987 s/s (platelet-derived growth
factor B chain) gene transcript levels are elevated in
human atherosclerotic lesions compared to normal artery.
Proc Natl Acad Sci USA 84:1099-1103
28. King GL, Kahn CR 1984 The growth-promoting effects of
insulin. IN: Guroff G (ed) Growth and Maturation Factors,
John Wiley and Sons, Inc, vol 2
29. Stout RW 1985 Overview of the association between
insulin and atherosclerosis. Metabolism 34 [Suppl 1]:712
30. Munkgaard-Rasmussen S, Heding LG, Parbst E, Velund
AA 1975 Serum IRI in insulin treated diabetics during a
24-hour period. Diabetologia 11:151 -158
31. Taub R, Roy A, Dieter R, Koontz J 1987 Insulin as a
growth factor in rat hepatoma cells: stimulation of protooncogene expression. J Biol Chem 262:10893-10897
32. Clemmons DR 1984 Interaction of circulating cell-derived
and plasma growth factors in stimulating cultured smooth
muscle cell replication. J Cell Physiol 121:425-430
33. Clemmons DR, Van Wyk J J1985 Evidence for a functional
MOL ENDO-1989
1190
34.
35.
36.
37.
38.
39.
40.
role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human
fibroblasts and porcine smooth muscle cells. J Clin Invest
75:1914-1918
Pfeifle B, Boeder H, Ditschuneit H 1987 Interaction of
receptors for insulin-like growth factor I, platelet-derived
growth factor and fibroblast growth factor in rat aortic
cells. Endocrinology 120:2251-2258
Kasuga M, Karlsson Fa, Kahn CR 1982 Insulin stimulates
the phosphorylation of the 95,000 dalton subunit of its
own receptor. Science 215:185-187
Jacobs S, Kull FC, Earp HS, Svoboda M, Van Wyk JJ,
Cuatrecasas P 1983 Somatomedin-C stimulates the
phosphorylation of the /3-subunit of its own receptor. J
Biol Chem 258:9581-9584
Frackelton AR, Tremble PM, William LT 1984 Evidence
for the platelet-derived growth factor-stimulated tyrosine
phosphorylation of the platelet-derived growth factor receptor in vivo. J Biol Chem 259:7909-7915
Kelly K 1986 The regulation and expression of c-myc in
normal and malignant cells. Annu Rev Immunol 4:317338
Watt RA, Shatzman AR, Rosenberg M 1985 Expression
and characterization of human c-myc DNA binding protein. Mol Cell Biol 5:448-456
Cole MD 1986 The myc oncogene: its role in transformation and differentiation. Annu Rev Genet 20:361-384
Vol 3 No. 8
41. Heikkila R, Schwab G, Wickstrom E, Loke SL, Pluznik
DH, Watt R, Neckers LM 1987 A c-myc antisense oligodeoxynucleotide inhibits entry into S-phase but not progress from Go to G L Nature 328:445-449
42. Rechler MM, Podskalny JM, Goldfine IM, Wells CA 1974
DNA synthesis in human fibroblasts: stimulation by insulin
and by nonsuppressible insulin-like activity (NSILA's). J
Clin Endocrinol Metab 39:512-521
43. Lowry DH, Rosebrough Nl, Farr AL, Randall RJ 1951
Protein measurement with the Folin phenol reagent. J Biol
Chem 193:265-275.
44. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ
1979 Isolation of biologically active ribonucleic acid from
sources enriched in ribonuclease. Biochemistry 18:52945299
45. Davis LG, Dibner MD, Battey JF 1986 In: Basic Methods
in Molecular Science. Elsevier Science Publishing Co,
New York
46. Spiegelman BM, Frank M, Green H 1983 Molecular cloning of mRNA from 3T3 adipocytes. J Biol Chem
258:10083-10089
47. Taub R, Moulding C, Battey J, Murphy W, Vasicek T,
Lenoir GM, Leder P1984 Activation and somatic mutation
of the translocated c-myc gene in Burkitt lymphoma cells.
Cell 36:339-348
48. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high
specific activity. Anal Biochem 132:6-13