Expression of Transforming Growth Factor beta  (TGFbeta ) Type III Receptor Restores Autocrine TGFbeta 1 Activity in Human Breast Cancer MCF-7 Cells*

(Received for publication, January 6, 1997, and in revised form, February 20, 1997)

Changguo Chen Dagger , Xiao-Fan Wang § and LuZhe Sun Dagger par

From the Dagger  Department of Pharmacology,  Member, Lucille P. Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536 and the § Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

While transforming growth factor beta  (TGFbeta ) type III receptor (RIII) is known to increase TGFbeta 1 binding to its type II receptor (RII), the significance of this phenomenon is not known. We used human breast cancer MCF-7 cells to study the role of RIII in regulating autocrine TGFbeta 1 activity because they express very little RIII and no detectable autocrine TGFbeta activity. A tetracycline-repressible RIII expression vector was stably transfected into this cell line. Expression of RIII increased TGFbeta 1 binding to TGFbeta type I receptor (RI) as well as RII. Treatment with tetracycline suppressed RIII expression and abolished TGFbeta 1 binding to RI and RII. Growth of RIII-transfected cells was reduced by 40% when plated at low density on plastic. This reduction was reversed by tetracycline treatment and was partially reversed by treatment with a TGFbeta 1 neutralizing antibody. The activity of a TGFbeta -responsive promoter construct when transiently transfected was more than 3-fold higher in the RIII-transfected cells than in the control cells. Treating the cells with tetracycline or the TGFbeta 1 neutralizing antibody also significantly attenuated the increased promoter activity. These results suggest that expression of RIII restored autocrine TGFbeta 1 activity in MCF-7 cells. The RIII-transfected cells were also much less clonogenic in soft agarose than the control cells indicating a reversion of progression. Thus, RIII may be essential for an optimal level of the autocrine TGFbeta activity in some cells, especially in the transformed cells with reduced RII expression.


INTRODUCTION

Transforming growth factor beta  (TGFbeta )1 isoforms are homodimer polypeptides of 25 kDa. They are multifunctional growth factors involved in the regulation of cell proliferation, differentiation, extracellular matrix formation, and immune response (1-3). Many studies have shown that TGFbeta can inhibit the growth of a variety cell types including epithelial, endothelial, lymphoid, and myeloid cells (4). Almost all types of cells express one or more of the three isoforms identified in mammals. Although in most systems, the majority of TGFbeta s secreted is in a latent form with no biological activity, a small percentage can be detected as mature, active TGFbeta s which may act to regulate cellular functions in an autocrine fashion. For example, neutralization of endogenous TGFbeta s with anti-TGFbeta antibodies was shown to stimulate proliferation of breast and colon cancer cells (5-7). Repression of TGFbeta expression by TGFbeta 1 antisense RNA in colon cancer cells was shown to increase clonogenicity in soft agarose and tumorigenicity in nude mice indicating that autocrine TGFbeta activity is tumor-suppressive (8, 9).

TGFbeta s elicit their effects by binding mainly to three cell surface proteins termed type I (RI), type II (RII), and type III (RIII) receptors. RI and RII are serine/threonine kinase receptors of 55 and 75 kDa, respectively, that form heteromeric complex, apparently at one to one stoichiometric ratio and necessary for TGFbeta signal transduction (3). It has been shown that RI requires RII for TGFbeta binding, whereas RII needs RI for signaling (10, 11). Recently, several studies showed that mutation or down-regulated expression of RI and/or RII is associated with loss of TGFbeta sensitivity and progression of human gastric, colorectal, and prostate cancers and T-cell lymphomas (12-17). Restoration of TGFbeta sensitivity by replacing the mutated or down-regulated receptor into colon and breast cancer cells reduced their malignancy (18-20). These results again indicate that autocrine TGFbeta s can act as negative growth regulators and disruption of the autocrine TGFbeta loop is probably a major event contributing to malignant progression.

TGFbeta RIII, also called beta -glycan, is a proteoglycan of 280-330 kDa. It is the most abundant TGFbeta binding molecule on the cell surface of a variety of cell types (21-23). Sequence analyses indicate that human RIII has a relatively small cytoplasmic domain of 41 amino acid residues that contains no consensus signaling motif (23, 24). Therefore, it is believed not to directly transduce TGFbeta signal. However, RIII binds all three TGFbeta isoforms with high affinity. The Kd ranges from 50 to 300 pM depending on cell types (21, 25, 26). Expression of RIII in the cells that lacks RIII significantly increased TGFbeta 1 binding to RII by 2.5-fold or more (24, 27). It was shown that in the absence of RIII, only a small population of RII in myoblasts could bind TGFbeta 1 with high affinity, while a larger population of RII had a much lower affinity for TGFbeta 1. However, expression of RIII converted all RII receptors into one population with high affinity for TGFbeta 1 (27). In the presence of ligands, RIII has been shown to form a heteromeric complex with RII suggesting that RIII enhances TGFbeta binding to RII by directly presenting the ligands to RII (27, 28).

While it is clear that RIII can enhance TGFbeta 1 binding to RII, the significance of this phenomenon remains to be elucidated. Since TGFbeta isoforms are produced mainly in a latent form and mature, activated TGFbeta levels are very low, if detectable, in most cell types, the majority of RII receptors with low affinity would probably not be occupied by the ligands in the absence of RIII. On the basis of what is known about the stoichiometric interaction between TGFbeta RI and RII, it appears that RIII should enhance autocrine TGFbeta 1 activity if a cell expresses a similar or lower level of RII than RI. Under this condition, autocrine TGFbeta activity is minimized without RIII because only a small percentage of RII is ligand-bound and can activate a small percentage of RI to transduce the signal. Thus, expression of RIII should increase the percentage of ligand-bound RII and consequently the percentage of activated RI. To test this hypothesis, we expressed RIII in human breast cancer MCF-7 cells which express a very low level of RII with no detectable autocrine TGFbeta activity. We show that expression of RIII inhibited both anchorage-dependent and anchorage-independent growth of MCF-7 cells and the inhibition could be reversed partially by a TGFbeta 1 neutralizing antibody, indicating that RIII-induced growth inhibition is at least partially due to restored autocrine negative activity of TGFbeta 1.


MATERIALS AND METHODS

Cell Culture

MCF-7 cells were originally obtained from the Michigan Cancer Foundation. The cell line was adapted to McCoy's 5A medium with 10% fetal bovine serum (FBS), pyruvate, vitamins, amino acids, and antibiotics (29). Working cultures were maintained at 37 °C in a humidified incubator with 5% CO2 and routinely checked for mycoplasma contamination. MCF-7 limiting dilution clones were obtained by plating parental cells into 96-well culture plates at 0.5 cell/well.

RIII Expression Vector Construction and Transfection

The full-length cDNA of rat TGFbeta RIII (24) was subcloned by blunt-ended ligation into a tetracycline-repressible expression system as described previously (20). The sense orientation of the RIII cDNA was confirmed by restriction digestion and agarose gel electrophoresis.

The expression vectors were linearized and transfected into one of MCF-7 limiting dilution clones with a BTX Electro Cell Manipulator at 250 V and 950 microfarads. The control cells were transfected with the empty vectors. The transfected cells were plated in 10-cm culture plates and maintained in the 10% FBS medium for 2 days. Selection of stable transfectants were accomplished by adding Geneticin (G418 sulfate; Life Technologies, Inc.) to the culture medium at 600 µg/ml. G418-resistant clones were ring-cloned and expanded for screening of RIII expression. Control clones were pooled and designated as Neo cell.

RNA Analysis

Total RNA from G418-resistant cells was isolated by guanidine thiocyanate homogenization and acidic phenol extraction (30). To measure the transfected rat RIII mRNA levels in the clones, we constructed a rat RIII riboprobe by inserting a 470-base pair BamHI fragment of the rat RIII cDNA into pBSK(-) plasmid (Stratagene Cloning Systems). The recombinant plasmid was then linearized with EcoRI and T3 RNA polymerase was used to synthesize a radioactive antisense RIII probe. RNase protection assays were performed using this radioactive antisense riboprobe to measure RIII mRNA levels in the transfected clones as described previously (20).

Receptor Cross-linking

Simian recombinant TGFbeta 1 was purified from conditioned media of transfected Chinese hamster ovary cells as described previously (31). Purified TGFbeta 1 was iodinated by the chloramine T method as described by Ruff and Rizzino (32). To measure the expression of cell surface TGFbeta receptors, [125I]TGFbeta 1 (200 pM) was incubated with a cell monolayer in 35-mm tissue culture wells and then cross-linked to its receptors as described by Segarini et al. (33). Labeled cell monolayers were solubilized in 200 µl of 1% Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride. Equal amounts of cell lysate protein were electrophoresed in 4-10% gradient SDS-polyacrylamide gel electrophoresis under reducing conditions and exposed for autoradiography.

Measurement of Secreted Mature TGFbeta 1

To measure the amount of active TGFbeta 1 in the media conditioned by control or RIII-transfected cells, monolayer cells in 6-well culture plates were cultured in the 10% FBS medium until confluence. The cells were washed twice with a serum-free McCoy's 5A medium and incubated in 1.5 ml of this serum-free medium for additional 72 h. The conditioned medium was then collected in siliconized microcentrifuge tubes and the cell number was counted with a hemocytometer. The amount of mature, activated TGFbeta 1 in the conditioned medium was determined with a TGFbeta 1 ELISA kit from Promega (Madison, WI) according to the manufacturer's instructions.

Plating Efficiency Assay

To study the effect of RIII expression on cell proliferation at low seeding densities, the control and RIII-transfected cells were plated at 200 or 400 cells per well in 24-well culture plates. After 9 days of incubation, relative cell number was determined with an MTT assay as described previously (20). To determine whether autocrine TGFbeta 1 activity was enhanced after RIII expression, a TGFbeta 1 neutralizing antibody (R&D Systems) was added to the cells after plating at a final concentration of 30 µg/ml.

Transient Transfection and Luciferase Assay

To determine whether RIII expression enhanced autocrine TGFbeta 1 activity, we measured a TGFbeta -responsive promoter activity using a plasmid called p3TP-Lux from Dr. J. Massague. The promoter activity is reported by luciferase (10). The p3TP-Lux (20 µg) and a beta -galactosidase expression plasmid (5 µg) were transiently co-transfected into the MCF-7 Neo control or RIII-expressing cells (107 cells) by electroporation in the same manner as the stable transfection described above. The cells were then equally divided into replicate culture dishes, part of which were treated with tetracycline (0.1 µg/ml) or the TGFbeta 1 neutralizing antibody (30 µg/ml) for 48 h. The treated and control cells were lysed in luciferase buffer (100 mM K2HPO4, pH 7.8, 1 mM dithiothreitol) using three cycles of freeze-thaw. The activities of luciferase and beta -galactosidase in the cell extract were assayed using published procedures (34, 35). Luciferase activity was normalized to beta -galactosidase activity and expressed as relative luciferase activity.

Soft Agarose Assay

To compare the clonogenic potential of the control and RIII-transfected cells in a semisolid medium, soft agarose assays were performed as described previously (20). Briefly, 6 × 103 cells were suspended in 1 ml of 0.4% low melting point agarose (Life Technologies) dissolved in the 10% FBS medium and plated on the top of a 1-ml underlayer of 0.8% agarose in the same medium in 6-well culture plates. For tetracycline treatment, we added tetracycline in soft agarose at 0.1 µg/ml as well as 200 µl of the medium containing 0.2 µg/ml tetracycline on the top of the solidified agarose. Control wells received 200 µl of the medium without tetracycline. The media were replenished every 3 days. After 3 weeks of incubation in the humidified incubator with 5% CO2 at 37 °C, the cell colonies were visualized by staining with 1 ml of p-iodonitrotetrazolium violet staining (Sigma). To quantitate the clonogenicity, the colonies in each well were counted under a magnifying lens.


RESULTS

Expression of RIII

MCF-7 cells express very low levels of RIII and undetectable RII on the cell surface using the receptor cross-linking method (20). However, they express low levels of RII mRNA and their growth can be slightly inhibited by high concentrations of exogenous TGFbeta 1 suggesting that they do have functional TGFbeta receptors and intracellular signaling components (20). In addition, this cell line expresses mainly TGFbeta 1 with no detectable TGFbeta 2 and TGFbeta 3 (36).2 Therefore, this cell line is ideal for the examination of whether TGFbeta RIII plays a role in regulating autocrine TGFbeta 1 activity and, consequently, cell growth.

Tetracycline-repressible RIII expression vectors were transfected into one of the MCF-7 limiting dilution clones. Using RNase protection assays and a rat RIII riboprobe, we identified two RIII-expressing clones. Both clones showed similar levels of RIII expression and growth properties on plastic and in soft agarose (data not shown). Therefore, we used one clone for the study. The mRNA level of the transfected RIII in this clone, designated as RIII cell, is shown in Fig. 1. Consistent with the mRNA level, RIII cells also expressed a higher level of cell surface RIII protein than the Neo cells by the receptor cross-linking assay (Fig. 2). The specificity of [125I]TGFbeta 1 binding to the receptor was confirmed by competition with a 100-fold excess of unlabeled TGFbeta 1 (Fig. 2, third lane). Treatment with tetracycline at 0.1 µg/ml for 4 days prior to the receptor cross-linking assay almost completely suppressed the expression of transfected RIII (Fig. 2, fourth lane). This reversible expression of RIII by tetracycline was particularly useful in the subsequent experiments for the demonstration of the specific effect of the RIII on the cell growth and autocrine TGFbeta activity.


Fig. 1. Expression of TGFbeta RIII mRNA in the MCF-7 transfectants. A typical MCF-7 limiting dilution clone was stably transfected with rat RIII expression plasmids and selected with Geneticin. The mRNA of the transfected RIII was detected in 20 µg of total RNA from an RIII-expressing clone by an RNase protection assay with a rat RIII riboprobe. The control cells were transfected with the plasmids without RIII cDNA and designated as Neo cells. Yeast tRNA was used as a negative control for the assay. Human actin mRNA levels were used for normalization.
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Fig. 2. Cell surface expression of TGFbeta receptors in the MCF-7 transfectants. Receptor cross-linking assays were used to examine the cell surface profiles of the TGFbeta receptors in the Neo and RIII cells. Confluent monolayer cultures of the Neo and RIII cells were incubated with 200 pM 125I-TGFbeta 1 alone (first and second lanes) or in the presence of 20 nM cold TGFbeta 1 (third lane) for 3 h at 4 °C. For the fourth lane, the RIII cells were cultured in the presence of 0.1 µg/ml tetracycline for 4 days prior to the receptor cross-linking assay. The receptor-bound 125I-TGFbeta 1 was cross-linked with disuccinimidyl suberate. Cell lysates containing equal amounts of protein were electrophoresed in 4-10% gradient SDS-polyacrylamide gel electrophoresis under reducing conditions. The 125I-TGFbeta 1-linked receptors were visualized after autoradiography.
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Consistent with a previous observation (20), TGFbeta RI and RII were not detectable by the receptor cross-linking assay on the Neo cell surface (Fig. 2). However, two bands corresponding to RI and RII were detected in the RIII cell samples. Since they were absent in the Neo cells and in the tetracycline-treated RIII cells (Fig. 2), the increased TGFbeta 1 binding to RI and RII appears to be specifically due to RIII expression. This is consistent with what was observed in myoblasts (24, 27).

Expression of Endogenous TGFbeta 1

While RIII cells showed increased TGFbeta 1 binding to RI and RII, their sensitivity to exogenous TGFbeta 1 remained relatively similar to that of the Neo cells in a growth inhibition assay (20) (data not shown). Therefore, we hypothesized that MCF-7 cells may express enough activated TGFbeta 1 such that the expression of RIII regenerated a maximal TGFbeta growth inhibition in an autocrine manner. To test this hypothesis, we first examined whether the cells produced mature, activated TGFbeta 1 in our system. Using a TGFbeta 1 ELISA kit we were able to detect a modest amount of activated TGFbeta 1 (200 pg/106 cells/72 h) in the medium conditioned by the Neo cells. The amount of activated TGFbeta 1 in the medium conditioned by the RIII cells was reduced by more than half (Fig. 3). This reduction could be reversed by treating the RIII cells with tetracycline, suggesting that the reduced amount of free activated TGFbeta 1 in the medium was due to its binding to RIII. This result led us to further examine whether the cell growth was inhibited by the autocrine TGFbeta 1 after RIII expression.


Fig. 3. Activated TGFbeta 1 in the conditioned media of the MCF-7 transfectants. The Neo and RIII cells in 6-well plates were cultured in the 10% FBS medium with or without 0.1 µg/ml tetracycline (Tet.) till confluence. The medium was then changed to a serum-free McCoy's 5A medium with or without tetracycline and incubated for 72 h. The conditioned media were collected from duplicate wells. Activated TGFbeta 1 in the media was determined as described under "Materials and Methods." The amount of activated TGFbeta 1 is normalized by the cell number and expressed as the mean ± S.E. of four measurements except the mean of RIII+Tet. which was the average of two measurements with identical values.
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Plating Efficiency Assay

It is well known that TGFbeta isoforms suppress cell cycle progression of newly plated, non-dividing cells more effectively than that of exponentially growing cells. Plating cells at low density is one way to ensure that the cells are maintained for a period of non- or slow-dividing state before entering exponential growth. This approach has been successfully employed previously to demonstrate the autocrine inhibitory activity of TGFbeta (18, 19). When Neo and RIII cells were plated in 24-well tissue culture plates at 200 and 400 cells/well, the RIII cells proliferated significantly slower than the Neo cells by about 40% after nine days of incubation (Fig. 4A). The growth reduction was due to reduced number of colonies as well as number of cells per colony. Treatment of the cells with tetracycline at 0.1 µg/ml prior to and during the plating efficiency assay completely reversed the growth property of the RIII cells back to that of the Neo cells as shown in Fig. 4B suggesting that the growth inhibition was specifically due to RIII expression. To assess whether the growth reduction was due to RIII-enhanced autocrine TGFbeta 1 activity, we treated the cells with a TGFbeta 1 neutralizing antibody or a control antibody after plating. While the growth of Neo cells was not affected by the antibody, the growth of the RIII cells was stimulated (Fig. 5). Even though the stimulation was only 15%, it is statistically significant (p < 0.05) by Student's t test. Since RIII expression reduced the growth by 40%, 15% growth stimulation represents a 37.5% recovery of the growth. The reason that tetracycline was more effective than the TGFbeta 1 neutralizing antibody to stimulate the growth of the RIII cells was probably due to the fact that tetracycline could completely suppress RIII expression whereas the antibody had to compete with the RIII for endogenous TGFbeta 1.


Fig. 4. Plating efficiency of the MCF-7 transfectants. A, the Neo and RIII cells were plated in a 24-well plate at 200 or 400 cells/well in the 10% FBS medium and incubated for 9 days. The relative cell numbers were determined with the MTT assay and expressed as optical density values at 590 nm. B, the Neo and RIII stock cells were first cultured in the absence or presence of 0.1 µg/ml tetracycline (Tet.) for 1 week and then plated in a 24-well plate at 400 cells/well in the 10% FBS medium with or without 0.1 µg/ml tetracycline. The media were changed every other day to replenish the tetracycline. The MTT assay was performed after 6 days of incubation and the OD values of each treatment were expressed as percent of the optical density value of the Neo cells. The values in both panels A and B are presented as means ± S.E. of six OD measurements from duplicate wells.
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Fig. 5. Effect of a TGFbeta 1 neutralizing antibody on the plating efficiency of the MCF-7 transfectants. The Neo and RIII cells were plated in 24-well plates at 400 cells/well in the presence of 30 µg/ml normal IgG (IgG) or 30 µg/ml TGFbeta 1 neutralizing antibody (Ab). The MTT assay was performed after 9 days of incubation. The OD values of TGFbeta 1 antibody treatment are presented as percent of the respective IgG-treated controls. The values are means ± S.E. determined in 6 samples from duplicate wells.
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Activity of a TGFbeta -responsive Promoter

To confirm our observation that RIII expression enhanced autocrine TGFbeta 1 activity, we measured the activity of a TGFbeta -responsive promoter/luciferase construct after being transiently transfected into the Neo and RIII cells. This construct has been widely used to measure the efficacy of TGFbeta in activating its signaling receptors and stimulating gene expression (10). While RIII expression inhibited the growth of the MCF-7 cells, it stimulated TGFbeta -responsive promoter activity as indicated by luciferase activity by more than 3-fold (Fig. 6A). Treatment of the transiently transfected cells with tetracycline for 48 h reduced the luciferase activity almost to the level of the Neo cells, again demonstrating the specificity of the RIII effect. Similar to the growth assay, the stimulated promoter activity could be partially, but significantly (p < 0.05) reduced by treating the transfected RIII cells with the TGFbeta 1 neutralizing antibody for 48 h. Thus, with two different approaches, we have showed that RIII can restore autocrine TGFbeta 1 activity in the MCF-7 cells.


Fig. 6. The activity of a TGFbeta -responsive promoter in the MCF-7 transfectants. The Neo and RIII cells were transiently co-transfected with p3TP-Lux and a beta -galactosidase expression plasmid by electroporation. Then, the transfected cells were equally divided into two (panel A) or six (panel B) 35-mm culture wells. In panel A, one of the two wells was treated with 0.1 µg/ml tetracycline (Tet.) during a 48-h incubation. In panel B, three of the six wells were treated with 30 µg/ml normal IgG (IgG), whereas the other three wells were treated with 30 µg/ml TGFbeta 1 neutralizing antibody (Ab) during a 48-h incubation. The cells were harvested 48 h after transfection. The activities of the luciferase and beta -galactosidase in the cell lysate were separately measured. The data are presented as relative luciferase activity after normalized to the beta -galactosidase activity. The experiment presented in panel A was repeated with similar results. The values in panel B are presented as means ± S.E. of three replicate wells.
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Clonogenicity in Soft Agarose

The growth inhibitory activity of autocrine TGFbeta is known to maintain cancer cells at a less malignant state as previously shown by its suppressive effect on the clonogenicity in soft agarose and the tumorigenicity in nude mice (8, 18, 20). Since RIII expression restored autocrine TGFbeta 1 activity, we compared the ability of Neo and RIII cells to form colonies in a soft agarose assay. As shown in Fig. 7A, the number of colonies formed by RIII cells were considerably less than that formed by Neo cells. Tetracycline had no effect on Neo cells, but significantly increased the number of colonies formed by RIII cells (Fig. 7B). These results suggest that by restoring autocrine TGFbeta 1 activity, RIII can also reduce the anchorage-independent growth of the MCF-7 cells in the same manner as RII (20).


Fig. 7. Anchorage-independent colony formation in soft agarose of the MCF-7 transfectants. Exponentially growing Neo and RIII cells (6,000 cells) were resuspended in 1 ml of 0.4% low melting point agarose in the 10% FBS medium and plated on top of a 1-ml underlayer of 0.8% agarose in a 6-well culture plate. Tetracycline treatment was carried out as described under "Materials and Methods." After 3 weeks of incubation, cell colonies were visualized by staining with 1 ml of p-iodonitrotetrazolium violet (panel A). The number of colonies were counted in three wells for each treatment and presented as means ± S.E. (panel B).
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DISCUSSION

Although RIII has been shown to enhance TGFbeta 1 binding to RII, this is the first report demonstrating an important role of RIII in regulating autocrine TGFbeta 1 activity. Expression of RIII in MCF-7 cells increased affinity labeling of both RI and RII by [125I]TGFbeta 1 to a detectable level. Increased TGFbeta 1 binding to RII after RIII expression is consistent with what was observed in myoblasts (24, 27). However, RIII expression in myoblasts had no effect on TGFbeta 1 binding to RI. The authors suspected that the amount of RII with high affinity for TGFbeta 1 in the myoblasts without RIII was sufficiently high enough to support TGFbeta 1 binding to all cell surface RI molecules (27). In contrast, MCF-7 cells expressed a very low level of RII mRNA and no detectable amount of cell surface RII protein with the affinity labeling (20). As a result, even though they express a relatively high level of RI mRNA (20), little RI could be affinity-labeled with TGFbeta 1 since RI requires TGFbeta -bound RII for ligand binding. RIII expression in MCF-7 cells had no effect on RII mRNA expression,2 suggesting that the increased TGFbeta 1 binding to RII in the RIII cells was due to increased binding affinity as observed in the myoblasts (27). It appears that the increased TGFbeta 1 binding to RI resulted from the increased TGFbeta 1 binding to RII. Therefore, whether RIII is necessary for maintaining an optimal autocrine TGFbeta 1 activity in a given cell type appears to be dependent on the ratio of RII to RI.

In the myoblasts that lack RIII expression, expression of RIII was shown to convert RII molecules into a single population with high affinity (27). Due to its low expression level, we do not know whether the two populations of RII also exist in MCF-7 cells. The fact that their growth can only be inhibited by high concentrations of exogenous TGFbeta 1 (20) suggests that they contain the low affinity RII. Since the TGFbeta 1 neutralizing antibody had no effect on the growth of the Neo cells, it appears that they contain no or little high affinity RII which does not confer a significant amount of autocrine TGFbeta 1 activity.

TGFbeta RI, RII, and RIII are widely expressed in many types of normal and transformed cells (37). Whether RIII plays a role in regulating autocrine TGFbeta activity in certain types of normal cells remains to be elucidated. However, our data would suggest that if a normal cell expresses less RII than RI, RIII could be essential to confer TGFbeta sensitivity. This could also be the case during development of a cell or an organ when the ratio of RII to RI may vary at different stages. In transformed cells including breast, colon, stomach, prostate cancer cells, retinoblastoma cells, and lymphoma cells, RII is often down-regulated (12-14, 17, 38-42). While in some cancers such as colon and gastric cancers, the down-regulation of RII can be due to gene mutation (12, 14, 15), in other cases, it may be due to reduced gene expression as in the MCF-7 cells. If the latter is true for a given type of transformed cells, RIII level will be critical to the maintenance of the autocrine TGFbeta activity and consequently a less malignant phenotype of the cells. The fact that the expression of RIII shown in this report can be as effective as the expression of a moderate level of RII (20) in suppressing the anchorage-independent growth of the MCF-7 cells demonstrates this important role of RIII in keeping the autocrine TGFbeta 1 activity at an optimal level. Since TGFbeta 3 behaves similarly to TGFbeta 1 in receptor binding and TGFbeta 2 requires RIII for binding to the signaling receptors (27), it is conceivable that RIII can also be critical to the maintenance of the autocrine activities of TGFbeta 2 and TGFbeta 3 in the cells that express these two isoforms.

While exogenous TGFbeta can inhibit the growth of many types of transformed cells (4), studies have shown that in some systems, autocrine TGFbeta can render cells insensitive to the growth inhibitory activity of exogenous TGFbeta 1 (18). This is likely due to the fact that the cells not only produce high levels of one or more TGFbeta isoforms, but also can activate them. In TGFbeta 1-transfected fibroblasts and fibrosarcoma cells, it was found that a significant amount of cell-activated TGFbeta 1 was bound to the cell surface even though very little activated TGFbeta 1 could be detected in the conditioned medium (43). The MCF-7 cells we used produced a readily detectable amount of mature, activated TGFbeta 1 in the conditioned medium as shown in Fig. 3. In addition, the cells express a low level of RII which should be easily saturable by the ligand. Therefore, we hypothesized that although RIII-enhanced TGFbeta 1 binding to the signaling receptor RII, the reason that the sensitivity of the MCF-7 cells to the growth inhibitory activity of the exogenous TGFbeta 1 was not increased after RIII expression was due to the fact that the autocrine TGFbeta 1 had generated a maximal level of growth inhibition by saturating RII and/or the intracellular signaling pathway. Since RIII is known to form a heteromeric complex with RII in the presence of activated TGFbeta (27, 28), its main role is probably to make the RII more accessible by the ligand. In addition, by capturing and retaining ligand, RIII may protect the ligand from cellular degradation and/or inactivation, rendering more ligand available for binding to the signaling receptors. The RIII-enhanced autocrine TGFbeta 1 activity was demonstrated in the MCF-7 cells by the fact that a TGFbeta 1 neutralizing antibody could partially reverse the RIII-induced growth inhibition and gene expression.

Because of its short cytoplasmic domain with no consensus signaling motif and its absence in some of the TGFbeta -sensitive cells, RIII has been regarded as a non-signaling, accessory TGFbeta receptor. As a result, the RIII-generated growth inhibition we observed in MCF-7 cells was solely attributed to the increased autocrine TGFbeta 1 binding to RII and, consequently the activation of RI·RII receptor complex even though TGFbeta 1 neutralizing antibody only abrogated about one-third of the RIII-induced growth inhibition as well as gene expression. However, our study cannot rule out the possibility that RIII may transduce signal by itself or in association with RII. We are currently attempting to address this possibility.

In summary, our study showed that the expression of RIII can lead to the inhibition of both anchorage-dependent and anchorage-independent growth in MCF-7 cells. This is at least in part mediated by RIII-restored autocrine inhibitory activity of the endogenous TGFbeta 1. RIII may be essential for an optimal level of autocrine TGFbeta activity in various types of normal cells. Its expression is likely to be critical for the maintenance of the growth inhibitory activity of autocrine TGFbeta isoforms and, consequently of a less malignant phenotype in other adenocarcinoma cells with reduced RII expression.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant CA63480 and the University of Kentucky Research Fund (to L.-Z. S.) and United States Army Grant DAMD17-94-J-4065 (to X.-F. W.).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.
par    To whom correspondence should be addressed. Tel.: 606-257-1404; Fax: 606-323-1981.
1   The abbreviations used are: TGFbeta , transforming growth factor beta ; RI, RII, and RIII, receptor types I, II, and III, respectively; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
2   C. Chen and L.-Z. Sun, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. H. Bujard at the University of Heidelberg, Germany, for the tetracycline-repressible expression plasmids and Dr. J. Massague at Memorial Sloan-Kettering Cancer Center, New York, for the p3TP-Lux plasmid. We also thank Dr. David Kaetzel at the University of Kentucky College of Medicine for critical reading of this manuscript.


REFERENCES

  1. Moses, H. L., Yang, E. Y., and Pietenpol, J. A. (1990) Cell 63, 245-247 [Medline] [Order article via Infotrieve]
  2. Roberts, A. B., and Sporn, M. B. (1991) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds), pp. 419-472, Springer-Verlag, Heidelberg
  3. Attisano, L., Wrana, J. L., Lopez-Casillas, F., and Massague, J. (1994) Biochim. Biophys. Acta 1222, 71-80 [Medline] [Order article via Infotrieve]
  4. Lyons, R. M., and Moses, H. L. (1990) Eur. J. Biochem. 187, 467-473 [Abstract]
  5. Arteaga, C. L., Coffey, R. J., Jr., Dugger, T. C., McCutchen, C. M., Moses, H. L., and Lyons, R. M. (1990) Cell Growth Differ. 1, 367-374 [Abstract]
  6. Hafez, M. M., Infante, D., Winawer, S., and Friedman, E. (1990) Cell Growth Differ. 1, 617-626 [Abstract]
  7. Sun, L., Wu, S., Coleman, K., Fields, K. C., Humphrey, L. E., and Brattain, M. G. (1994) Exp. Cell Res. 214, 215-224 [CrossRef][Medline] [Order article via Infotrieve]
  8. Wu, S. P., Theodorescu, D., Kerbel, R. S., Willson, J. K., Mulder, K. M., Humphrey, L. E., and Brattain, M. G. (1992) J. Cell Biol. 116, 187-196 [Abstract]
  9. Wu, S. P., Sun, L. Z., Willson, J. K., Humphrey, L., Kerbel, R., and Brattain, M. G. (1993) Cell Growth Differ. 4, 115-123 [Abstract]
  10. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X. F., and Massague, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  11. Vivien, D., Attisano, L., Wrana, J. L., and Massague, J. (1995) J. Biol. Chem. 270, 7134-7141 [Abstract/Free Full Text]
  12. Park, K., Kim, S. J., Bang, Y. J., Park, J. G., Kim, N. K., Roberts, A. B., and Sporn, M. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8772-8776 [Abstract]
  13. Kadin, M. E., Cavaille-Coll, M. W., Gertz, R., Massague, J., Cheifetz, S., and George, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6002-6006 [Abstract]
  14. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., Vogelstein, B., Brattain, M., and Willson, J. K. V. (1995) Science 268, 1336-1338 [Medline] [Order article via Infotrieve]
  15. Myeroff, L. L., Parsons, R., Kim, S. J., Hedrick, L., Cho, K. R., Orth, K., Mathis, M., Kinzler, K. W., Lutterbaugh, J., Park, K., Bang, Y. J., Lee, H. Y., Park, J. G., Lynch, H. T., Roberts, A. B., Vogelstein, B., and Markowitz, S. D. (1995) Cancer Res. 55, 5545-5547 [Abstract]
  16. Kim, I. Y., Ahn, H. J., Zelner, D. J., Shaw, J. W., Sensibar, J. A., Kim, J. H., Kato, M., and Lee, C. (1996) Cancer Res. 56, 44-48 [Abstract]
  17. Kim, I. Y., Ahn, H. J., Zelner, D. J., Shaw, J. W., Lang, S., Kato, M., Oefelein, M. G., Miyazono, K., Nemeth, J. A., Kozlowski, J. M., and Lee, C. (1996) Clin. Cancer Res. 2, 1255-1261 [Abstract]
  18. Wang, J., Sun, L., Myeroff, L., Wang, X., Gentry, L. E., Yang, J., Liang, J., Zborowska, E., Markowitz, S., Willson, J. K. V., and Brattain, M. G. (1995) J. Biol. Chem. 270, 22044-22049 [Abstract/Free Full Text]
  19. Wang, J., Han, W., Zborowska, E., Liang, J., Wang, X., Willson, J. K. V., Sun, L., and Brattain, M. G. (1996) J. Biol. Chem. 271, 17366-17371 [Abstract/Free Full Text]
  20. Sun, L., Wu, G., Willson, J. K. V., Zborowska, E., Yang, J., Rajkarunanayake, I., Wang, J., Gentry, L. E., Wang, X.-F., and Brattain, M. G. (1994) J. Biol. Chem. 269, 26449-26455 [Abstract/Free Full Text]
  21. Cheifetz, S., Bassols, A., Stanley, K., Ohta, M., Greenberger, J., and Massague, J. (1988) J. Biol. Chem. 263, 10783-10789 [Abstract/Free Full Text]
  22. Segarini, P. R., Rosen, D. M., and Seyedin, S. M. (1989) Mol. Endocrinol. 3, 261-272 [Abstract]
  23. Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massague, J. (1991) Cell 67, 785-795 [Medline] [Order article via Infotrieve]
  24. Wang, X. F., Lin, H. Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and Weinberg, R. A. (1991) Cell 67, 797-805 [Medline] [Order article via Infotrieve]
  25. Cheifetz, S., Hernandez, H., Laiho, M., ten Dijke, P., Iwata, K. K., and Massague, J. (1990) J. Biol. Chem. 265, 20533-20538 [Abstract/Free Full Text]
  26. Andres, J. L., Ronnstrand, L., Cheifetz, S., and Massague, J. (1991) J. Biol. Chem. 266, 23282-23287 [Abstract/Free Full Text]
  27. Lopez-Casillas, F., Wrana, J. L., and Massague, J. (1993) Cell 73, 1435-1444 [Medline] [Order article via Infotrieve]
  28. Moustakas, A., Lin, H. Y., Henis, Y. I., Plamondon, J., O'Connor-McCourt, M. D., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22215-22218 [Abstract/Free Full Text]
  29. Mulder, K. M., and Brattain, M. G. (1989) Mol. Endocrinol. 3, 1215-1222 [Abstract]
  30. Xie, W. Q., and Rothblum, L. I. (1991) Biotechniques 11, 324 [Medline] [Order article via Infotrieve] , 326-327
  31. Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell Biol. 8, 4162-4168 [Medline] [Order article via Infotrieve]
  32. Ruff, E., and Rizzino, A. (1986) Biochem. Biophys. Res. Commun. 138, 714-719 [Medline] [Order article via Infotrieve]
  33. Segarini, P. R., Roberts, A. B., Rosen, D. M., and Seyedin, S. M. (1987) J. Biol. Chem. 262, 14655-14662 [Abstract/Free Full Text]
  34. Schmidt, J. V., Carver, L. A., and Bradfield, C. A. (1993) J. Biol. Chem. 268, 22203-22209 [Abstract/Free Full Text]
  35. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  36. Arrick, B. A., Korc, M., and Derynck, R. (1990) Cancer Res. 50, 299-303 [Abstract]
  37. Massague, J. (1990) Annu. Rev. Cell Biol. 6, 597-641 [CrossRef]
  38. Kimchi, A., Wang, X. F., Weinberg, R. A., Cheifetz, S., and Massague, J. (1988) Science 240, 196-199 [Medline] [Order article via Infotrieve]
  39. Arteaga, C. L., Tandon, A. K., Von Hoff, D. D., and Osborne, C. K. (1988) Cancer Res. 48, 3898-3904 [Abstract]
  40. Kalkhoven, E., Roelen, B. A., de Winter, J. P., Mummery, C. L., van den Eijnden-van Raaij, A. J., van der Saag, P. T., and van der Burg, B. (1995) Cell Growth Differ. 6, 1151-1161 [Abstract]
  41. Hoosein, N. M., McKnight, M. K., Levine, A. E., Mulder, K. M., Childress, K. E., Brattain, D. E., and Brattain, M. G. (1989) Exp. Cell Res. 181, 442-453 [CrossRef][Medline] [Order article via Infotrieve]
  42. Filmus, J., Zhao, J., and Buick, R. N. (1992) Oncogene 7, 521-526 [Medline] [Order article via Infotrieve]
  43. Beauchamp, R. D., Sheng, H. M., Bascom, C. C., Miller, D. A., Lyons, R. M., Torre-Amione, G., and Moses, H. L. (1992) Endocrinology 130, 2476-2486 [Abstract]

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