The Type V Transforming Growth Factor beta  Receptor Is the Putative Insulin-like Growth Factor-binding Protein 3 Receptor*

(Received for publication, May 28, 1997)

Sandra M. Leal , Qianjin Liu , Shuan Shian Huang and Jung San Huang Dagger

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)-beta 1 binding to the type V TGF-beta 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-beta 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-beta 1 peptide antagonist. The 125I-IGFBP-3 affinity-labeled putative receptor can only be detected in cells expressing the type V TGF-beta receptor, but not in cells lacking the type V TGF-beta receptor. These results indicate that the type V TGF-beta receptor is the putative IGFBP-3 receptor and that IGFBP-3 is a functional ligand for the type V TGF-beta receptor.


INTRODUCTION

The type V transforming growth factor beta  (TGF-beta )1 receptor is a 400-kDa non-proteoglycan membrane glycoprotein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types (1-5). The type V TGF-beta receptor as well as the type I and type II TGF-beta 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-beta receptors have been shown to be important in TGF-beta -induced cellular responses (1-6), but the role of the type V TGF-beta receptor in these responses has not been defined (3-5). Recently, we have demonstrated that the type V TGF-beta receptor mediates TGF-beta -induced growth inhibition and that both type I and type II TGF-beta 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-beta , retinoic acid, and the tumor suppressor gene p53 (19-21). Since the type V TGF-beta receptor appears to play an important role in TGF-beta -induced growth inhibition (7), we tested the hypothesis that IGF-independent actions of IGFBP-3 are mediated by the type V TGF-beta receptor. In this communication, we demonstrate that IGFBP-3 inhibits 125I-labeled TGF-beta 1 (125I-TGF-beta 1) binding to the type V TGF-beta 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-beta receptor and that the 125I-IGFBP-3-putative receptor complex is detected only in cells expressing the type V TGF-beta 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-beta 1 peptide antagonist.


EXPERIMENTAL PROCEDURES

Materials

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; beta -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-beta 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-beta 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-beta 1 and 125I-IGFBP-3 was 1-4 × 105 cpm/ng. The antigen used to prepare specific rabbit antiserum to the type V TGF-beta receptor was thyroglobulin-conjugated to a hexadecapeptide whose amino acid sequence was derived from the partial amino acid sequence of bovine type V TGF-beta receptor (7). The antiserum specifically reacted with the type V TGF-beta 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-beta receptors on Western blot analysis and in immunoprecipitation (7). TGF-beta 1 and TGF-beta 3 peptide antagonists were synthetic pentacosapeptides whose amino acid sequences were derived from those of TGF-beta 1 and TGF-beta 3, respectively.2 The IC50 values of TGF-beta 1 and TGF-beta 3 peptide antagonists for inhibiting 125I-TGF-beta 1 (0.1 nM) binding to TGF-beta 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-beta 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-beta 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.

125I-TGF-beta 1 Binding and Affinity Labeling in Mink Lung Epithelial Cells and Human Colorectal Carcinoma Cells

The 125I-TGF-beta binding and affinity labeling were carried out as described previously (3, 26). The specific binding of 125I-TGF-beta 1 was calculated by subtracting the total binding from the nonspecific binding obtained in the presence of 100-fold excess of unlabeled TGF-beta 1 or 10 µM TGF-beta 1 peptide antagonist. The 125I-TGF-beta 1 affinity labeling of cell-surface TGF-beta receptors was carried out using DSS as the cross-linking agent (3, 26).

125I-IGFBP-3 Binding and Affinity Labeling in Mink Lung Epithelial Cells and Human Colorectal Carcinoma Cells and NIH 3T3 Cells

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-beta 1, and TGF-beta 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.

Immunoprecipitation of the 125I-IGFBP-3-Putative Receptor Complex with Specific Antiserum to the Type V TGF-beta 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-beta receptor complex on the autoradiogram was quantitated by a PhosphorImager.

[methyl-3H]Thymidine Incorporation Assay and RNA Analysis

Cells were plated on 24-well clustered dishes at near confluence and incubated with various concentrations of IGFBP-3 or 10 pM of TGF-beta 1 ± 10 µM TGF-beta 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-beta (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-beta 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-beta receptor, a 400-kDa membrane glycoprotein which co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types (2-4, 26), plays an important role in mediating TGF-beta -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-beta receptor or other TGF-beta receptor types, we investigated the effect of IGFBP-3 on the binding of 125I-TGF-beta 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-beta 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-beta 1 for binding to specific TGF-beta receptor types. To identify which TGF-beta receptor types are responsible for IGFBP-3 binding, we performed 125I-TGF-beta 1 affinity labeling of cell-surface TGF-beta receptors after incubation of the cells with 125I-TGF-beta 1 in the presence of 16 µg/ml unlabeled IGFBP-3 or 10 µM of TGF-beta 1 peptide antagonist. The TGF-beta 1 peptide antagonist is a synthetic pentacosapeptide whose amino acid sequence was derived from that of TGF-beta 1.2 As shown in Fig. 1B, the type I, type II, type III, and type V TGF-beta receptors were all affinity-labeled with 125I-TGF-beta 1 in the presence of the cross-linking agent DSS (lane 2). Unlabeled IGFBP-3 (~500 nM) appeared to completely block 125I-TGF-beta 1 affinity labeling of the type V TGF-beta receptor, and to a much lesser extent (30-40% inhibition), the type III TGF-beta receptor (Fig. 1B, lane 3). In the control experiment, the TGF-beta 1 peptide antagonist completely blocked 125I-TGF-beta 1 affinity labeling of all TGF-beta receptor types (Fig. 1B, lane 1). These results suggest that IGFBP-3 strongly competes with 125I-TGF-beta 1 for binding to the type V TGF-beta receptor.


Fig. 1. Effect of IGFBP-3 on 125I-TGF-beta 1 binding (A) and 125I-TGF-beta 1 affinity labeling (B) of the type V TGF-beta receptor in mink lung epithelial cells. A, cells were incubated with 0.1 nM 125I-TGF-beta 1 in the presence of various concentrations of IGFBP-3 with or without 100-fold excess of unlabeled TGF-beta 1. The specific binding of 125I-TGF-beta 1 to the cells was determined. The specific binding of 125I-TGF-beta 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-beta 1 in the absence (lane 2) and presence of 16 µg/ml of IGFBP-3 (lane 3) or 10 µM TGF-beta 1 peptide antagonist (lane 1), the cell-surface TGF-beta receptors were affinity-labeled and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. The brackets indicate the locations of the 125I-TGF-beta 1 affinity-labeled type I, type II, and type III TGF-beta receptors (Tbeta R-I, Tbeta R-II, and Tbeta R-III). The arrow indicates the location of the 125I-TGF-beta 1 affinity-labeled type V TGF-beta receptor (Tbeta R-V).
[View Larger Version of this Image (21K GIF file)]

To further confirm that IGFBP-3 binds to the type V TGF-beta receptor with high affinity or that the type V TGF-beta 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-beta receptor (7). At 5 nM, 125I-IGFBP-3 was found to bind to the type V TGF-beta receptor but not other TGF-beta 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-beta 1 peptide antagonist but not by 10 µM TGF-beta 3 peptide antagonist (Fig. 2A, lanes 2, 3, and 4, respectively). The TGF-beta 3 peptide antagonist, a pentacosapeptide whose amino acid sequence was derived from TGF-beta 3, has a lower affinity to the type V TGF-beta receptor.2 The antiserum to the type V TGF-beta 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-beta 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-beta receptor in mink lung epithelial cells. To further characterize the binding of 125I-IGFBP-3 to the type V TGF-beta receptor, we determined the specific binding of various concentrations of 125I-IGFBP-3 to the type V TGF-beta receptor in mink lung epithelial cells. As shown in Fig. 2B, 125 I-IGFBP-3 bound to the type V TGF-beta 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-beta 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-beta 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-beta receptor was inhibited. Heparin at 100 µg/ml inhibited ~80% of the 125I-IGFBP-3 binding to the type V TGF-beta 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-beta 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-beta receptor. As a control, TGF-beta 1 peptide antagonist (3 µM) strongly inhibited >95% of the 125I-IGFBP-3 binding to the type V TGF-beta receptor (Fig. 2D, lane 2 versus lane 1). To further demonstrate that the type V TGF-beta 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-beta receptor. As shown in Fig. 3, human colorectal carcinoma cells (RII-37 cells and HCT 116 Neo cells), which lack the type V TGF-beta 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-beta 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-beta 1 peptide antagonist (data not shown).


Fig. 2. 125I-IGFBP-3 affinity labeling of the type V TGF-beta receptor in mink lung epithelial cells (A and B) and inhibition of 125I-IGFBP-3 binding to the type V TGF-beta receptor in these cells by IGF-I (C), heparin, and TGF-beta 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-beta 1 peptide antagonist (lanes 3 and 6), or 10 µM TGF-beta 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-beta 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-beta receptor (Tbeta R-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-beta 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-beta receptor (Tbeta R-V) complex. The relative intensity of the 125I-IGFBP-3-type V TGF-beta receptor (Tbeta R-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-beta 1 peptide antagonist (for estimation of nonspecific or non-type V TGF-beta 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-beta 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-beta 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-beta receptor (Tbeta R-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-beta receptor complex in lanes 2, 3, and 9. The arrow indicates the location of the 125I-IGFBP-3-type V TGF-beta receptor (Tbeta R-V) complex. The relative intensity of the 125I-IGFBP-3-type V TGF-beta receptor (Tbeta R-V) complex was quantitated by a PhosphorImager.
[View Larger Version of this Image (62K GIF file)]


Fig. 3. 125I-IGFBP-3 affinity labeling of the putative cell-surface IGFBP-3 receptor in hereditary human colorectal carcinoma cells, mink lung epithelial cells, and NIH 3T3 cells. Cell-surface receptors were affinity-labeled with 125I-IGFBP-3 and DSS. The 125I-IGFBP-3 affinity-labeled receptors were then analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. Prior to affinity labeling (cross-linking), the binding of 125I-IGFBP-3 to HCT 116 Neo, R II-37, and NIH 3T3 cells was carried out in the absence (lanes 1-6) and presence of 10 µM TGF-beta 1 peptide antagonist (data not shown). The arrow indicates the location of 125I-IGFBP-3-type V TGF-beta receptor (Tbeta R-V) complex.
[View Larger Version of this Image (81K GIF file)]

Since the type V TGF-beta 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-beta 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-beta receptor (7). IGFBP-3 should be a specific ligand to test the function of the type V TGF-beta receptor, because it does not bind to the type I, type II, or type III TGF-beta 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-beta receptor-defective mutant mink lung epithelial cells (DR26 cells), but to a lesser extent (~20% inhibition) in type I TGF-beta 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-beta 1 peptide antagonist (Fig. 4). These results indicate that IGFBP-3 induces a growth inhibitory response in cells expressing the type V TGF-beta receptor. These results also support the hypothesis that the type V TGF-beta receptor can mediate the growth inhibitory response (7).


Fig. 4. IGFBP-3-induced growth inhibition as measured by DNA synthesis in wild-type, type I, and type II TGF-beta receptor-defective mutant mink lung epithelial cells. Wild-type and type I and type II TGF-beta 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-beta 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.
[View Larger Version of this Image (28K GIF file)]

In a previous study (26), we reported that many types of carcinoma cells lacked the type V TGF-beta receptor and that such cells do not respond to TGF-beta 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-beta receptor (27). Stable transfection of these carcinoma cells with the type II TGF-beta receptor cDNA was found to rescue the transcriptional response but failed to restore the growth inhibitory response to exogenous TGF-beta stimulation (27). This appears to be due to the lack of the type V TGF-beta receptor expression in cells stably transfected with the neo vector only (HCT 116 Neo cells) or with vector expressing the type II TGF-beta 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-beta receptor (data not shown).

TGF-beta elicits a variety of biological activities in different cell types (23-25). In addition to growth inhibitory activity, the other prominent activity of TGF-beta is transcriptional activation of fibronectin, collagen, and PAI-1 genes (23-25). To see if IGFBP-3 and TGF-beta 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-beta 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-beta 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-beta . These results also support the hypothesis that the type V TGF-beta receptor preferentially mediates the growth inhibitory response in responsive cells (7).

IGFBP-3 has been implicated as a mediator of the actions of TGF-beta , retinoic acid, and p53 (19-21). Antisense deoxyoligonucleotide to IGFBP-3 has been shown to diminish the growth inhibitory response induced by TGF-beta and retinoic acid in human mammary carcinoma cells (19-21). The functional role of IGFBP-3 in TGF-beta -induced growth inhibition in other cell types is unknown. We speculate that the IGFBP-3 expression induced by TGF-beta 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-beta receptor in IGF-unresponsive cells. It is important to note that upon ligand activation, the type V TGF-beta 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-beta receptor.4


FOOTNOTES

*   This work was supported by National Institutes of Health Grant CA 38808.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. Section 1734 solely to indicate this fact.
Dagger    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.
1   The abbreviations used are: TGF, transforming growth factor; IGFBP-3, insulin-like growth factor-binding protein 3; DSS, disuccinimidyl suberate; PAI-1, plasminogen activator inhibitor 1.
2   S. S. Huang, Q. Liu, F. E. Johnson, Y. Konish, and J. S. Huang, submitted for publication.
3   S. M. Leal, Q. Liu, S. S. Huang, and J. S. Huang, unpublished results.
4   Q. Liu, J. H. Grubb, S. S. Huang, W. S. Sly, and J. S. Huang, submitted for publication.

ACKNOWLEDGEMENTS

We thank Celtrix Pharmaceutical Inc. for providing recombinant nonglycosylated human IGFBP-3; Drs. Joan Massagué and Michael G. Brattain for providing TGF-beta 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.


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