(Received for publication, October 12, 1995)
From the
Previous studies have shown that porcine aortic smooth muscle cells (SMCs) secrete two insulin-like growth factor-binding proteins (IGFBP), IGFBP-2 and -4, and that these IGFBPs modulate IGF-I-stimulated SMC proliferation and migration. In this study we demonstrate that porcine SMCs express IGFBP-5 mRNA and synthesize and secrete the protein. In this cell type, the biosynthesis of IGFBP-5 is up-regulated by IGF-I. This increase in IGFBP-5 synthesis is accompanied by an increase in the steady-state mRNA levels. The induction of IGFBP-5 mRNA by IGF-I is time- and dose-dependent and requires de novo protein synthesis. IGF-II and insulin also increase IGFBP-5 mRNA levels at high doses. An IGF-I analog with normal affinity for the IGF-I receptor but reduced affinity for IGFBPs evokes a similar increase. Another analog that binds to IGFBPs but not to the receptor has no effect, indicating that this effect of IGF-I is mediated through the IGF-I receptor. The IGF-I-induced IGFBP-5 gene expression is cell type-specific because IGF-I had no such effect in other cell types examined. Nuclear run-on assays revealed that IGF-I increased transcription rate of the IGFBP-5 gene, while IGF-I did not change the IGFBP-5 mRNA stability. Furthermore, the IGFBP-5 promoter was 3.5-fold more active in directing expression of the luciferase reporter gene in IGF-I-treated aortic SMCs as compared to control cells, whereas the luciferase activity remained the same in control- and IGF-I-treated fibroblasts. These results suggest that IGF-I up-regulates IGFBP-5 synthesis by transcriptionally activating the IGFBP-5 gene in aortic SMCs.
Many studies have linked the accumulation of aortic smooth
muscle cells (SMCs) ()to the development of atherosclerotic
lesions. This accumulation is due to a combination of SMC
proliferation, directed migration from the arterial media into the
intima(1, 2) , and inhibition of
apoptosis(3) . All of these events are modulated by a number of
peptide growth factors including insulin-like growth factors (IGFs).
SMCs in culture have been shown to synthesize IGF-I, and this
endogenously produced IGF-I stimulates SMC proliferation in an
autocrine fashion(4, 5, 6) . In
vivo, IGF-I mRNA and immunoreactive IGF-I are detected in intimal
lesions that develop after angioplasty(7) . IGF-I mRNA and
immunoreactive IGF-I levels both increase severalfold after balloon
denudation injury, and these increases temporally precede an associated
increase in SMC proliferation(8, 9) . Likewise, SMCs
possess IGF-I receptors and selective inhibition of the receptors by
antisense targeting results in marked reduction in SMC
proliferation(9, 10) . These observations together
with the well established fact that IGF-I is a mitogen for SMCs suggest
that the local production of IGF-I plays an important role in SMC
proliferation(11) . In addition to its role in mitogenesis,
IGF-I has recently been shown to stimulate SMC migration. Bornfeldt et al.(12) showed that IGF-I stimulates SMC directed
migration using a Boyden chamber assay. Studies from our laboratory
have shown that IGF-I and IGF-II stimulate SMC migration in a monolayer
wounding assay, and this response is mediated by the IGF-I receptor (13) . Thus, IGF-I is important for SMC proliferation and
migration and may therefore play an important role in the development
of atherosclerotic lesions.
The bioactivities of IGFs are modulated by a group of high affinity specific binding proteins (IGFBPs). Six distinct IGFBPs, designated as IGFBP-1 to IGFBP-6, have been identified in mammalian systems to date (14, 15) . These proteins share relatively high amino acid sequence similarity, but each has distinct structural and biochemical properties that partially determine whether they act to inhibit or potentiate IGF bioactivity. Previous studies from our laboratory have shown that porcine aortic SMCs secrete IGFBP-2 and -4, and that they both modulate IGF-I-stimulated DNA synthesis and cell migration in this cell type (13, 16, 17, 18) . The availability of IGFBP-2 and -4 in SMCs is regulated by IGFs, PDGF, insulin, and other factors(17, 18) . While PDGF or insulin treatment results in moderate increases in IGFBP-2 and -4 synthesis, IGFs accelerate the degradation of the inhibitory IGFBP-4 by activating specific proteases. These findings indicate that the presence of IGFBPs in the area of the vascular lesion may play a role in modifying IGF activity, potentially resulting in modulation of SMC proliferation and migration.
In the present study, we report that porcine aortic SMCs express IGFBP-5 mRNA and synthesize and secrete IGFBP-5. Our data indicate that the synthesis of IGFBP-5 is up-regulated by its own ligand, IGF-I. Of particular interest to us was that IGF-I increased the transcription rate of the IGFBP-5 gene without significantly affecting the mRNA stability and that this response is specific for aortic SMCs.
Figure 1: Porcine aortic SMCs express IGFBP-5 mRNA. A, Northern blot analyses of IGFBP-5 mRNA levels. Ten µg of total RNA isolated from A673 human rhabdomyosarcoma cells (lane 1), GM-10 human fibroblasts (lane 2), and porcine aortic SMCs (lane 3) was loaded and subjected to Northern blotting using a human IGFBP-5 cDNA probe and a GAPDH cDNA probe. The arrows denote the 6-kb IGFBP-5 message and 1.4-kb GAPDH message. B, RT-PCR amplification of porcine IGFBP-5 mRNA. One µg of total RNA isolated from GM-10 human fibroblasts (lane 2) or porcine aortic SMCs (lane 3) was reverse transcribed into cDNA followed by PCR amplification, as described under ``Experimental Procedures.'' Lane 1 is the 1-kb DNA ladder.
To determine if porcine aortic SMCs synthesize and secrete IGFBP-5, conditioned media (CM) from porcine SMC cultures were subjected to Western ligand blot and immunoblot analysis. As reported previously (17) , ligand blotting and immunoblotting of CMs from confluent SMCs failed to detect IGFBP-5 (data not shown). This is likely due to the fact that porcine SMC-CM contains abundant proteolytic activity for IGFBP-5(22, 23) . Since IGF-I and heparin have been shown to inhibit IGFBP-5 proteolytic degradation in human fibroblasts(19, 24) , IGF-I and/or heparin were added to cell cultures prior to the collection of medium. When heparin and IGF-I were added to subconfluent SMC cultures, an IGFBP at the size of 31 kDa, which comigrated with purified human IGFBP-5, was observed by ligand blotting (Fig. 2A). The identity of this 31-kDa IGFBP as IGFBP-5 was confirmed by immunoblotting using two different antibodies to human IGFBP-5 (Fig. 2B). The addition of IGF-I increased both intact IGFBP-5 and a 22-kDa IGFBP-5 fragment, suggesting that the IGF-I-induced porcine IGFBP-5 increase may not be simply due to a decrease in degradation.
Figure 2: Porcine aortic SMCs secrete IGFBP-5. A, ligand blot analysis of porcine SMC conditioned media. The 24-h conditioned medium (0.5 ml) from control (lane 2) or IGF-I (100 ng/ml)-treated SMC cultures (lane 3) was concentrated 20 times and separated by 12.5% SDS-PAGE gel. Heparin (100 µg/ml) was added 6 h prior to the collection. Lane 1 contains human IGFBP-5 (100 ng). B, immunoblot analysis of porcine SMC conditioned media. The same 24-h conditioned medium samples shown in panel A from control (lanes 2 and 4) or IGF-I (100 ng/ml)-treated SMCs (lanes 3 and 5) were immunoblotted with a human IGFBP-5 antibody prepared in rabbit (lanes 2 and 3) or in guinea pig (lanes 4 and 5). Lane 1 contains purified human IGFBP-5 (100 ng).
Figure 3:
IGF-I stimulates IGFBP-5 synthesis in
porcine aortic SMCs. A, autoradiogram showing the effect of
IGF-I and/or heparin on newly synthesized IGFBP-5. Porcine SMCs were
preincubated without (lanes 1-3) or with IGF-I (100
ng/ml, lanes 4 and 5) for 18 h followed by a 1-h
incubation in methionine-free medium before the addition of 50 µCi
of [S]methionine without (lanes 1, 2, and 4) or with heparin (100 µg/ml, lanes 3 and 5). After 6 h, culture media were collected and
immunoprecipitated using normal guinea pig serum (lane 1) or
human IGFBP-5 antiserum prepared in guinea pig (lanes
2-5). The pellets were boiled in sample buffer for 10 min
and analyzed by SDS-PAGE followed by autoradiography. B,
phosphorimager analysis. Values are means of two immunoprecipitation
experiments as described in A.
Figure 4: IGF-I increases the steady-state levels of IGFBP-5 mRNA in porcine aortic SMCs. A, autoradiogram showing dose-dependent effect of IGF-I. Porcine SMC cultures were treated without (lane 1) or with 1 (lane 2), 5 (lane 3), 50 (lane 4), and 250 ng/ml IGF-I (lane 5) for 24 h. Total RNA was isolated from SMC cultures and subjected to Northern blot with cDNA probes for IGFBP-5, IGFBP-2, and GAPDH. B, autoradiogram showing the time-course effect of IGF-I. Porcine SMC cultures were preincubated in serum-free medium for 24 h and then treated without (lanes 1, 2, 4, 6, 8, and 10) or with 100 ng/ml IGF-I (lanes 3, 5, 7, 9, and 11) for 0 (lane 1), 1 (lanes 2 and 3), 3 (lanes 4 and 5), 6 (lanes 6 and 7), 12 (lanes 8 and 9), and 24 h (lanes 10 and 11). C and D, phosphorimager analyses of the concentration dependence and time-course experiments, respectively. Values are means ± S.E. of four (C) or three (D) separate experiments. They are expressed as a percentage of mRNA levels in the control, untreated samples. *, significantly different from the controls (p < 0.05).
Figure 5: A, autoradiogram showing the effects of PDGF and FGF on IGF-I-induced IGFBP-5 mRNA expression. Porcine aortic SMCs were incubated in serum-free medium without (lane 1) or with IGF-I (100 ng/ml, lane 2), PDGF (5 ng/ml, lane 3), FGF (50 ng/ml, lane 4), IGF-I plus PDGF (lane 5), or IGF-I plus FGF (lane 6) for 24 h. Total RNA was isolated from SMC cultures and subjected to Northern blotting with cDNA probes for IGFBP-5 and GAPDH. B, phosphorimager analysis of three experiments as described in A. Values are means ± S.E. expressed as a percentage of mRNA levels in the control, untreated samples. *, significantly different from the controls (p < 0.05).
Figure 6:
The
effect of IGF-I on IGFBP-5 gene expression is mediated through the
IGF-I receptor. A, autoradiogram showing the effects of IGF-I,
IGF-II, and insulin. Porcine aortic SMCs were incubated with serum-free
medium (lane 1), or serum-free medium plus IGF-I (10 ng/ml, lane 2; 50 ng/ml, lane 3), IGF-II (50 ng/ml, lane
4), or insulin (1 µg/ml, lane 5) for 24 h. B, autoradiogram showing the effects of IGF analogs and the
IGF-I receptor blocking antibody IR3. Porcine SMCs were incubated
with serum-free medium (lane 1), or serum-free medium with
IGF-I (100 ng/ml, lane 2),
IR3 (10 µg/ml, lane
3), IGF-I (100 ng/ml) plus
IR3 (10 µg/ml, lane
4), Des(1-3)-IGF-I (100 ng/ml, lane 5), or
[Leu
]IGF-I
(100 ng/ml, lane 6) for 24 h. C and D, phosphorimager
analyses of three experiments as described in A and B, respectively. Values are means ± S.E. expressed as a
percentage of mRNA levels in the control, untreated samples. *,
significantly different from the controls (p <
0.05).
Figure 7:
IGF-I
stimulates the transcription rate of the IGFBP-5 gene in porcine aortic
SMCs. A, autoradiogram showing the effect of IGF-I. Nuclei
from control and IGF-I-treated cultures were isolated and nuclear
run-on assays performed in the presence of
[P]UTP for 30 min. The nascent
P-labeled transcripts were hybridized to slots of
filter-bound IGFBP-2 (lane 1), IGFBP-5 (lane 2), and
pBluescript DNA (lane 3). B, phosphorImager analysis
of two separate experiments. Values are means expressed as a percentage
of mRNA levels in the control, untreated
samples.
We also sought to
determine if IGF-I treatment affects IGFBP-5 mRNA stability. Porcine
aortic SMCs were incubated with or without IGF-I for 18 h, and then
treated with actinomycin D. Although there was no difference in the
calculated t of IGFBP-5 mRNA in the control and
IGF-I-treated groups, treatment of actinomycin D caused a transient
rise in IGFBP-5 mRNA levels (data not shown). This actinomycin
D-associated increase in IGFBP-5 mRNA levels has previously been
observed in human breast carcinoma cells (25) and complicated
the interpretation of these data. We next performed similar experiments
using the RNA polymerase II inhibitor DRB. Addition of DRB to porcine
SMCs led to a progressive decline in IGFBP-5 abundance (Fig. 8A) with a calculated t
for
both control and IGF-I-treated groups of approximately 18 h (Fig. 8B). Thus, IGF-I treatment does not cause an
alternation in IGFBP-5 mRNA stability.
Figure 8: IGF-I does not cause alteration in the stability of IGFBP-5 mRNA in porcine aortic SMCs. A, autoradiogram showing a representative Northern blot. Porcine aortic SMCs were incubated without (lanes 1-9) or with IGF-I (lanes 10-18) for 18 h, followed by the addition of vehicle (lanes 2-5 and 11-14) or DRB at 75 µM concentration (lanes 6-9 and 15-18). The cells were harvested at 0 (lanes 1 and 10), 3 (lanes 2, 6, 11, and 15), 6 (lanes 3, 7, 12, and 16), 12 (lanes 4, 8, 13, and 17), and 24 h (lanes 5, 9, 14, and 18) after the addition of DRB or vehicle. Total RNA was isolated and subjected to Northern blotting with cDNA probes for IGFBP-5 and 18 S rRNA. B, effect of IGF-I on IGFBP-5 mRNA decay in transcriptionally blocked porcine SMCs. Values are means of two separate experiments expressed as a percentage of levels in the control, untreated samples.
These results indicate that the increase in the levels of IGFBP-5 mRNA induced by IGF-I is primarily due to the activation of the IGFBP-5 gene. We wondered whether IGF-I modulates IGFBP-5 transcripts by direct interaction with the 5`- or 3`-regulatory sequences or, alternatively, by inducing the synthesis of an intermediate regulatory factor(s). Accordingly, porcine SMCs were treated with IGF-I in the presence and absence of cycloheximide. As shown in Fig. 9, while IGF-I alone induced a 656% increase, co-incubation with cycloheximide completely blocked the IGF-I-induced IGFBP-5 gene expression (151% of the controls), suggesting this effect of IGF-I requires de novo protein synthesis.
Figure 9: The protein synthesis inhibitor cycloheximide abrogates the IGF-I-induced IGFBP-5 expression. A, autoradiogram showing the inhibitory effect of cycloheximide. Porcine aortic SMC cultures were incubated without (lanes 1 and 3) or with IGF-I (100 ng/ml, lanes 2 and 4) in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of cycloheximide (10 µg/ml) for 24 h. Total RNA was isolated from SMC cultures and subjected to Northern blotting with cDNA probes for IGFBP-5 and GAPDH. B, phosphorimager analysis. Values are means of two separate experiments expressed as a percentage of levels in the control, untreated samples.
In order to gain insight into the promoter region(s) of the IGFBP-5 gene responsive to IGF-I, we transfected SMCs with a 1278-bp segment of human IGFBP-5 promoter fused to the reporter luciferase gene. This segment of the IGFBP-5 promoter contains identical sequences of a number of well defined regulatory elements, including a TATA box, a CAAT box, and several AP-2 elements, which previously have been shown to be responsible for the cAMP-induced activation of this gene(21) . As shown in Fig. 10A, relative luciferase activity in IGF-I-treated SMCs was 345% higher than those of the control SMCs (p < 0.05). This indicates that this 1278-base pair promoter region contains a cis-acting element(s) that is responsible for the IGFBP-5 gene response to IGF-I.
Figure 10: Effect of IGF-I on human IGFBP-5 promoter activity in aortic SMCs (A) and fibroblasts (B). A 1278-bp DNA fragment of the human IGFBP-5 gene 5`-flanking region was fused to a luciferase reporter gene (pGL2-Basic) and transiently transfected into porcine SMCs and GM-10 fibroblasts. After growing in complete medium for 72 h, cells were incubated in serum-free medium with or without IGF-I (100 ng) for another 6 h. The cellular extracts were prepared and the luciferase activity was measured as described under ``Experimental Procedures.'' The relative luciferase activities represent the relative value normalized by galactosidase activity. Values are means ± S.E. expressed as a percentage of the levels in the controls. * Significantly different from the controls (p < 0.05).
Figure 11: IGF-I stimulates IGFBP-5 gene expression in human aortic SMCs (A) but not in human fibroblasts (B), glioblastoma (C), and human intestinal SMCs (D). Confluent cells were incubated without (lane 1) or with IGF-I (100 ng/ml, lane 2) for 24 h. Total RNA was isolated, and 15-µg RNA aliquots were loaded and subjected to Northern blotting with cDNA probes for IGFBP-5 and GAPDH.
The present study demonstrates that porcine as well as human aortic SMCs express IGFBP-5 mRNA and secrete the protein. In this cell type, the biosynthesis of IGFBP-5 is stimulated by IGF-I. IGF-I regulates IGFBP-5 synthesis at the mRNA level. This effect of IGF-I appears to be mediated by the IGF-I receptor and requires de novo protein synthesis. The increase in IGFBP-5 mRNA levels that is induced by IGF-I is primarily due to an elevation in the transcription rate of the IGFBP-5 gene rather than an alteration in the stability of the transcript, suggesting that IGF-I regulates IGFBP-5 expression primarily by transcriptional activation of the gene in aortic SMCs.
The expression of the IGFBP-5 gene is cell type-specific. High
levels of IGFBP-5 mRNA have been found in fibroblasts, glioblastoma
cells, skeletal muscle cells, osteoblasts, chondrocytes, granulosa
cells, and thyroid cells but not in hepatoma or rhabdomyosarcoma cells
(19, 21, 26, 28-30, 55, 56). Although the aortic SMC has been
extensively used as a model to study the IGF system, there was no
previous report regarding the expression of IGFBP-5 in this cell type.
Several experimental difficulties seem to be partly responsible. First,
aortic SMC-CM contains abundant proteolytic activity for IGFBP-5.
SMC-CM has been shown to rapidly degrade exogenously added human
IGFBP-5(22, 23) . As shown in this study, the
endogenously secreted IGFBP-5 was completely degraded yielding small
fragments under basal conditions unless IGF-I and/or heparin was added
to the culture medium ( Fig. 2and Fig. 3). Second,
endogenously produced IGFBP-5 (31 kDa) was difficult to distinguish
from 32-kDa IGFBP-2, which is the predominant form of IGFBP secreted by
porcine SMCs that is detected by Western ligand blotting. Third, the
IGFBP-5 production by SMCs is inversely correlated to cell density.
IGFBP-5 mRNA levels were 4-5-fold lower in postconfluent cultures
as compared with subconfluent SMC cultures. ()In this study,
we used subconfluent cultures, whereas in previous studies confluent
cultures were used(17, 31) .
The fact that aortic SMCs synthesize IGFBP-5 and that its synthesis is under the regulation of IGF-I implies that this binding protein may play an important role in modulating IGF-induced SMC proliferation and migration. IGFBP-5 has been shown to have the unique property of adhering to extracellular matrix(32) . When associated with the extracellular matrix, it has been shown to potentiate the effect of IGFs on fibroblast growth. In addition, IGFBP-5 may also be involved in muscle cell differentiation. The expression of IGFBP-5 is greatly increased during terminal differentiation of the mouse myoblast cell lines(33, 34, 35) . This process is also stimulated by IGFs. The exact physiological function(s) of IGFBP-5 in aortic SMC proliferation and migration is currently under investigation.
The abundance of IGFBP-5 is influenced by a number of factors. Of the substances studied, IGF-I was the most potent regulator in aortic SMCs. IGF-I increased the IGFBP-5 protein as well as IGFBP-5 mRNA levels. The effect of IGF-I in inducing IGFBP-5 expression is specific. IGF-I had no effect on IGFBP-2 mRNA level. There was a small increase in GAPDH mRNA, but this change was negligible in comparison to a severalfold increase in IGFBP-5. Two other SMC mitogenic factors, FGF and PDGF, had no stimulatory effect. In fact, PDGF acts as a inhibitor of IGFBP-5 expression either added alone or in combination with IGF-I. This inhibitory effect of PDGF was previously observed in rat osteoblasts(36) . An increase in IGFBP-5 abundance in CM induced by IGF-I was previously reported in human fibroblasts and other cells(19, 26, 27, 28, 29, 37, 55, 56) . However, inconsistent and confusing results have been documented regarding the mechanisms accounting for this increase. In human fibroblasts, U2 osteosarcoma cells and breast carcinoma cells, IGF-I treatment significantly increases the IGFBP-5 protein concentrations without affecting the IGFBP-5 mRNA abundance(19, 26, 27, 37, 38) . This effect of IGF-I may be attributed to the inhibition of IGFBP-5 proteolysis rather than an alteration of the biosynthesis in these cells. In rat FRTL-5 thyroid cells and osteoblasts, on the other hand, IGF-I stimulated IGFBP-5 concentrations with a concomitant increase in IGFBP-5 mRNA levels, suggesting that IGF-I may regulate the IGFBP-5 abundance by simulating its synthesis (28, 29) .
In this study, we have directly demonstrated that IGF-I induces an increase in IGFBP-5 synthesis in aortic SMCs by using metabolic labeling of porcine SMCs coupled with immunoprecipitation. Although porcine SMC conditioned medium contains IGFBP-5 protease activity, IGF-I does not appear to greatly affect proteolysis. IGF-I treatment resulted in an increase in the newly synthesized intact IGFBP-5 as well as the proteolytically degraded fragment, while heparin only increased the intact protein levels by inhibiting the degradation (Fig. 3). Therefore IGF-I regulates the IGFBP-5 abundance in porcine SMCs primarily by increasing the biosynthesis. These results are consistent with the previous data obtained in rat thyroid cells and osteoblasts, but different from those of human fibroblasts, human osteosarcoma, and breast carcinoma cells. Since the cell models used in the above studies are derived from different tissues and species, this discrepancy may reflect cell-specific regulation or species differences. A recent study using human and bovine fibroblasts suggested a possible species difference may exist(27) . We addressed this question by examining IGF-I-induced IGFBP-5 gene expression in a number of human cell types. Our data with human aortic SMCs, human skin fibroblasts, human glioblastoma cells, and intestinal SMCs (Fig. 11) indicate that the stimulation of IGFBP-5 gene transcription by IGF-I is a cell type-specific event. This conclusion is in agreement with the fact that the structure of the IGFBP-5 promoter is highly conserved among mammalian species. The proximal 200 bp of the IGFBP-5 gene promoter, which has been shown to contain the primary promoter activity(39) , is more than 90% identical among human, mouse, and rat(40, 41, 42) . Therefore, it is unlikely species difference is the sole factor accounting for the different regulation of IGFBP-5 synthesis.
Although previous studies have demonstrated IGF-I-induced IGFBP-5
mRNA steady-state levels in other cell
types(27, 28, 29) , the pathways and
mechanisms responsible for the rise in IGFBP-5 mRNA levels have not
been established. In this study, we attempted to delineate whether
IGF-I operates through the IGF-I receptor to induce IGFBP-5 gene
expression. Because the anti-IGF-I receptor antibody (IR3) behaved
as a partial agonist in stimulating IGFBP-5 expression, definite proof
that type I IGF-I receptor stimulation accounts for IGFBP-5 induction
could not be obtained. The partial agonist activity seen here with
IR3 has been reported previously with IGF-I-induced c-myc induction in human SMCs(43) . Our experiments using
IGF-II, insulin, and IGF analogs, however, strongly suggest that the
stimulation of IGFBP-5 expression by IGF-I is mediated through the
IGF-I receptor.
The data presented in this study demonstrate that
IGF-I treatment transcriptionally activates the IGFBP-5 gene without
altering the transcript stability. The IGFBP-5 gene expression is
activated within 6 h of exposure to IGF-I, as indicated by Northern
blotting and transient gene transfer studies. Coupled with the
relatively long half-life of IGFBP-5 mRNA in porcine aortic SMCs (18
h), the progressive acceleration of the transcription rate is
sufficient to result in a substantial increase in mRNA expression,
protein synthesis, and secretion into the culture medium. The
transcriptional activation of the IGFBP-5 gene by IGF-I in aortic SMCs
was further ascertained by the results of gene transfer studies in
SMCs. A fusion plasmid containing 1278 bp of 5`-flanking region of
human IGFBP-5 gene showed an IGF-I-induced rise in directing reporter
gene expression (Fig. 10). These observations indicate that
IGF-I is able to activate the IGFBP-5 gene through cis-acting
element(s) residing on this 1278-bp region. IGF-I has been shown to
regulate transcription of a number of genes, e.g. c-myc(43) , growth hormone(44) ,
thyroglobulin(45) , 1-crystallin(46) ,
elastin(47) , P-450 cholesterol side chain cleavage
gene(48) , and others(49) . However, little is known
regarding the cis-DNA sequences responsive to IGF-I. Recently,
IGF-I was shown to regulate chicken
1-crystallin gene expression
through a GC-rich sequence that binds to a Sp-1-like
protein(46) . In the rat elastin promoter, a similar but not
identical GC-rich sequence is capable of binding to IGF-I-regulated
proteins and responsible for the IGF-I-responsiveness of this
gene(47) . One of the proteins has been shown to be
Sp-1(50) . The IGFBP-5 promoter contains several GC-rich
regions superficially resembling the Sp-1 element. In particular, the
DNA sequence 5`-CCCCACCCCCACCC-3` at position -147 to -134
has this potential. Although this highly conserved sequence contains
two overlapping AP-2 elements (5`-CCCCACCC-3`) and is capable of
binding to AP-2 in vitro, it does not appear to mediate the
AP-2 regulation under basal condition in vivo(21) .
This sequence contains sequences identical to the retinoblastoma (Rb)
control element 5`-CCACCC-3`. The Rb control element motif has been
identified as a Sp-1-binding sequence responsible for Rb-induced trans-activation(51) . A recent study by Jensen et
al.(50) suggested that IGF-I may disrupt Sp-1 binding to
the GC-rich domain of the elastin gene by affecting the phosphorylation
state of Rb in rat SMCs. Studies using transient transfection assays
are currently under way to determine if this sequence and/or other
sequence(s) is required for IGFBP-5 gene to be activated in response to
IGF-I.
Our results have shown that IGF-I up-regulates IGFBP-5 gene
expression in a dose-dependent fashion and in a time frame consistent
with that of an intermediate effect. The finding that cycloheximide
abrogates IGF-I-induced IGFBP-5 transcription suggests a requirement
for the synthesis of an intermediate protein(s). It has been reported
that IGF-I stimulates the expression the immediate-early gene c-fos in several cell types(52, 59) . The encoded
proteins Fos can dimerize with Jun and form the AP-1 complex, a
transcriptional activator that regulates many genes(53) . An
elevation in AP-1 transcriptional activity induced by IGF-I has
recently been observed in IEC-6 intestinal epithelial
cells(60) . Although a consensus AP-1 element is present in the
rat IGFBP-5 promoter region (-314 to -320 bp; (42) ), there is no evidence that this AP-1 element is
functional in rat. Moreover, this sequence is not conserved even among
mammalian species; a single residue alteration from T to C leads to the
ablation of the potential AP-1 element in the human IGFBP-5
promoter(40) . One of the other genes that responds rapidly to
IGF-I is the prereplicative (G) phase-specific cyclin D1.
This gene is activated by IGF-I treatment within 1 h in MG63 human
osteosarcoma cells(54) . D-type cyclins are known to be able to
form complexes with Rb and affect Rb phosphorylation
status(61, 62) . The phosphorylation states of Rb are
reported to be affected by IGF-I in aortic SMCs, and this change
appears to be related to the IGF-I-induced disruption of Sp-1 binding
of the rat elastin gene(50) . Further studies are needed to
determine which mechanism(s) IGF-I uses to activate IGFBP-5 gene
transcription in aortic SMCs.
In summary, aortic SMCs synthesize IGFBP-5 in addition to previously identified IGFBP-2 and -4. The abundance of this important modulator of IGF activity is regulated by its own ligand, IGF-I. IGF-I regulates IGFBP-5 synthesis by activating transcription of the IGFBP-5 gene rather than altering the stability of the transcript. This effect of IGF-I appears to be mediated by the IGF-I receptor and is aortic SMC-specific. Since IGF-I is important for aortic SMC proliferation and migration, analysis of the regulation of IGFBP-5 gene expression and action in SMCs should provide insight into the role of the IGF system in the development of atherosclerotic lesions.