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Receptor Is the
Putative Insulin-like Growth Factor-binding Protein 3 Receptor*
(Received for publication, May 28, 1997)
From the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ABSTRACT 
Insulin-like growth factor-binding protein
3 (IGFBP-3) has been shown to inhibit cell growth by
IGF-dependent and -independent mechanisms. The
putative cell-surface IGFBP-3 receptor that mediates the
IGF-independent growth inhibition has not been identified. Here we show
that recombinant human IGFBP-3 inhibits
125I-transforming growth factor
(TGF)-
1 binding to the type V TGF- receptor
(Mr 400,000) in mink lung epithelial
cells. We also demonstrate that the ~400-kDa
125I-IGFBP-3 affinity-labeled putative IGFBP-3
receptor is immunoprecipitated by specific antiserum to the type V
TGF- receptor. The 125I-IGFBP-3 affinity labeling of the
putative receptor and IGFBP-3-induced growth inhibition as measured by
DNA synthesis in these cells is blocked by a TGF-1
peptide antagonist. The 125I-IGFBP-3 affinity-labeled
putative receptor can only be detected in cells expressing the type V
TGF- receptor, but not in cells lacking the type V TGF- receptor.
These results indicate that the type V TGF- receptor is the putative
IGFBP-3 receptor and that IGFBP-3 is a functional ligand for the
type V TGF- receptor.
INTRODUCTION
The type V transforming growth factor (TGF-)1 receptor is a
400-kDa non-proteoglycan membrane glycoprotein that co-expresses with
the type I, type II, and type III TGF- receptors in most cell types
(1-5). The type V TGF- receptor as well as the type I and type II
TGF- receptors are Ser/Thr-specific protein kinases and belong to
the new class of membrane receptors associated with a Ser/Thr-specific
protein kinase activity (1-6). The type I and type II TGF-
receptors have been shown to be important in TGF--induced cellular
responses (1-6), but the role of the type V TGF- receptor in these
responses has not been defined (3-5). Recently, we have demonstrated
that the type V TGF- receptor mediates TGF--induced growth
inhibition and that both type I and type II TGF- receptors are
required for mediating maximal growth inhibition (7).
Insulin-like growth factor-binding protein 3 (IGFBP-3) is the most abundant insulin-like growth factor-binding protein in the circulation (8-11). In human plasma, IGFBP-3 forms an ~140-kDa ternary complex with IGFs and an acid-labile subunit (12). This complex serves as a reservoir for IGFs (12). IGFBP-3 is produced by a variety of cell types (12) and appears to inhibit cell growth by IGF-dependent and -independent mechanisms (13-15). Although several small cell membrane-associated IGFBP-3 binding proteins have recently been reported (16-18), the putative IGFBP-3 receptor that mediates the IGF-independent growth inhibition has not been identified.
IGFBP-3 has been implicated as a mediator of the actions of TGF-,
retinoic acid, and the tumor suppressor gene p53 (19-21). Since the
type V TGF- receptor appears to play an important role in
TGF--induced growth inhibition (7), we tested the hypothesis that
IGF-independent actions of IGFBP-3 are mediated by the type V TGF-
receptor. In this communication, we demonstrate that IGFBP-3 inhibits
125I-labeled TGF-1
(125I-TGF-1) binding to the type V TGF-
receptor in mink lung epithelial cells. We also show that
125I-labeled IGFBP-3 (125I-IGFBP-3)
affinity-labeled putative cell-surface IGFBP-3 receptor is
immunoprecipitated by specific antiserum to the type V TGF- receptor
and that the 125I-IGFBP-3-putative receptor complex is
detected only in cells expressing the type V TGF- receptor. Finally,
we show that 125I-IGFBP-3 affinity labeling of the putative
IGFBP-3 receptor and IGFBP-3-induced growth inhibition can be blocked
by a TGF-1 peptide antagonist.
EXPERIMENTAL PROCEDURES
Na125I (17 Ci/mg) and
[methyl-3H]Thymidine (67 Ci/mmol) were
purchased from ICN Biochemicals Inc. (Costa Mesa, CA). High molecular mass protein standards (myosin, 205 kDa; -galactosidase, 116 kDa;
phosphorylase, 97 kDa; bovine serum albumin, 66 kDa) and other chemical
reagents were obtained from Sigma. Disuccinimidyl suberate (DSS) was
obtained from Pierce. TGF-1 was purchased from Austral
Biologicals (San Ramon, CA). Recombinant nonglycosylated human IGFBP-3
(expressed in Escherichia coli) was provided by Celtrix
Pharmaceutical Inc. (Santa Clara, CA).
125I-TGF-1 and 125I-IGFBP-3 were
prepared as described previously (3, 5, 26) except 0.2 M
sodium phosphate buffer, pH 7.4, was used as the solvent for Sephadex
G-25 column chromatography to separate 125I-IGFBP-3 from
free 125I. The specific radioactivity of
125I-TGF-1 and 125I-IGFBP-3 was
1-4 × 105 cpm/ng. The antigen used to prepare
specific rabbit antiserum to the type V TGF- receptor was
thyroglobulin-conjugated to a hexadecapeptide whose amino acid sequence
was derived from the partial amino acid sequence of bovine type V
TGF- receptor (7). The antiserum specifically reacted with the type
V TGF- receptor from different species, including mink, rat, mouse,
cow, and human (7). This antiserum did not react with the type I, type
II, and type III TGF- receptors on Western blot analysis and in
immunoprecipitation (7). TGF-1 and TGF-3
peptide antagonists were synthetic pentacosapeptides whose amino acid
sequences were derived from those of TGF-1 and TGF-3,
respectively.2 The
IC50 values of TGF-1 and
TGF-3 peptide antagonists for inhibiting
125I-TGF-1 (0.1 nM) binding to
TGF- receptors in mink lung epithelial cells are ~1-2 and
~20-30 µM, respectively.2 Human colorectal
carcinoma cells transfected with neo vector only and with
vector expressing type II TGF- receptor cDNA (HCT-116 and RII-37
cells) were provided by Dr. Michael G. Brattain. (Department of
Biochemistry and Molecular Biology, Medical College of Ohio, Toledo,
OH)The type I and type II TGF- receptor-defective mutant mink lung
epithelial cells (R1-B and DR 26 cells) were provided by Dr. Joan
Massagué (Sloan-Kettering Cancer Center, New York). Wild-type and
mutant mink lung epithelial cells and other cell types were maintained
in Dulbecco's modified Eagle medium containing 10% fetal calf
serum.
1 Binding and Affinity
Labeling in Mink Lung Epithelial Cells and Human Colorectal Carcinoma
Cells
The 125I-TGF- binding and affinity labeling
were carried out as described previously (3, 26). The specific binding
of 125I-TGF-1 was calculated by subtracting
the total binding from the nonspecific binding obtained in the presence
of 100-fold excess of unlabeled TGF-1 or 10 µM TGF-1 peptide antagonist. The
125I-TGF-1 affinity labeling of cell-surface
TGF- receptors was carried out using DSS as the cross-linking agent
(3, 26).
Cells grown on 60-mm Petri dishes were incubated with 5 nM 125I-IGFBP-3 (specific radioactivity:
~1 × 105 cpm/ng) in the presence of 100-fold excess
of unlabeled IGFBP-3 or inhibitors, including heparin, IGF-I,
TGF-1, and TGF-3 peptide antagonists.
After 125I-IGFBP-3 affinity labeling in the presence of DSS
(3, 26), the 125I-IGFBP-3-putative receptor complex was
analyzed by 5% SDS-polyacrylamide gel electrophoresis under reducing
conditions and autoradiography.
Receptor
After 125I-IGFBP-3 affinity labeling, the
cells were detached and lysed in 100 µl of 1% Triton X-100 in 10 mM Tris-HCl, pH 7.0, 125 mM NaCl, and 1 mM EDTA. After centrifugation, the Triton X-100 extracts
were then diluted 10-fold with Triton X-100-free buffer and incubated
with antiserum or non-immune serum (1:100 dilution) at 4 °C
overnight. The immunocomplexes were precipitated with 20 µl of
protein A-Sepharose (50%, v/v). After washing with 20 mM
Tris-HCl, pH 7.4, 0.2% Triton X-100, the immunoprecipitates were
analyzed by 5% SDS-polyacrylamide gel electrophoresis under reducing
conditions and autoradiography. The relative intensity of
125I-IGFBP-3-type V TGF- receptor complex on the
autoradiogram was quantitated by a PhosphorImager.
Cells were plated on 24-well clustered dishes at near
confluence and incubated with various concentrations of IGFBP-3 or 10 pM of TGF-1 ± 10 µM
TGF-1 peptide antagonist in Dulbecco's modified Eagle
medium containing 0.1% fetal calf serum. After incubation at 37 °C
for 16 h, the cells were pulse-labeled with 1 µCi/ml of
[methyl-3H]thymidine at 37 °C for 4 h.
The [methyl-3H]thymidine incorporation into
cellular DNA was determined by a liquid scintillation counter. For RNA
analysis, cells grown on 12-well cluster dishes were treated with
various concentrations (0, 0.2, 0.4, 0.8, and 5 µg/ml) of IGFBP-3 or
with 0.1 nM TGF- (as a positive control) for 2.5 h
at 37 °C in 0.1% fetal calf serum. Total cellular RNA was extracted
with RNAzol B (Tel-Test, Inc.) according to the manufacturer's
protocol. RNA was electrophoresed in 1.2% formaldehyde-agarose gel and
transferred to Duralon UV membrane using 10 × SCC. The Northern
blot was probed at 42 °C with a random-primed, radiolabeled
1-kilobase fragment of HindIII and NcoI digests
of plasminogen activator inhibitor 1 (PAI-1) cDNA. The blots were
washed with 0.1 × SCC containing 0.1% SDS at room
temperature.
RESULTS AND DISCUSSION
TGF- is the most potent known polypeptide growth inhibitor for
epithelial cells and other cell types (23-25). Our recent studies have
indicated that the type V TGF- receptor, a 400-kDa membrane glycoprotein which co-expresses with the type I, type II, and type III
TGF- receptors in most cell types (2-4, 26), plays an important
role in mediating TGF--induced growth inhibition in mink lung
epithelial cells (7). To see if the IGF-independent growth inhibitory
action of IGFBP-3 is mediated by the type V TGF- receptor or other
TGF- receptor types, we investigated the effect of IGFBP-3 on the
binding of 125I-TGF-1 to mink lung
epithelial cells, for which IGFBP-3 is also a growth inhibitor. As
shown in Fig. 1A, IGFBP-3
inhibited the specific binding of 125I-TGF-1
in a concentration-dependent manner. At 16 µg/ml (~500 nM) or higher of IGFBP-3, a maximal ~50% inhibition was
observed. This partial inhibition implies that IGFBP-3 competes with
125I-TGF-1 for binding to specific TGF-
receptor types. To identify which TGF- receptor types are
responsible for IGFBP-3 binding, we performed
125I-TGF-1 affinity labeling of cell-surface
TGF- receptors after incubation of the cells with
125I-TGF-1 in the presence of 16 µg/ml
unlabeled IGFBP-3 or 10 µM of TGF-1
peptide antagonist. The TGF-1 peptide antagonist is a
synthetic pentacosapeptide whose amino acid sequence was derived from
that of TGF-1.2 As shown in Fig.
1B, the type I, type II, type III, and type V TGF-
receptors were all affinity-labeled with
125I-TGF-1 in the presence of the
cross-linking agent DSS (lane 2). Unlabeled IGFBP-3 (~500
nM) appeared to completely block
125I-TGF-1 affinity labeling of the type V
TGF- receptor, and to a much lesser extent (30-40% inhibition),
the type III TGF- receptor (Fig. 1B, lane 3). In the
control experiment, the TGF-1 peptide antagonist
completely blocked 125I-TGF-1 affinity
labeling of all TGF- receptor types (Fig. 1B, lane 1).
These results suggest that IGFBP-3 strongly competes with
125I-TGF-1 for binding to the type V TGF-
receptor.
1 binding (A) and
125I-TGF-1 affinity labeling (B)
of the type V TGF- receptor in mink lung epithelial cells.
A, cells were incubated with 0.1 nM
125I-TGF-1 in the presence of various
concentrations of IGFBP-3 with or without 100-fold excess of unlabeled
TGF-1. The specific binding of
125I-TGF-1 to the cells was determined. The
specific binding of 125I-TGF-1 in the
absence of IGFBP-3 was taken as 0% inhibition (4,537 ± 250 cpm/well). The error bars are means ± S.D. of
triplicate cultures. B, after incubation of cells with 0.1 nM 125I-TGF-1 in the absence
(lane 2) and presence of 16 µg/ml of IGFBP-3 (lane
3) or 10 µM TGF-1 peptide antagonist
(lane 1), the cell-surface TGF- receptors were
affinity-labeled and analyzed by 5% SDS-polyacrylamide gel
electrophoresis and autoradiography. The brackets indicate the locations of the 125I-TGF-1
affinity-labeled type I, type II, and type III TGF- receptors
(TR-I, TR-II, and TR-III). The
arrow indicates the location of the
125I-TGF-1 affinity-labeled type V TGF-
receptor (TR-V).
To further confirm that IGFBP-3 binds to the type V TGF- receptor
with high affinity or that the type V TGF- receptor is the putative
receptor for IGFBP-3, we performed the binding and cross-linking of
125I-labeled recombinant nonglycosylated human IGFBP-3
(125I-IGFBP-3, 5 nM) to its putative
cell-surface receptor, followed by immunoprecipitation with specific
antiserum to the type V TGF- receptor (7). At 5 nM,
125I-IGFBP-3 was found to bind to the type V TGF-
receptor but not other TGF- receptor types. As shown in Fig.
2A, 125I-IGFBP-3
was cross-linked to an ~400-kDa putative receptor on the cell surface
of mink lung epithelial cells (lane 1). This 125I-IGFBP-3 binding and subsequent cross-linking was
blocked by 100-fold excess of unlabeled IGFBP-3 or 10 µM
TGF-1 peptide antagonist but not by 10 µM
TGF-3 peptide antagonist (Fig. 2A, lanes 2, 3, and 4, respectively). The TGF-3
peptide antagonist, a pentacosapeptide whose amino acid sequence was
derived from TGF-3, has a lower affinity to the type V
TGF- receptor.2 The antiserum to the type V TGF-
receptor specifically immunoprecipitated the ~400-kDa
125I-IGFBP-3-putative receptor complex (Fig. 2A,
lanes 5 and 7). Two, ~70-kDa and ~64 kDa,
125I-IGFBP-3 complexes were also found in the cell lysates
and in the immunoprecipitates (Fig. 2A, lanes 1, 4, 5, and
7). Since the preparation of 125I-IGFBP-3
(apparent Mr ~35,000 on SDS-polyacrylamide gel
electrophoresis) used in the experiments was found to contain
proteolytic products (apparent Mr 
32,000),
and since 125I-IGFBP-3 has been shown to form a dimer in
solution (17),3 these
125IGFBP-3 complexes may be cross-linked dimers of
125I-IGFBP-3 and its proteolytic products. In the control
experiments, no 125I-IGFBP-3-putative receptor complex was
found in the immunoprecipitates when the cells were incubated with
125I-IGFBP-3 in the presence of 10 µM
TGF-1 peptide antagonist prior to cross-linking and
immunoprecipitation (Fig. 2A, lane 6). Non-immune serum did
not immunoprecipitate the 125I-IGFBP-3-putative receptor
complex (Fig. 2A, lane 8). These results suggest that
125I-IGFBP-3 specifically binds to the type V TGF-
receptor in mink lung epithelial cells. To further characterize the
binding of 125I-IGFBP-3 to the type V TGF- receptor, we
determined the specific binding of various concentrations of
125I-IGFBP-3 to the type V TGF- receptor in mink lung
epithelial cells. As shown in Fig. 2B, 125 I-IGFBP-3 bound to the type V TGF- receptor in these cells in a
concentration dependent manner (lanes 1-5). The
Scatchard plot analysis of the binding revealed that the apparent
Kd for 125I-IGFBP-3 binding to the type
V TGF- receptor was 6 ± 2 nM (data not shown).
Since IGFBP-3 is known to bind IGFs with high affinity, and since it
contains a heparin-binding site near its C-terminal end (8-11), we
determined the effects of the IGF-I complex and heparin on the binding
of 125I-IGFBP-3 to type V TGF- receptor in mink lung
epithelial cells. As shown in Fig. 2C, at 1 mol:1 mol
stoichiometry of IGF-I and 125I-IGFBP-3, approximately 80%
of the 125I-IGFBP-3 specific binding to the type V TGF-
receptor was inhibited. Heparin at 100 µg/ml inhibited ~80% of the
125I-IGFBP-3 binding to the type V TGF- receptor (Fig.
2D, lane 9 versus lane 1). The control (Fig. 2D, lane
1) was overexposed to show the 125I-IGFBP-3-type V
TGF- receptor complex in lanes 2, 3, and 8. These results suggest that both the 125I-IGFBP-3-IGF-I
complex and the 125I-IGFBP-3-heparin complex are not
capable of binding to the type V TGF- receptor. As a control,
TGF-1 peptide antagonist (3 µM) strongly
inhibited >95% of the 125I-IGFBP-3 binding to the type V
TGF- receptor (Fig. 2D, lane 2 versus lane 1). To further
demonstrate that the type V TGF- receptor is the putative IGFBP-3
receptor, we performed the 125I-IGFBP-3 affinity labeling
of its putative cell-surface receptor in cells expressing and lacking
the type V TGF- receptor. As shown in Fig.
3, human colorectal carcinoma cells
(RII-37 cells and HCT 116 Neo cells), which lack the type V TGF-
receptor (7, 27), did not show the ~400-kDa
125I-IGFBP-3-putative receptor complex (Fig. 3, lanes
3-6). In contrast, NIH 3T3 cells, which are known to express the
type V TGF- receptor (26), showed the ~400-kDa
125I-IGFBP-3-putative receptor complex (Fig. 3, lanes
1 and 2). The formation of the ~400-kDa
125I-IGFBP-3-putative receptor complex in NIH 3T3 cells was
blocked in the presence of 100-fold excess of unlabeled IGFBP-3 or 10 µM TGF-1 peptide antagonist (data not
shown).
receptor in mink lung epithelial cells
(A and B) and inhibition of
125I-IGFBP-3 binding to the type V TGF- receptor in
these cells by IGF-I (C), heparin, and TGF-1
peptide antagonist (D). A, cells were incubated
with 5 nM 125I-IGFBP-3 in the absence
(lane 1) and presence of 100-fold excess of unlabeled
IGFBP-3 (lane 2), 10 µM TGF-1
peptide antagonist (lanes 3 and 6), or 10 µM TGF-3 peptide antagonist (lanes
4 and 5). The cell-surface putative receptor was then
affinity-labeled. After affinity labeling, the cell lysates were
directly analyzed by 5% SDS-polyacrylamide gel electrophoresis and
autoradiography (lanes 1-4) or subjected to
immunoprecipitation with specific antiserum to the type V TGF-
receptor (lanes 5-7) or with non-immune serum (lane
8). The immunoprecipitates were then analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. The arrow indicates the location of the
125I-IGFBP-3-type V TGF- receptor (TR-V)
complex. The arrowheads indicate the locations of two,
~70-kDa and ~64-kDa, 125I-IGFBP-3 complexes, which are
likely the cross-linked dimers of 125I-IGFBP-3
(Mr ~35,000) and its degradation products.
B, cells were incubated with various concentrations of
125I-IGFBP-3 (lanes 1-5). After affinity
labeling, the 125I-IGFBP-3-type V TGF- receptor complex
was analyzed by 5% SDS-polyacrylamide gel electrophoresis and
autoradiography. The arrow indicates the location of the
125I-IGFBP-3-type V TGF- receptor (TR-V)
complex. The relative intensity of the 125I-IGFBP-3-type V
TGF- receptor (TR-V) complex on the autoradiogram was
quantitated by a PhosphorImager. C, cells were incubated
with 5 nM of 125I-IGFBP-3 and various
concentrations of IGF-I or 10 µM TGF-1 peptide antagonist (for estimation of nonspecific or non-type V TGF-
receptor-mediated binding). 125I-IGFBP-3 and IGF-I were
preincubated on ice for 10 min prior to the binding assay. The specific
binding of 125I-IGFBP-3 to the type V TGF- receptor was
then determined. The specific binding of 125I-IGFBP-3
obtained in the absence of IGF-I was taken as 100% binding (4,657 ± 321 cpm/well). D, after 125I-IGFBP-3 binding
in the absence (lane 1) and presence of 10 and 100 µg/ml
of heparin (lanes 8 and 9) or various
concentrations of TGF-1 peptide antagonist (lanes
2-7), 125I-IGFBP-3 affinity labeling was carried out
in the presence of DSS. The 125I-IGFBP-3-type V TGF-
receptor (TR-V) complex was then analyzed by 5%
SDS-polyacrylamide gel electrophoresis and autoradiography. The control
(lane 1) was overexposed to show the
125I-IGFBP-3-type V TGF- receptor complex in lanes
2, 3, and 9. The arrow indicates the
location of the 125I-IGFBP-3-type V TGF- receptor
(TR-V) complex. The relative intensity of the
125I-IGFBP-3-type V TGF- receptor (TR-V)
complex was quantitated by a PhosphorImager.
1 peptide antagonist (data not shown). The
arrow indicates the location of
125I-IGFBP-3-type V TGF- receptor (TR-V)
complex.
Since the type V TGF- receptor has been shown to mediate the growth
inhibitory response in mink lung epithelial cells (7), we examined the
effect of IGFBP-3 on the proliferation of wild-type and type I and type
II TGF- receptor-defective mutant mink lung epithelial cells (Mv1Lu,
R-1B, and DR26 cells, respectively) (22, 28-30). All Mv1Lu, R-1B, and
DR26 cells have been shown to express the type V TGF- receptor (7).
IGFBP-3 should be a specific ligand to test the function of the type V
TGF- receptor, because it does not bind to the type I, type II, or
type III TGF- receptor with high affinity. As shown in Fig.
4, IGFBP-3 (0.6 µg/ml or ~20
nM) induced a similar growth inhibitory response as
measured by DNA synthesis (~60% inhibition) in either wild-type
(Mv1Lu cells) or type II TGF- receptor-defective mutant mink lung
epithelial cells (DR26 cells), but to a lesser extent (~20%
inhibition) in type I TGF- receptor-defective mutant cells (R-1B
cells). The growth inhibitory response induced by IGFBP-3 in these
cells could be blocked in the presence of TGF-1 peptide
antagonist (Fig. 4). These results indicate that IGFBP-3 induces a
growth inhibitory response in cells expressing the type V TGF-
receptor. These results also support the hypothesis that the type V
TGF- receptor can mediate the growth inhibitory response (7).
receptor-defective mutant mink lung epithelial cells. Wild-type
and type I and type II TGF- receptor-defective mutant mink lung
epithelial cells (Mv1Lu, R-1B, and DR26 cells) were incubated with
various concentrations of IGFBP-3 in the presence and absence of 10 µM TGF-1 peptide antagonist. The
[methyl-3H]thymidine incorporation into DNA of
Mv1Lu, R-1B, and DR26 cells treated without IGFBP-3 was taken as 0%
inhibition (22,500 ± 1,063 cpm/well, 18,775 ± 595 cpm/well,
and 25,615 ± 757 cpm/well, respectively). The error
bars are means ± S.D. of triplicate cell cultures.
In a previous study (26), we reported that many types of carcinoma
cells lacked the type V TGF- receptor and that such cells do not
respond to TGF-1 stimulation, as measured by growth inhibition (7). Recently, hereditary human colorectal carcinoma cells
(HCT 116 cells) were shown to be deficient in the type II TGF-
receptor (27). Stable transfection of these carcinoma cells with the
type II TGF- receptor cDNA was found to rescue the
transcriptional response but failed to restore the growth inhibitory
response to exogenous TGF- stimulation (27). This appears to be due
to the lack of the type V TGF- receptor expression in cells stably
transfected with the neo vector only (HCT 116 Neo cells) or
with vector expressing the type II TGF- receptor cDNA (RII-37
cells) (7, 27). As would be expected, IGFBP-3 also failed to inhibit
growth in these HCT 116 Neo and RII-37 cells that do not express the
type V TGF- receptor (data not shown).
TGF- elicits a variety of biological activities in different cell
types (23-25). In addition to growth inhibitory activity, the other
prominent activity of TGF- is transcriptional activation of
fibronectin, collagen, and PAI-1 genes (23-25). To see if IGFBP-3 and
TGF- share similar activities, we determined the effect of IGFBP-3
on the transcriptional expression of PAI-1 in mink lung epithelial
cells. IGFBP-3 showed little if any effect on the transcription of
PAI-1 in these epithelial cells (data not shown). TGF- has been
shown to be a bifunctional growth regulator: a growth inhibitor for
epithelial cells, endothelial cells, and other cell types, and a
mitogenic factor for mesenchymal cells (23-25). We therefore determined the effect of IGFBP-3 on DNA synthesis in NIH 3T3 cells, for
which TGF- is a mitogen. IGFBP-3 did not stimulate DNA synthesis of
NIH 3T3 cells at concentrations of 0.1-100 nM, suggesting
that IGFBP-3 is a partial agonist of TGF-. These results also
support the hypothesis that the type V TGF- receptor preferentially
mediates the growth inhibitory response in responsive cells (7).
IGFBP-3 has been implicated as a mediator of the actions of TGF-,
retinoic acid, and p53 (19-21). Antisense deoxyoligonucleotide to
IGFBP-3 has been shown to diminish the growth inhibitory response induced by TGF- and retinoic acid in human mammary carcinoma cells
(19-21). The functional role of IGFBP-3 in TGF--induced growth
inhibition in other cell types is unknown. We speculate that the
IGFBP-3 expression induced by TGF- and retinoic acid in these
mammary carcinoma cells may cause the growth inhibition by sequestering
IGFs from binding to IGF-I receptor in IGF-responsive cells and by
propagating the growth inhibitory response mediated by the type V
TGF- receptor in IGF-unresponsive cells. It is important to note
that upon ligand activation, the type V TGF- receptor may also
decrease IGF-II concentration in the extracellular compartment by
increasing the internalization and recycling of the cell surface
mannose 6-phosphate/IGF-II
receptor.4 This effect on
IGF-II may also contribute to the growth inhibitory response mediated
by the type V TGF- receptor.4
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, St. Louis University School of Medicine, St.
Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-577-8156; E-mail: huangjs{at}wpogate.slu.edu.
ACKNOWLEDGEMENTS
We thank Celtrix Pharmaceutical Inc. for
providing recombinant nonglycosylated human IGFBP-3; Drs. Joan
Massagué and Michael G. Brattain for providing TGF-
receptor-defective mutant mink lung epithelial cells (R-1B and DR26
cells) and human colorectal carcinoma cells (HCT 116 Neo and RII-37
cells), respectively; Drs. William S. Sly and Frank E. Johnson for
critical comments and review of the manuscript; and Maggie Klevorn for
editorial assistance in the preparation of this manuscript.
REFERENCES
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M. Oufattole, S. W.-J. Lin, B. Liu, D. Mascarenhas, P. Cohen, and B. D. Rodgers Ribonucleic Acid Polymerase II Binding Subunit 3 (Rpb3), a Potential Nuclear Target of Insulin-Like Growth Factor Binding Protein-3 Endocrinology, May 1, 2006; 147(5): 2138 - 2146. [Abstract] [Full Text] [PDF]
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J. V. Silha, P. C. Sheppard, S. Mishra, Y. Gui, J. Schwartz, J. G. Dodd, and L. J. Murphy Insulin-Like Growth Factor (IGF) Binding Protein-3 Attenuates Prostate Tumor Growth by IGF-Dependent and IGF-Independent Mechanisms Endocrinology, May 1, 2006; 147(5): 2112 - 2121. [Abstract] [Full Text] [PDF]
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 S. H. Lee, M. Takahashi, K. Honke, E. Miyoshi, D. Osumi, H. Sakiyama, A. Ekuni, X. Wang, S. Inoue, J. Gu, et al. Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in fut8-/- mice. J. Biochem., March 1, 2006; 139(3): 391 - 398. [Abstract] [Full Text] [PDF]
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 S.-H. Oh, W.-Y. Kim, J.-H. Kim, M. N. Younes, A. K. El-Naggar, J. N. Myers, M. Kies, P. Cohen, F. Khuri, W. K. Hong, et al. Identification of Insulin-Like Growth Factor Binding Protein-3 as a Farnesyl Transferase Inhibitor SCH66336-Induced Negative Regulator of Angiogenesis in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., January 15, 2006; 12(2): 653 - 661. [Abstract] [Full Text] [PDF]
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 L O'Rear, L Longobardi, M Torello, B K Law, H L Moses, F Chiarelli, and A Spagnoli Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-{beta} in mesenchymal chondroprogenitor cell growth J. Mol. Endocrinol., June 1, 2005; 34(3): 723 - 737. [Abstract] [Full Text] [PDF]
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J. V. Silha, Y. Gui, S. Mishra, A. Leckstrom, P. Cohen, and L. J. Murphy Overexpression of Gly56/Gly80/Gly81-Mutant Insulin-Like Growth Factor-Binding Protein-3 in Transgenic Mice Endocrinology, March 1, 2005; 146(3): 1523 - 1531. [Abstract] [Full Text] [PDF]
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 Y. Li, J. Xiang, and C. Duan Insulin-like Growth Factor-binding Protein-3 Plays an Important Role in Regulating Pharyngeal Skeleton and Inner Ear Formation and Differentiation J. Biol. Chem., February 4, 2005; 280(5): 3613 - 3620. [Abstract] [Full Text] [PDF]
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X. Yan, B. E. Forbes, K. A. McNeil, R. C. Baxter, and S. M. Firth Role of N- and C-terminal Residues of Insulin-like Growth Factor (IGF)-binding Protein-3 in Regulating IGF Complex Formation and Receptor Activation J. Biol. Chem., December 17, 2004; 279(51): 53232 - 53240. [Abstract] [Full Text] [PDF]
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 M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]
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 H.-Y. Lee, H. Moon, K.-H. Chun, Y.-S. Chang, K. Hassan, L. Ji, R. Lotan, F. R. Khuri, and W. K. Hong Effects of Insulin-like Growth Factor Binding Protein-3 and Farnesyltransferase Inhibitor SCH66336 on Akt Expression and Apoptosis in Non-Small-Cell Lung Cancer Cells J Natl Cancer Inst, October 20, 2004; 96(20): 1536 - 1548. [Abstract] [Full Text] [PDF]
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 J. F. Kuemmerle, K. S. Murthy, and J. G. Bowers IGFBP-3 activates TGF-{beta} receptors and directly inhibits growth in human intestinal smooth muscle cells Am J Physiol Gastrointest Liver Physiol, October 1, 2004; 287(4): G795 - G802. [Abstract] [Full Text] [PDF]
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T.-Y. Ling, C.-L. Chen, Y.-H. Huang, I-H. Liu, S. S. Huang, and J. S. Huang Identification and Characterization of the Acidic pH Binding Sites for Growth Regulatory Ligands of Low Density Lipoprotein Receptor-related Protein-1 J. Biol. Chem., September 10, 2004; 279(37): 38736 - 38748. [Abstract] [Full Text] [PDF]
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 L. Peng, P. J. Malloy, and D. Feldman Identification of a Functional Vitamin D Response Element in the Human Insulin-Like Growth Factor Binding Protein-3 Promoter Mol. Endocrinol., May 1, 2004; 18(5): 1109 - 1119. [Abstract] [Full Text] [PDF]
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