1 Department of Pediatrics, Mattel Children's Hospital at University of California, Los Angeles, California 90095-1752; and 2 Department of Anatomy, University of Pennsylvania, Children's Hospital, Philadelphia, Pennsylvania 19104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both insulin-like growth factor binding protein-3
(IGFBP-3) and transforming growth factor- (TGF-
) have been
separately shown to have cell-specific growth-inhibiting or
growth-potentiating effects. TGF-
stimulates IGFBP-3 mRNA and
peptide expression in several cell types, and TGF-
-induced growth
inhibition and apoptosis have been shown to be mediated through the
induction of IGFBP-3. However, a link between the growth stimulatory
effects of TGF-
and IGFBP-3-induction has not been shown. In this
study, we investigated the role of IGFBP-3 in mediating
TGF-
1-induced cell growth using human airway smooth muscle (ASM)
cells as our model. TGF-
1 (1 ng/ml) treatment induced a 10- to
20-fold increase in the levels of expression of IGFBP-3 mRNA and
protein. Addition of either IGFBP-3 or TGF-
1 to the growth medium
resulted in an approximately twofold increase in cell proliferation.
Coincubation of ASM cells with IGFBP-3 antisense (but not sense)
oligomers as well as with an IGFBP-3 neutralizing antibody (but not
with control IgG) blocked the growth induced by TGF-
1 (P < 0.001). Several IGFBP-3-associated proteins were observed in ASM cell lysates, which may have a role in the cellular responses to IGFBP-3. These findings demonstrate that IGFBP-3 is capable of mediating the
growth stimulatory effect of TGF-
in ASM cells.
insulin-like growth factor binding protein; cell proliferation; receptor; transforming growth factor.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INSULIN-LIKE GROWTH FACTOR (IGF) binding protein-3 (IGFBP-3) belongs to a family of high-affinity IGFBPs, which bind to IGFs and modulate their actions. IGFBP-3 modulates IGF action at the cellular level through either inhibition (4, 9, 18, 28) or potentiation (5, 6) of cell growth, and it also has an intrinsic activity, which is independent of its binding to IGFs (29). IGF-independent antiproliferative effects of IGFBP-3, such as cell growth arrest, have been demonstrated previously in cancer epithelial cell lines (22, 24). In addition, we recently demonstrated a novel apoptosis-inducing effect of IGFBP-3 in prostate cancer cells and in IGF receptor-null fibroblasts (25).
Transforming growth factor-1 (TGF-
1) is a pluripotent cytokine
capable of inhibiting or stimulating cell growth depending on the
nature of the target cell (19). TGF-
1 is a potent growth inhibitor
of a variety of epithelial cell types, whereas it stimulates cell
growth in stromal cells. TGF-
1 and TGF-
2 enhance IGFBP-3 mRNA and
protein in both epithelial and stromal cell types (10, 15, 23). The
role of IGFBP-3 as a mediator of TGF-
1-induced antiproliferative
effects has been demonstrated in several epithelial cell types (10,
23). We have shown that IGFBP-3 stimulation is required for the
apoptosis-inducing effects of TGF-
1 in prostate cancer cells (25).
The secretion and mitogenic action of TGF-1 have been demonstrated
in bovine airway smooth muscle (ASM) cells (2) and have been suggested
to be associated with the hyperplastic nature of ASM cells in chronic
asthma and bronchopulmonary dysplasia. However, the mechanisms
underlying the mitogenic effect of TGF-
1 on ASM cells are not
clearly understood.
We therefore hypothesized that the growth stimulatory effect of
TGF-1 in stromal cells may be related to its ability to induce IGFBP-3 expression. To test this hypothesis and to determine the role
of IGFBP-3 as a mediator of cell growth induced by TGF-
1, we
investigated the ability of IGFBP-3 to induce cell growth and to
mediate TGF-
1-induced cellular proliferation in human ASM cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. 125I-IGF-I and 125I-IGF-II
were purchased from Amersham Life Sciences (Arlington Heights, IL).
Recombinant human IGFBP-3 was obtained from Celtrix Pharmaceuticals
(Santa Clara, CA). 125I-IGFBP-3 and
antibodies to IGFBP-3 purified on an IGFBP-3 affinity column (25) were
purchased from Diagnostic Systems Laboratories (Webster, TX). Smooth
muscle basal medium, fetal bovine serum, insulin, human recombinant
epidermal growth factor (EGF), basic human recombinant fibroblast
growth factor, gentamicin and amphotericin B were purchased from
Clonetics (San Diego, CA). Recombinant human TGF-1 was purchased
from R&D Systems (Minneapolis, MN). SDS-PAGE reagents were purchased
from Bio-Rad (Richmond, CA). The nonradioactive CellTiter 96 assay kit
was purchased from Promega Biological Research Products (Madison, WI).
Phosphothiolated IGFBP-3-specific sense (5'-CCC CGG TTG CAG GCG
T-CATG-3') and antisense (5'-CAT GAC GCC TGC AAC CGG
GG-3') 20 mers, originally published by Oh et al. (23), were
purchased from OLIGOS (Wilsonville, OR).
Phenylmethylsulfonyl fluoride, EDTA, pepstatin, and aprotinin were
obtained from Sigma (St. Louis, MO).
ASM cells. The human bronchial ASM cell strains used in this
study were obtained from Clonetics and were derived from two healthy
male donors (37 and 16 years old). The cells were grown in medium
consisting of smooth muscle basal medium supplemented with 5% fetal
bovine serum, insulin (5 mg/ml), human recombinant EGF (10 ng/ml),
human recombinant fibroblast growth factor (2 ng/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml). The standard experimental
protocol involved growing the cells in the complete medium
(serum-containing medium, SCM) and treating for various times with and
without recombinant human TGF-1 in SCM. Both cell strains were used
for all experiments.
Cell growth assays. For each experimental condition, ASM cells were plated at 1 × 104 cells/cm2 in 96-well plates. The nonradioactive CellTiter 96 assay was used to measure cell proliferation. Samples were treated in multiples of eight for each condition. This method measures the cellular conversion of the tetrazolium salt MTS into a formazan, which is measured at 490 nm directly in the plate. The absorbance reading is directly proportional to the number of viable cells per well, and means ± SD were determined. Absorbance values were significantly correlated to cell number measurements made with a Coulter counter (data not shown).
RNA analysis. Total RNA was isolated from 75-cm2 flasks of ASM cells (in duplicate) using the acid guanidium thiocyanate-phenol-chloroform extraction method but modified to include a proteinase K (in 0.5% SDS) digestion of proteins in the initial RNA pellet. For Northern blots, 20-µg samples were fractionated on 1% agarose formaldehyde gels, transferred to Zeta-Probe membranes (Bio-Rad), and probed with a 440-bp human IGFBP-3 cDNA and a 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment labeled with 32P by random oligo priming. The level of IGFBP-3 mRNA was normalized to the GAPDH signal. Specific mRNA levels were quantified using a Molecular Dynamics PhosphorImager.
Western ligand blots. This technique was performed as
previously described (1, 4-6, 9, 10, 12, 15, 22-26, 29). IGFBP protein levels were measured using conditioned medium from ASM
cells incubated for 72 h with serum-free medium with and without 1 ng/ml TGF-1. Samples of 50 µl of conditioned medium were separated by nonreducing 10% SDS-PAGE overnight at constant voltage and electroblotted onto nitrocellulose. The membranes were then
sequentially washed with Nonidet P-40, 1% BSA, and Tween 20, incubated
with 106 counts/min each of 125I-IGF-I and
125I-IGF-II for 12 h (specific activity of 2,000 Ci/mmol),
washed with Tween-Tris buffered saline (TBS) × 3, dried, and
exposed to film for 5 days.
Western immunoblots. Samples of conditioned medium (50 µl) from ASM cells treated as described for the Western ligand blots were subjected to electrophoresis overnight through 10% nonreducing SDS-PAGE at constant voltage. Gels were electroblotted onto nitrocellulose, blocked with 5% nonfat dry milk in TBS, probed with an affinity-purified IGFBP-3 antibody (1:5,000 dilution), and detected using a peroxidase-linked enhanced chemiluminescence detection system (Amersham). IGFBP-3 normally appears as a doublet due to the existence of two glycosylation states at 40 and 44 kDa. Both bands were included in the quantification
IGFBP-3 ELISA assays. Samples of conditioned media were assayed for IGFBP-3 levels by ELISA kits [Diagnostic Systems Laboratories (DSL), Webster, TX] according to the manufacturer's recommendations.
ASM cell proliferation assay with IGFBP-3-specific
oligodeoxynucleotides or IGFBP-3 neutralizing antibodies. ASM cells
seeded at 1 × 104/cm2 were incubated on
96-well plates for 5 days in SCM with and without 1 ng/ml TGF-1 in
the presence of no oligomer or 20 µg/ml thiolated IGFBP-3-specific
sense or antisense oligomers (23). All residues of the oligomers were
modified per the manufacturer. Oligomers were dosed once at the
beginning of the experiment. Conditioned media from identical wells
were assayed at 3 days for IGFBP-3 levels to verify oligomer activity
on inhibiting IGFBP-3 secretion. Similar experiments were set up with
control IgG or IGFBP-3 neutralizing antibodies (8 µg/ml) (25). These
experiments were set up for 5 days. Antibodies were dosed once at the
beginning of the experiment. A nonradioactive CellTiter 96 assay was
used to measure cell growth.
Preparation of cell lysates. Confluent ASM cells were briefly
washed with cold PBS and allowed to dissociate in dispersion buffer (1 mM EDTA in PBS, pH 7.4). Free-floating cells were collected and
centrifuged (2,000 rpm = 200 g in an Eppendorf C-5415
centrifuge for 5 min), suspended in cold lysis buffer containing 10 mM
HEPES, 1.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µM aprotinin, and 1 mM pepstatin in PBS (pH 7.4), vortexed, and boiled at 100°C for 5 min. Aliquots were
stored at 70°C until further use.
Western ligand blot with 125I-IGFBP-3 (reverse Western ligand blot). ASM cell lysates were subjected to electrophoresis through 10% nonreducing SDS-PAGE overnight at constant voltage and electroblotted onto nitrocellulose, blocked with 1% BSA in TBS and incubated with 5 × 106 counts/min of 125I-IGFBP-3 (DSL) for 12 h. The membranes were exposed to film for 3 days and visualized by autoradiography.
Densitometric and statistical analysis. Densitometric
measurement of immunoblots, Western ligand blots, and reverse
ligand blots were performed using a Bio-Rad GS-670 imaging densitometer (Bio-Rad, Melville, NY). Protein levels were estimated by comparing the
optical density of each specific protein band from control conditions
to that of the TGF-1-treated conditions. All experiments were
repeated at least three times. When applicable, means ± SE are shown.
Unpaired two-tailed Student t-tests were used for statistical analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of TGF-1 on ASM cell growth. Figure
1 shows the additive effect of complete SCM
and TGF-
1 on ASM cell growth. Treatment with SCM for 2 days induced
a twofold increase in cell growth, whereas treatment for 5 or 7 days
resulted in a 2.5-fold increase in cell growth compared with that on
day 0. Treatment with medium containing both serum and TGF-
1
(1 ng/ml) for 7 days resulted in an additive effect on ASM cell growth
and elicited a significant stimulation (4-fold) at day 7 relative to day 0 (P < 0.001), with a lesser but
still significant effect (P < 0.01) seen on days 2 and 5. Thus addition of 1 ng/ml TGF-
1 to SCM results in a
nearly twofold (90%) increase in ASM cell growth after 7 days relative to SCM alone (P < 0.001). Results represent mean values of
two experiments each in two cell strains performed on sets of eight samples. The effects of TGF-
1 in the absence of serum were slightly stimulatory, although the results did not achieve significance and
therefore are not shown. Thus a yet to be identified serum factor
contributes to the TGF-
effect on growth stimulation.
|
Effect of TGF-1 on IGFBPs secreted by ASM cells.
We examined the levels of IGFBPs secreted into ASM serum-free
conditioned medium in the presence and absence of TGF-
1. Figure
2A is a Western ligand blot, which
shows that IGFBP-2, -3, and -4 (validated by specific immunoblots; see
Ref. 25) are expressed at a low concentration by ASM cells in
serum-free conditions. Exposure to 1 ng/ml TGF-
1 selectively
increased IGFBP-3 protein secreted into conditioned medium at 72 h.
Other IGFBPs did not change significantly. Figure 2B shows that the rise in IGFBP-3 was 10-fold at 72 h as
measured by densitometry (P < 0.001).
|
Induction of IGFBP-3 secretion by TGF-1 in ASM
cells. We confirmed the selective induction of IGFBP-3 by TGF-
1
in ASM cells by measuring the levels of secreted IGFBP-3 protein using
specific IGFBP-3 antibodies. Figure
3B shows a 10-fold increase in
IGFBP-3 protein in the TGF-
1-treated ASM cell-conditioned medium
relative to control conditions (P < 0.001). Results represent
mean values of three experiments each on two cell strains performed on
sets of four samples. Immunodetection of IGFBP-3 in ASM
cell-conditioned medium also revealed the presence of IGFBP-3 fragments
in both control and TGF-
1-treated conditions (Fig. 3A). This
is known because these bands are seen on an IGFBP-3 immunoblot but not on a Western ligand blot and have been characterized previously (25).
With use of a specific ELISA assay, seen in Fig. 3C, IGFBP-3 level in conditioned medium was 85 ± 15 ng/ml in the absence of TGF-
1 and rose to 1,080 ± 220 ng/ml in the presence of 1 ng/ml of
TGF-
1 (P < 0.001).
|
Stimulation of IGFBP-3 mRNA levels by TGF-1 in ASM
cells. To determine the level of IGFBP-3 mRNA expression under
control (SCM) and TGF-
1-treated conditions, total RNA samples from
ASM cells were analyzed by Northern blotting. The levels of the 2.6-kb IGFBP-3 mRNA and 1.4-kb GAPDH were quantified using a phosphorimager. Figure 4A shows the autoradiograph
of the RNA from ASM cells probed with 32P-labeled IGFBP-3
cDNA. Figure 4B shows that 48-h exposure of ASM cells to 1 ng/ml concentration of TGF-
1 induces a 20-fold increase in IGFBP-3
mRNA relative to treatments either with SCM. This increase in
expression of IGFBP-3 mRNA in TGF-
1-treated ASM cells was observed
as early as 24 h after treatment (data not shown).
|
Effects of IGFBP-3 on ASM cell growth. Figure
5 shows the effect of IGFBP-3 (0-1,000
ng/ml) on ASM cell growth in the presence of SCM. These concentrations
were chosen because they are similar to the levels of IGFBP-3 achieved
in conditioned medium in response to TGF-1 treatment. Treatment of
ASM cells for 5 days with SCM and IGFBP-3 induced a dose-dependent
increase in cell growth, with a twofold stimulation at 1,000 ng/ml of
IGFBP-3 (P < 0.001). Results represent mean values of two
experiments each on two cell strains performed on sets of eight
samples. The effects of IGFBP-3 were slightly less than those of
TGF-
; we suspect that this may be the result of uncharacterized
effects of TGF-
1 on IGFBP-3 degradation, which may occur through
proteases that are TGF inhibited.
|
Selective inhibition of TGF-1-induced IGFBP-3
protein secretion by IGFBP-3 antisense oligonucleotides. To
demonstrate the effectiveness of the IGFBP-3 antisense reagents in our
system, we examined if their addition selectively inhibits
TGF-
1-induced IGFBP-3 protein secretion from ASM cells. Cells were
simultaneously treated with TGF-
1 peptides at a concentration of 1 ng/ml, and sense or antisense oligomers against IGFBP-3 for 72 h and
the levels of IGFBP-3 in conditioned medium were determined using IGFBP-3 immunoblotting. Figure 6 shows the
10-fold increase in IGFBP-3 in TGF-
1-treated ASM cell-conditioned
medium relative to serum-free conditions. This induction of IGFBP-3
protein by TGF-
1 was fully blocked when cells were treated with
IGFBP-3 antisense oligomers but not with IGFBP-3 sense oligomers. The levels of the other IGFBPs were not changed by oligomer treatment (data
not shown).
|
TGF-1-induced cell growth is inhibited by blocking
IGFBP-3. We determined the requirement for IGFBP-3 in the TGF-
1
stimulation of ASM cell by evaluating ASM cell proliferation after
treating the cells simultaneously with TGF-
1 and IGFBP-3 sense or
antisense oligomers. Figure 7 shows that
IGFBP-3 antisense oligodeoxynucleotides selectively inhibited
TGF-
1-stimulated cell growth after 5 days of exposure of ASM cells
to TGF-
1 and SCM. Treatment with IGFBP-3 sense oligomers did not
have any effect on TGF-
1-induced ASM cell proliferation. To further
substantiate the observations made using the IGFBP-3 antisense
oligomers and to confirm that the secreted IGFBP-3 plays a role in cell
growth stimulation, we evaluated cell growth in ASM cells
simultaneously treated for 5 days with TGF-
1 in SCM and IGFBP-3
neutralizing antibodies. Figure 8
demonstrates that IGFBP-3 neutralizing antibodies (but not control IgG)
selectively inhibited TGF-
1-stimulated ASM cell growth. Results
represent mean values of three experiments performed on sets of eight
samples.
|
|
IGFBP-3 association proteins (receptors) in ASM
cells. Because IGFBP-3 may be regulating cell growth by
directly binding to specific receptors, we examined the presence of
IGFBP-3 association proteins by visualizing the cellular proteins,
which bind to 125I-IGFBP-3, using reverse-ligand blotting
(Fig. 9). IGFBP-3 association proteins of
approximately 150, 100, and 68 kDa were observed in both control and
TGF-1-treated conditions. However, the 68-kDa protein is elevated in
the TGF-
1-treated conditions. In addition, two new IGFBP-3
association proteins of approximately 58 and 16 kDa were detected only
in TGF-
1-treated cell extracts.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IGFBP-3 belongs to a family of high-affinity IGFBPs, which bind to IGFs and modulate their actions. IGFBPs regulate the availability of free IGFs and therefore their mitogenic activity (1, 4, 8, 12, 13, 28). It has been suggested previously that cell-associated IGFBP-3 is involved in the presentation of IGF-I to its receptor as well as a means to heighten receptor reactivity to IGF-I and related peptides (5, 6). IGFBPs also play an important role in directly regulating cell growth. These cell growth regulatory effects of IGFBPs have been shown to be either growth inducing (1, 5, 6,) or growth inhibiting (10, 18, 23, 25).
The antiproliferative and apoptosis-inducing effects of IGFBP-3 have a
key role in TGF-1-induced growth inhibition in human cancer cells.
We have demonstrated that IGFBP-3 suppresses epithelial cell number by
inducing apoptosis and have identified putative cell surface receptors
in these epithelial cells (25). In the p53-negative prostate cancer
cell line PC-3, addition of recombinant IGFBP-3 resulted in a
dose-dependent induction of apoptosis. 125I-IGFBP-3 bound
with high affinity to specific proteins in PC-3 cell lysates and plasma
membrane preparations. These membrane-associated molecules may serve as
receptors that mediate the direct effect of IGFBP-3 on apoptosis. In
addition, in an IGF receptor-negative mouse fibroblast cell line,
treatment with recombinant IGFBP-3, as well as transfection of the
IGFBP-3 gene, induced apoptosis, suggesting that neither IGFs nor IGF
receptors are required for this action. Furthermore, treatment with
TGF-
1, a known apoptosis-inducing agent, resulted in the induction
of IGFBP-3 expression 6-12 h before the onset of apoptosis. This
effect of TGF-
1 was prevented by cotreatment with IGFBP-3
neutralizing antibodies or IGFBP-3-specific antisense phosphothiolated
oligonucleotides. These findings led us to conclude that IGFBP-3
induces apoptosis through a novel pathway independent of either p53 or
the IGF-IGF receptor-mediated cell survival pathway and that IGFBP-3
mediates TGF-
1-induced apoptosis in PC-3 cells. We and others have
previously demonstrated the effects of IGFBP-3 as a negative regulator
of cell proliferation in various types of epithelial cancer cells (10,
23, 25). However, this study is the first demonstration of the
involvement of IGFBP-3 in the stimulation of cell growth by TGF-
1.
We have evaluated apoptosis in these ASM cells using several methods
(fluorescence-activated cell sorting, ELISA, and terminal
deoxynucleotidyltransferase dUTP nick end labeling). We detected a
minimal number of cells undergoing apoptosis and showed no effect of
TGF or IGFBP-3.
TGF-1 is a pluripotent modulator of cell function and an important
suppressor of epithelial cell proliferation. The predominant effect of
TGF-
1 on cell proliferation is inhibitory. However, epithelial cell
lines transformed by SV40 and human papilloma virus (HPV-16 and HPV-18)
are no longer susceptible to TGF-
1-induced growth inhibition (2).
Furthermore, TGF-
1 has been shown to induce cell growth and
proliferation in ASM cells (16) as well as in vascular smooth muscle
cells (7). These observations suggest an existence of a dual pathway
for TGF-
1-regulated cell growth, which may be dependent on the
specific cell type.
Several mechanisms have been suggested for the TGF-1-induced cell
growth in transformed cells. Earlier studies demonstrated that the
inhibition of epithelial cell proliferation by TGF-
1 involves
suppression of c-myc transcription. Epithelial cells transformed by SV40 or human papilloma virus resisted growth inhibition and suppression of c-myc mRNA by TGF-
1 (7). Other studies showed that growth stimulation by TGF-
1 occurred indirectly via establishment of an autocrine loop that involves an increase in EGF
receptors. In this study, we demonstrate another novel mechanism (via
inducing IGFBP-3) by which TGF-
1 may induce cell growth and proliferation.
Our observation of increased expression of IGFBP-3 mRNA and protein by
TGF-1 in ASM cells led us to examine the role of IGFBP-3 in
TGF-
1-induced cell growth in ASM cells. Confirmation for the role of
IGFBP-3 in TGF-
1-induced cell growth was obtained by preventing the
increase in ASM cell growth stimulated by TGF-
1 by exposing the ASM
cells to IGFBP-3-specific antisense oligomers or IGFBP-3 neutralizing
antibodies along with TGF-
1. Blocking IGFBP-3 protein synthesis
almost completely blocked the cell growth stimulatory effect of
TGF-
1. This observation suggests that IGFBP-3 may be a key mediator
of TGF-
1-induced cell proliferation. Furthermore, ASM cells also
have specific IGFBP-3 association proteins that are potential IGFBP-3
receptors, suggesting a possible IGF-independent action of IGFBP-3 in
mediating ASM cell growth. IGFBP-3 binding studies, affinity
cross-linking with recombinant 125I-IGFBP-3, and
immunoprecipitation of cell monolayers and cell lysates with
anti-IGFBP-3 antibodies have revealed the presence of IGFBP-3
association proteins in several cell types. Cell surface association
proteins ranging from 20 to 200 kDa that are specific for IGFBP-3 have
been demonstrated in human breast cancer epithelial cells (24),
prostate cancer epithelial cells (25), and human fetal lung fibroblasts
(Rajah and Cohen, unpublished data). In this study, we have
demonstrated the presence of several such IGFBP-3 association proteins
in ASM cells, some of which may be IGFBP-3-specific receptors (Fig. 9).
TGF-1 and IGFBP-3 have been suggested to have a shared receptor
(14). However, the biological actions of IGFBP-3, which are mediated
through this putative TGF-
1 receptor, have not been demonstrated.
This TGF-
type V receptor is approximately 400 kDa in size and
appears to bind both TGF-
1 and IGFBP-3 (11, 20, 21), but the cDNA
for this receptor has not yet been cloned. Loss of this receptor has
been suggested to potentially contribute to the transformed state of
certain epithelial tumor cells (14). However, the presence of several
cell membrane association proteins for IGFBP-3 in epithelial
and stromal cells suggests the possibility that there may be more than
one IGFBP-3 receptor.
TGF-1 is formed in the airways and has been previously suggested to
have a role in airway remodeling in asthma and bronchopulmonary dysplasia (3, 16, 27). This pleiomorphic growth factor induces cell
growth in bovine ASM cells and is a potent stimulator of ASM cell
growth in the presence of low concentrations of serum (9, 10). Our
study demonstrates a similar growth-inducing effect of TGF-
1 in
human ASM cells and further provides a possible mechanism by which
TGF-
1 may induce ASM cell proliferation and hyperplasia in human ASM
cells. Varying degrees of both smooth muscle cell hypertrophy and
hyperplasia occur in asthma. The results obtained from our study
suggest that the inflammation-associated cytokine TGF-
1 has a
complex mechanism of action on the induction of ASM cell growth and
that IGFBP-3 plays a significant role in TGF-
1 action and in the
structural changes seen in airways of asthmatic subjects.
In summary, human ASM cells treated with the growth factor TGF-1
exhibit significant increase in cell proliferation. Treatment with
IGFBP-3 results in a similar increase in cell growth. The ASM cells
treated with TGF-
1 display an increased IGFBP-3 mRNA and protein
expression. Treatment with either IGFBP-3 antisense oligonucleotide or
neutralizing antibodies blocks the TGF-
1-induced increase in ASM
cell growth. These results provide evidence that TGF-
1 enhances ASM
cell growth through a mechanism that requires IGFBP-3 expression as
presented in the cartoon in Fig. 10,
depicting a hypothetical model of TGF-IGFBP-3 cascade of action.
|
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported in part by the National Institutes of Health Grants 1P50-HL-56401 and 1R01-AI-40203 (P. Cohen) and an National Research Service Award (R. Rajah).
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Cohen, Div. of Endocrinology, Dept. of Pediatrics, Mattel Children's Hospital at UCLA, 10833 Le Conte Ave., MDCC 22-315, Los Angeles, CA 90095-1752 (E-mail: hassy{at}mednet.ucla.edu).
Received 5 November 1998; accepted in final form 27 September 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Angelloz-Nicoud, P.,
and
M. Binoux.
Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3).
Endocrinology
136:
5485-5492,
1995[Abstract].
2.
Black, P. N.,
P. G. Young,
and
S. J. Skinner.
Response of airway smooth muscle cells to TGF-1: effects on growth and synthesis of glycosaminoglycans.
Am. J. Physiol. Lung Cell. Mol. Physiol.
271:
L910-L917,
1996
3.
Cohen, M. D.,
V. Ciocca,
and
R. A. Panettieri, Jr.
TGF-beta 1 modulates human airway smooth-muscle cell proliferation induced by mitogens.
Am. J. Respir. Cell Mol. Biol.
16:
85-90,
1997[Abstract].
4.
Cohen, P.,
D. M. Peehl,
H. C. Graves,
and
R. G. Rosenfeld.
Biological effects of prostate specific antigen as an insulin-like growth factor binding protein-3 protease.
J. Endocrinol.
142:
407-415,
1994[Abstract].
5.
Conover, C. A.
Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.
Endocrinology
129:
3259-3268,
1991[Abstract].
6.
Conover, C. A.
Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.
Endocrinology
130:
3191-3199,
1992[Abstract].
7.
Gibbons, G. H.,
R. E. Pratt,
and
V. J. Dzau.
Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II.
J. Clin. Invest.
90:
456-461,
1992[ISI][Medline].
8.
Giudice, L. C.,
J. C. Irwin,
B. A. Dsupin,
E. M. Pannier,
I. H. Jin,
T. H. Vu,
and
A. R. Hoffman.
Insulin-like growth factor (IGF), IGF binding protein (IGFBP), and IGF receptor gene expression and IGFBP synthesis in human uterine leiomyomata.
Hum. Reprod.
8:
1796-1806,
1993[Abstract].
9.
Goldstein, S.,
E. J. Moerman,
R. A. Jones,
and
R. C. Baxter.
Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts.
Proc. Natl. Acad. Sci. USA
88:
9680-9684,
1991[Abstract].
10.
Gucev, Z. S.,
Y. Oh,
K. M. Kelley,
and
R. G. Rosenfeld.
Insulin-like growth factor binding protein 3 mediates retinoic acid- and transforming growth factor beta-2-induced growth inhibition in human breast cancer cells.
Cancer Res.
56:
1545-1550,
1996[Abstract].
11.
Huang, L. Q.,
S. S. Huang,
and
J. S. Huang.
Kinase activity of the type V transforming growth factor beta receptor.
J. Biol. Chem.
269:
9221-9226,
1994
12.
Jones, J. I.,
and
D. R. Clemmons.
Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I.
Endocr. Rev.
16:
3-34,
1995[ISI][Medline].
13.
Kaicer, E.,
C. Blat,
J. Imbenotte,
F. Troalen,
O. Cussenot,
F. Calvo,
and
L. Harel.
IGF binding protein-3 secreted by the prostate adenocarcinoma cells (PC-3): differential effect on PC-3 and normal prostate cell growth.
Growth Regul.
3:
180-189,
1993[ISI][Medline].
14.
Leal, S. M.,
Q. Liu,
S. S. Huang,
and
J. S. Huang.
The type V transforming growth factor beta receptor is the putative insulin-like growth factor-binding protein 3 receptor.
J. Biol. Chem.
272:
20572-20576,
1997
15.
Martin, J. L.,
M. Ballesteros,
and
R. C. Baxter.
Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.
Endocrinology
131:
1703-1710,
1992[Abstract].
16.
McKay, S.,
J. C. de Jongste,
P. R. Saxena,
and
H. S. Sharma.
Angiotensin II induces hypertrophy of human airway smooth muscle cells: expression of transcription factors and transforming growth factor-beta1.
Am. J. Respir. Cell Mol. Biol.
18:
823-833,
1998
17.
Minshall, E. M.,
D. Y. Leung,
R. J. Martin,
Y. L. Song,
L. Cameron,
P. Ernst,
and
Q. Hamid.
Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma.
Am. J. Respir. Cell Mol. Biol.
17:
326-333,
1997
18.
Moerman, E. J.,
R. Thweatt,
A. M. Moerman,
R. A. Jones,
and
S. Goldstein.
Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.
Exp. Gerontol.
28:
361-370,
1993[ISI][Medline].
19.
Moses, H. L.
TGF-beta regulations of epithelial cell proliferation.
Mol. Reprod. Dev.
32:
179-184,
1992[ISI][Medline].
20.
O'Grady, P.,
S. S. Huang,
and
J. S. Huang.
Expression of a new type high molecular weight receptor (type V receptor) of transforming growth factor beta in normal and transformed cells.
Biochem. Biophys. Res. Commun.
179:
378-385,
1991[ISI][Medline].
21.
O'Grady, P.,
Q. Liu,
S. S. Huang,
and
J. S. Huang.
Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.
J. Biol. Chem.
267:
21033-21037,
1992
22.
Oh, Y.,
H. L. Muller,
G. Lamson,
and
R. G. Rosenfeld.
Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition.
J. Biol. Chem.
268:
14964-14971,
1994
23.
Oh, Y.,
H. L. Muller,
L. Ng,
and
R. G. Rosenfeld.
Transforming growth factor-beta-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action.
J. Biol. Chem.
270:
13589-13592,
1995
24.
Oh, Y.,
H. L. Muller,
H. Pham,
and
R. G. Rosenfeld.
Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells.
J. Biol. Chem.
268:
26045-26048,
1993
25.
Rajah, R.,
S. Nunn,
D. Herrick,
M. M. Grunstein,
and
P. Cohen.
LTD-4 induces matrix metalloproteinase-1, which functions as an IGFBP protease in airway smooth muscle cells.
Am. J. Physiol. Lung Cell. Mol. Physiol.
271:
L1014-L1022,
1996
26.
Rajah, R.,
B. Valentinis,
and
P. Cohen.
IGF binding protein-3 induces apoptosis and mediates the effects of TGF on programmed cell death.
J. Biol. Chem.
272:
12221-12228,
1997
27.
Redington, A. E.,
J. Madden,
A. J. Frew,
R. Djukanovic,
W. R. Roche,
S. T. Holgate,
and
P. H. Howarth.
Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid.
Am. J. Respir. Crit. Care Med.
156:
642-647,
1997
28.
Rosenfeld, R. G.,
H. Pham,
P. Cohen,
P. Fielder,
S. E. Gargosky,
H. Muller,
L. Nonoshita,
and
Y. Oh.
Insulin-like growth factor binding proteins and their regulation.
Acta Pediatr. Suppl.
399:
154-158,
1994[Medline].
29.
Valentinis, B.,
A. Bhala,
T. DeAngelis,
R. Baserga,
and
P. Cohen.
The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.
Mol. Endocrinol.
9:
361-367,
1995[Abstract].