IGFBP-3 mediates TGF-beta 1-induced cell growth in human airway smooth muscle cells

Pinchas Cohen1, Roopmathy Rajah1, Joel Rosenbloom2, and David J. Herrick2

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Both insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor-beta (TGF-beta ) have been separately shown to have cell-specific growth-inhibiting or growth-potentiating effects. TGF-beta stimulates IGFBP-3 mRNA and peptide expression in several cell types, and TGF-beta -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-beta and IGFBP-3-induction has not been shown. In this study, we investigated the role of IGFBP-3 in mediating TGF-beta 1-induced cell growth using human airway smooth muscle (ASM) cells as our model. TGF-beta 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-beta 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-beta 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-beta in ASM cells.

insulin-like growth factor binding protein; cell proliferation; receptor; transforming growth factor.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 1 (TGF-beta 1) is a pluripotent cytokine capable of inhibiting or stimulating cell growth depending on the nature of the target cell (19). TGF-beta 1 is a potent growth inhibitor of a variety of epithelial cell types, whereas it stimulates cell growth in stromal cells. TGF-beta 1 and TGF-beta 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-beta 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-beta 1 in prostate cancer cells (25).

The secretion and mitogenic action of TGF-beta 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-beta 1 on ASM cells are not clearly understood.

We therefore hypothesized that the growth stimulatory effect of TGF-beta 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-beta 1, we investigated the ability of IGFBP-3 to induce cell growth and to mediate TGF-beta 1-induced cellular proliferation in human ASM cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta 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-beta 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-beta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of TGF-beta 1 on ASM cell growth. Figure 1 shows the additive effect of complete SCM and TGF-beta 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-beta 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-beta 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-beta 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-beta effect on growth stimulation.


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Fig. 1.   Cell growth induction by transforming growth factor-beta (TGF-beta ). Cells were grown in supplemented serum-containing medium (SCM) in presence and absence of 1 ng/ml TGF-beta 1, and cell proliferation was evaluated with nonradioactive CellTiter 96 assay using 96-well culture plates. * P < 0.01 and ** P < 0.01 relative to SCM treatment at that incubation time.

Effect of TGF-beta 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-beta 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-beta 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).


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Fig. 2.   Regulation of insulin-like growth factor binding proteins (IGFBPs) in airway smooth muscle (ASM)-conditioned medium by TGF-beta 1. A: secreted IGFBP levels were examined using conditioned medium from ASM cells incubated for 72 h with human bronchial smooth muscle (HBSM) serum-free medium with and without 1 ng/ml TGF-beta 1. Samples of 50 µl were separated by nonreducing 12.5% SDS-PAGE and electroblotted onto nitrocellulose. IGFBPs were visualized by Western ligand blotting (WLB) by incubating membrane with 106 counts/min each of 125I-IGF-I and IGF-II followed by autoradiography. B: data from densitometric analysis of WLBs for IGFBP-3 levels are presented as bar graph. * P < 0.001.

Induction of IGFBP-3 secretion by TGF-beta 1 in ASM cells. We confirmed the selective induction of IGFBP-3 by TGF-beta 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-beta 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-beta 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-beta 1 and rose to 1,080 ± 220 ng/ml in the presence of 1 ng/ml of TGF-beta 1 (P < 0.001).


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Fig. 3.   Induction of IGFBP-3 secretion in ASM-conditioned medium by TGF-beta 1. A: conditioned-medium samples (50 µl) from ASM cells treated as described for WLB were subjected to electrophoresis, electroblotted onto nitrocellulose, blocked with 5% nonfat dry milk in Tris-buffered saline, probed with affinity-purified IGFBP-3 antibody, and detected using peroxidase-linked enhanced chemiluminescence detection system. B: data from densitometric analysis of IGFBP-3 immunoblots. Results are expressed as densitometric band intensity arbitrary units (AU). * P < 0.0001. C: IGFBP-3 levels in ASM-conditioned medium as measured by ELISA assay. * P < 0.0001.

Stimulation of IGFBP-3 mRNA levels by TGF-beta 1 in ASM cells. To determine the level of IGFBP-3 mRNA expression under control (SCM) and TGF-beta 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-beta 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-beta 1-treated ASM cells was observed as early as 24 h after treatment (data not shown).


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Fig. 4.   Induction of IGFBP-3 mRNA by TGF-beta 1. A: ASM cells were grown for 48 h in SCM in presence and absence of TGF-beta 1. Total RNA was isolated from 75-cm2 flasks of ASM cells using the acid guanidium thiocyanate-phenol-chloroform extraction method. For Northern blots, 20-µg total RNA 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 or 1.3-kb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment labeled with 32P by random oligo priming. B: specific mRNA levels were quantified (and normalized for GAPDH mRNA levels) using a Molecular Dynamics PhosphorImager, and data from Northern blots were plotted in bar graph. Treatment with TGF-beta 1 shows 20-fold increase in IGFBP-3 mRNA. Results are expressed as densitometric band intensity AU.

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-beta 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-beta ; we suspect that this may be the result of uncharacterized effects of TGF-beta 1 on IGFBP-3 degradation, which may occur through proteases that are TGF inhibited.


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Fig. 5.   Cell growth induction by IGFBP-3. Cells were grown for 5 days in SCM in presence and absence of IGFBP-3 (0-1,000 ng/ml), and cell numbers were evaluated with nonradioactive CellTiter 96 assay using 96-well culture plates. * P < 0.001 relative to SCM.

Selective inhibition of TGF-beta 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-beta 1-induced IGFBP-3 protein secretion from ASM cells. Cells were simultaneously treated with TGF-beta 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-beta 1-treated ASM cell-conditioned medium relative to serum-free conditions. This induction of IGFBP-3 protein by TGF-beta 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).


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Fig. 6.   Inhibition of TGF-beta 1-stimulated IGFBP-3 protein secretion by IGFBP-3 antisense oligodeoxynucleotides. Conditioned-medium samples (50 µl) from ASM cells treated with 1 ng/ml TGF-beta 1 in presence of 20 µg/ml sense or antisense IGFBP-3 oligomers were subjected to electrophoresis and electroblotted onto nitrocellulose. IGFBP-3 was detected using an affinity-purified IGFBP-3 antibody and peroxidase-linked enhanced chemiluminescence detection system. Densitometric analysis of immunoblots is presented as bar graph and demonstrates 10-fold increase in IGFBP-3 peptide secretion in response to TGF-beta 1, which is fully suppressed by antisense oligomer treatment. * P < 0.001 relative to TGF-beta 1-treated conditions.

TGF-beta 1-induced cell growth is inhibited by blocking IGFBP-3. We determined the requirement for IGFBP-3 in the TGF-beta 1 stimulation of ASM cell by evaluating ASM cell proliferation after treating the cells simultaneously with TGF-beta 1 and IGFBP-3 sense or antisense oligomers. Figure 7 shows that IGFBP-3 antisense oligodeoxynucleotides selectively inhibited TGF-beta 1-stimulated cell growth after 5 days of exposure of ASM cells to TGF-beta 1 and SCM. Treatment with IGFBP-3 sense oligomers did not have any effect on TGF-beta 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-beta 1 in SCM and IGFBP-3 neutralizing antibodies. Figure 8 demonstrates that IGFBP-3 neutralizing antibodies (but not control IgG) selectively inhibited TGF-beta 1-stimulated ASM cell growth. Results represent mean values of three experiments performed on sets of eight samples.


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Fig. 7.   Inhibition of TGF-beta 1-stimulated ASM cell growth by IGFBP-3 antisense oligodeoxynucleotides. Cells were grown for 5 days in SCM and 1 ng/ml TGF-beta 1 in presence and absence of 20 µg/ml IGFBP-3 sense or antisense oligomers. Cell numbers were evaluated using nonradioactive CellTiter 96 assay using 96-well culture plates. * P < 0.001 for TGF-beta 1 + antisense (but not sense) treatment relative to TGF-beta 1-treated condition alone.



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Fig. 8.   Inhibition of TGF-beta 1-stimulated ASM cell growth by IGFBP-3 neutralizing antibodies. Cells were grown for 5 days in supplemented SCM in presence of 1 ng/ml TGF-beta 1 and 8 µg/ml neutralizing IGFBP-3 antibodies or control IgG. Cell numbers were evaluated by nonradioactive CellTiter 96 assay using 96-well culture plates. * P < 0.001 relative to control IgG.

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-beta 1-treated conditions. However, the 68-kDa protein is elevated in the TGF-beta 1-treated conditions. In addition, two new IGFBP-3 association proteins of approximately 58 and 16 kDa were detected only in TGF-beta 1-treated cell extracts.


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Fig. 9.   Putative IGFBP-3 receptors in ASM cells. Cell lysates were prepared from confluent ASM cells grown for 72 h in SCM with (+) and without (-) 1 ng/ml TGF-beta 1, subjected to electrophoresis, and electroblotted onto nitrocellulose. Membranes were incubated with 2 × 106 counts/min of 125I-IGFBP-3 for 12 h, exposed to film for 3 days, and visualized by autoradiography. Note induction IGFBP-3-binding bands at 16 and 58 kDa (bottom 2 arrows) in TGF-beta 1-treated lanes. Top 3 arrows, IGFBP-3 association proteins of ~150, 100, and 68 kDa. Nos. on left, molecular mass in kDa.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 1 is a pluripotent modulator of cell function and an important suppressor of epithelial cell proliferation. The predominant effect of TGF-beta 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-beta 1-induced growth inhibition (2). Furthermore, TGF-beta 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-beta 1-regulated cell growth, which may be dependent on the specific cell type.

Several mechanisms have been suggested for the TGF-beta 1-induced cell growth in transformed cells. Earlier studies demonstrated that the inhibition of epithelial cell proliferation by TGF-beta 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-beta 1 (7). Other studies showed that growth stimulation by TGF-beta 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-beta 1 may induce cell growth and proliferation.

Our observation of increased expression of IGFBP-3 mRNA and protein by TGF-beta 1 in ASM cells led us to examine the role of IGFBP-3 in TGF-beta 1-induced cell growth in ASM cells. Confirmation for the role of IGFBP-3 in TGF-beta 1-induced cell growth was obtained by preventing the increase in ASM cell growth stimulated by TGF-beta 1 by exposing the ASM cells to IGFBP-3-specific antisense oligomers or IGFBP-3 neutralizing antibodies along with TGF-beta 1. Blocking IGFBP-3 protein synthesis almost completely blocked the cell growth stimulatory effect of TGF-beta 1. This observation suggests that IGFBP-3 may be a key mediator of TGF-beta 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-beta 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-beta 1 receptor, have not been demonstrated. This TGF-beta type V receptor is approximately 400 kDa in size and appears to bind both TGF-beta 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-beta 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-beta 1 in human ASM cells and further provides a possible mechanism by which TGF-beta 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-beta 1 has a complex mechanism of action on the induction of ASM cell growth and that IGFBP-3 plays a significant role in TGF-beta 1 action and in the structural changes seen in airways of asthmatic subjects.

In summary, human ASM cells treated with the growth factor TGF-beta 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-beta 1 display an increased IGFBP-3 mRNA and protein expression. Treatment with either IGFBP-3 antisense oligonucleotide or neutralizing antibodies blocks the TGF-beta 1-induced increase in ASM cell growth. These results provide evidence that TGF-beta 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.


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Fig. 10.   Hypothetical diagrammatic representation of TGF-beta -induced IGFBP-3-mediated cell growth in ASM cells. See text for details.


    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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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