©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Transforming Growth Factor Type III Receptor in Vascular Endothelial Cells Increases Their Responsiveness to Transforming Growth Factor 2 (*)

Sabita Sankar , Negar Mahooti-Brooks , Michael Centrella (1), Thomas L. McCarthy (1), Joseph A. Madri (§)

From the (1) Departments of Pathology and Plastic Surgery, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bovine aortic endothelial cells (BAECs) express both type I and type II receptors for transforming growth factor (TGF). These cells respond to TGF1 but are relatively refractory to another isoform of TGF, termed TGF2. TGFs are thought to signal through receptor complexes composed of type I and/or type II receptors, both of which appear to be functional serine-threonine kinases. The TGF type III receptor, on the other hand, does not seem to have any direct signaling capacity. We have now stably transfected BAECs with the type III receptor cDNA. These cells displayed surface expression of the type III receptor protein, as determined by cross-linking with iodinated TGF1 and immunoprecipitation with antibodies to the type III receptor protein. Transfected BAECs exhibit increased responsiveness to TGF2 by several different criteria including an increase in plasminogen activator inhibitor-1 protein and inhibition of migration and proliferation. Thus, the type III receptor protein may play a role in presenting TGF2 to the type II receptor and increase responsiveness to TGF2 to a level comparable to that of TGF1.


INTRODUCTION

Transforming growth factor s (TGFs)() are multifunctional proteins that regulate cell proliferation, differentiation, and biosynthesis of the extracellular matrix (1, 2, 3) . The five known mammalian TGF isoforms (TGF1, 2, 3, 1.2, and 1.3) are homo- or heterodimers linked by a single disulfide bond. When studied under in vitro cell culture conditions, they have overlapping functions; however it remains unclear whether the five isoforms exert distinct physiological effects during differentiation (4) . Most types of cultured cells possess three distinct high affinity cell surface receptors for TGF as judged by chemical cross-linking to I-TGF1; types I (55 kDa), II (80 kDa), and III (280 kDa) (3, 5, 6) . Cloning of the cDNA encoding both type I and type II receptors revealed a transmembrane serine-threonine kinase (7, 8, 9, 10, 11) . In many studies using chemically mutagenized mink lung epithelial cells (12-15) and tumor cell lines that are resistant to the growth inhibitory effects of TGF, the results most consistently correlated with the loss of TGF type I receptor or the loss of both the type I and type II receptors. These observations support the notion that TGF-mediated signaling might involve both the TGF type I and type II receptors (15, 16, 17, 18) . Expression of the recombinant human TGF type II receptor in certain mutant-resistant cell lines restores sensitivity to TGF and results in the appearance of cell surface type II receptor as well as the type I receptor (8, 19) . Recently, Wrana and co-workers (20) provided evidence in these cell models that type II receptor is required to activate the type I receptor which subsequently sends the downstream signals, although these results may not be universal (21, 22, 23) .

The function of the type III receptor is unclear. The TGF type III receptor is the most abundant TGF receptor subtype in most cells (16, 24). Cloning of the cDNA encoding the type III receptor, a transmembrane proteoglycan, revealed a short highly conserved cytoplasmic domain with no apparent signaling motif (25, 26, 27) . Consequently, the type III receptor and its soluble secreted ectodomain may act as reservoir molecules for ligand retention or presentation of TGF to the signaling type I and type II receptors. Consistent with this, stable transfection of recombinant rat type III receptor in myoblasts resulted in increase in binding of TGF1 to the type II receptor (26) .

Although most of the biological activities are shared among TGF1, TGF2, and TGF3, there are some differences. TGF1 and 3 elicit their effects at about 100-fold lower protein concentration compared to TGF2 in certain cell types that lack type III receptor. Indeed vascular endothelial cells and hematopoietic progenitor cell lines that lack type III receptor are relatively resistant to TGF2 (17, 24, 28-32). Expression of type III receptor in LE myoblasts that endogenously lack the protein results in enhanced binding of TGF2 to the type II receptor and suggests interactions between the types II- and III-binding sites (33, 34). Based on the structural features of the type III receptor and the observation that cells that lack this receptor still respond to TGF, the role of type III receptor is most likely in modulating the TGF isoform response (16, 28) .

Our laboratory has spent several years characterizing the TGF responses of vascular endothelial cells, comparing the effects of TGF1, TGF2, and TGF3 on bovine aortic endothelial cells (BAECs). TGF1 and TGF3 profoundly inhibit BAEC proliferation and migration, while TGF2 has little or no effect. To test the hypothesis that the type III receptor regulates the binding of TGF2 to the signaling receptors and increases the TGF2 responsiveness in BAECs, we generated stable transfectants of BAECs expressing the type III receptor cDNA in both the sense and antisense orientations. We now present changes in BAEC responsiveness to TGF2 that are comparable to TGF1 effects when type III receptors are present.


EXPERIMENTAL PROCEDURES

Materials

Human TGF1 and porcine TGF2 were purchased from R & D Systems Inc., Minneapolis. Iodinated TGF1 was obtained from Biomedical Technologies Inc., Stoughton, MA. Bovine type I collagen was isolated and purified as described (35, 36, 37) . Tissue culture plates were coated with type I collagen at a concentration of 12.5 µg/ml.

Plasmids

The vector used for transfection of the type III receptor into bovine aortic endothelial cells was obtained from Wang and co-workers (26) . Basically the HindIII fragment of the rat type III receptor cDNA (R3-OFF) was subcloned into pcDNA I NEO (Invitrogen, San Diego, CA) which is under the control of the cytomegalovirus transcriptional promoter and the SV40 origin of replication. In addition, two other constructs were generated by subcloning a 3.9-kilobase EcoRI fragment from the rat type III receptor cDNA (a gift from J. Massague, Memorial Sloan Kettering Cancer Center, NY) into the pSV7d expression vector (38) which is under the control of the Rous sarcoma virus transcriptional promoter. The orientation of the EcoRI fragment was checked by restriction digests with AflIII and SalI. Thus the rat type III receptor cDNA was subcloned into pSV7d in both the sense and antisense orientations. A control vector pcDNA3 (Invitrogen, San Diego, CA) containing the neomycin resistance marker was cotransfected into the cells along with the type III receptor cDNA subcloned into the pSV7d.

Cell Culture and Transfection

Bovine aortic endothelial cells were isolated and cultured according to Madri and Furthmayr (36) . For the transfection experiments, cells were plated on 60-mm dishes at subconfluence. The cells were transfected with 10 µg of plasmid DNA and 25 µg of lipofectAMINE (Life Technologies, Inc.) per plate and incubated with serum-free media (Life Technologies, Inc.) for several hours before growth media was added. Selection for transfectants with 400 µg/ml G418 (Geneticin, Life Technologies, Inc.) was carried out 48 h after the transfection. Neomycin -resistant colonies were selected using cloning rings and expanded before analysis for surface expression of the receptors.

Receptor Binding Autoradiography Assay

The assay was a modification of our earlier studies (32, 39) . Briefly, confluent monolayers grown on 35 mm, 1.5% gelatin-coated tissue culture plates were washed with cold binding buffer (Dulbecco's modified essential media, 25 mM HEPES, pH 7.4, 0.1% bovine serum albumin) and then allowed to equilibrate with binding buffer for 30 min at 4 °C on a rotating platform. The buffer was aspirated, and 250 µl of ice-cold binding buffer containing 100 pMI-labeled TGF1 (220 µCi/µg) was added to each 35-mm dish and incubated on a rotary platform at 4 °C for 3 h. After washing at 4 °C, ice-cold binding buffer lacking bovine serum albumin was added to the plates. 5 µl of 27 mM disuccinimidyl suberate (Pierce) was added to each plate. The plates were swirled immediately after addition of the disuccinimidyl suberate. The dishes were allowed to shake on the rotary platform for 15 min. The medium was removed and washed several times with binding buffer before lysis with Laemmli loading buffer containing 30 µl of 1 mM dithiothreitol. The cell lysate was boiled and electrophoresed according to Laemmli (40) . Linear gradient resolving gels of 5-10% polyacrylamide were constructed with a 3.5% stacking gel. The gels were fixed, dried, and exposed to Amersham Hyperfilm -MP at -80 °C. The films were developed using a Kodak X-OMAT M20 processor (Kodak).

Surface Biotinylation

Cells plated on 100-mm dishes were washed with PBS containing 0.2 mM calcium chloride and 2 mM magnesium chloride and incubated for 30 min at 4 °C with 1 mg of NHS-Long Chain Biotin (Pierce) in 2 ml of PBS containing calcium chloride and magnesium chloride. The cells were washed with PBS and lysed with 0.05% Triton X-100, 120 mM Tris-HCl, pH 8.7. The cell lysates were spun at 14,000 revolutions/min in an Eppendorf centrifuge for 10 min at 4 °C, and the supernatant was used for immunoprecipitation.

Immunoprecipitation

Cell lysates obtained from I-TGF1 cross-linking experiments and surface biotinylation experiments were incubated with primary antibodies (anti-type III receptor and anti-type II receptor; gifts from F. Lopez-Casillas, Memorial Sloan-Kettering Cancer Center, New York and H. Y. Lin, Whitehead Institute for Biomedical Research, Cambridge, MA, respectively) overnight on a rotary shaker. 50 µl of protein-A Sepharose CL 4B (Pharmacia) was added to the cell lysate and further incubated for 2 h before spinning the beads down and washing the Sepharose beads with Tris-buffered saline containing 0.5% Triton X-100 (TBS-T) several times. The beads were resuspended in sample loading buffer and boiled for several minutes before loading onto the gel.

Immunoblot Analysis

Cell protein was extracted with 0.05% Triton X-100 and 120 mM Tris-HCl, pH 8.7, normalized for total protein using the bicinchonic acid assay (Pierce), and electrophoresed through a 10% non-reducing polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell), blocked with 8% nonfat milk in PBS, and incubated with mouse anti-bovine PAI-1 IgG (American Diagnostica Inc.) at 1:750 dilution and then goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Promega, Madison, WI) at 1:10 000 dilution. The blot was developed using the Enhanced Chemiluminescence method (ECL, Amersham Corp.) with Hyperfilm-MP. Bands were quantitated using a Molecular Dynamics densitometer (Molecular Dynamics Scanner, Sunnyvale, CA).

Reverse Zymography

Cell extracts were normalized for cell protein using the bicinchonic acid assay and then electropheresed in a 10% acrylamide gel. The gel was then washed in 2.5% Triton X-100 for 1 h followed by two 20-min washes in water. The gel was then overlaid on a thin agar gel with final concentrations of 4% nonfat milk, 0.1 M Tris-,HCl pH 8.0, 8 µg/ml plasminogen, 1.25% agar. The gel and overlay were incubated at 37 °C until the generated plasmin had diffused throughout the entire gel, and all the casein was degraded except where PAI-1 was located. The gels were then examined and photographed with darkfield illumination.

Proliferation Assays

Collagen I-coated bacteriological cultures dishes were washed with PBS before the addition of cell suspension (1 10 cells/dish). The cells were allowed to attach to the coated dishes for several hours. At this point, fresh medium with or without TGF was added to the cultures. The medium and factors were replaced once again on the third day. Cell numbers were determined by lifting the cells off the culture dishes with trypsin/EDTA and counting quadruplicate samples using a Coulter Counter (Coulter Electronics Inc, Hialech, FL). The mean number of cells/dish was then calculated.

Migration Assay

Cell migration was evaluated as described previously (41, 42) . Briefly, after cells plated within fences located in the center of the cultures achieved confluence (about 6 h), migration was induced by the removal of the fences and the cells migrated outward in a radial fashion. After 6 days, the cultures were washed with PBS and fixed with 10% neutral buffered formalin. Cultures were stained with Ham's hematoxylin. Net increase in surface area covered was assessed using the NIH Image Program. Maximum response was obtained from untreated control BAECs, and this was defined as 100% relative migration.


RESULTS

Expression of TGF Type III Receptor in BAECs

In order to screen for potential clones expressing the TGF type III receptor, we incubated the BAEC transfectants with radioiodinated TGF1, added chemical cross-linker, and resolved the labeled receptor by polyacrylamide gel electrophoresis as in previous studies (32) . Fig. 1A shows two predominant TGF-binding proteins on the surface of the control BAECs (lane 1). BAECs transfected with pcDNA 3 containing the neomycin resistance marker (lane 3) exhibited a similar binding profile. By relative migration and comparison with other culture models, these bands correspond to TGF bound to the type I receptor (65 kDa) and the type II receptor (85 kDa) complexes. Introduction of the TGF type III receptor cDNA in the forward orientation led to the expression of type III receptor protein in the BAECs (lane 5). The TGF type III receptor migrated as a diffuse band of 280-330 kDa. The identity of the molecules within the diffuse band in the BAECIIIR cells was confirmed by immunoprecipitation with antibodies to the TGF type III receptor protein (lane 6). Furthermore, introduction of the TGF type III receptor cDNA in the reverse orientation (pIIIRAS) profoundly reduced the surface expression of both the type I and type II receptors (Fig. 1B, lane 3).


Figure 1: A, chemical cross-linking of I-TGF1 to stably transfected BAECs and immunoprecipitation of cell lysates with antibodies to TGFIIIR. TGF type III receptor cDNA was stably transfected independently into BAECs either in the forward or reverse orientations relative to the promoter of the vectors. Clonal cell lines were incubated with I-TGF1, cross-linked, and analyzed by SDS-polyacrylamide gel electrophoresis as described under ``Experimental Procedures.'' Lanes 1, 3, and 5 show total lysates. Lanes 2, 4, and 6 show results from immunoprecipitation with antibodies to type III receptor. The different TGF receptor types are indicated in the margin. Lanes 1 and 2, control BAECs; lanes 3 and 4, BAECs transfected with pcDNA3; lanes 5 and 6, BAECs transfected with type III cDNA in the forward orientation. B, stable transfection of the TGF type IIIR cDNA in the reverse orientation significantly reduces surface expression of the TGF receptors. BAEC transfectants were labeled with iodinated TGF1, cross-linked, and analyzed as in A. Lane 1, BAECs transfected with vector alone; lane 2, BAECs transfected with the TGF type III cDNA in the forward orientation; lane 3, BAECs transfected with the TGF type III cDNA in the reverse orientation.



Further Characterization of the Transfected TGF Type III Receptor in BAECs

Our initial immunoprecipitation studies in Fig. 1 showed very little type II receptor protein co-immunoprecipitated with the type III receptor protein similar to the results reported by Moustakas and co-workers (34) when they precipitated type III receptor protein from Rat-1 cells. Therefore, to verify that negligible levels of type II receptor were associated with the type III receptor, we carried out immunoprecipitation reactions with antibodies to the type II receptor protein also. Immunoprecipitation of BAEC(V) cell lysates, which had been cross-linked to iodinated TGF1, with antibodies to the TGF type II receptor, co-precipitated the type I receptor (Fig. 2, lane 2). Immunoprecipitation of these lysates with the type IIIR antibodies did not precipitate any protein (Fig. 2, lane 3), indicating the absence of the TGF type IIIR protein in these cells. Interestingly, immunoprecipitation of BAECIIIR OE cell lysates with antibodies to the type II receptor co-precipitated a significant amount of the type III receptor protein (Fig. 2, lane 5) as well, indicating a complex formation between the TGF type III and type II receptor proteins in the BAECIIIR cells. Nonetheless, incubation with anti-type III receptor antibody still failed to co-precipitate type II receptor complexes.


Figure 2: Immunoprecipitation of BAEC TGFIIIR with anti-TGFIIIR and anti-TGFIIR antibodies. BAEC transfectants were labeled with I-TGF1, cross-linked, and analyzed as in Fig. 1A. Lanes 1-3, BAECs transfected with pcDNA3; lanes 4, 5, and 6, BAECs transfected with the type III receptor. Lanes 1 and 4 show binding in cell lysates before immunoprecipitation. Lanes 2 and 5 show results from immunoprecipitation with antibodies to type II receptor. Lanes 3 and 6 show results from immunoprecipitation with antibodies to type III receptor.



Secondly, we carried out immunoprecipitation reactions using cell lysates that had been surface-biotinylated. Control BAECs, BAEC(V), and BAECIIIR OE cells were surface biotinylated and equal amounts of cell lysate incubated with antibodies to the type III receptor protein. The immunoprecipitated proteins were detected using the streptavidin-horseradish peroxidase and ECL detection system. Several protein bands were detected only in BAECIIIR OE cells as shown in Fig. 3 . Bands in the region of 280 kDa are likely to represent the type III receptor, while the other bands noted in this immunoprecipitation are likely to represent surface molecules that co-precipitated with the type III receptor. Hence, immunoprecipitation with both I-TGF-labeled cell lysates and biotinylated lysates indicated that BAECIIIR OE cells were indeed expressing the type III receptor protein.


Figure 3: Immunoprecipitation of BAEC TGFIIIR with anti-TGFRIII antibodies following surface biotinylation. Control BAECs and BAEC transfectants were surface-biotinylated. Detergent extracts were then immunoprecipitated with anti-rat TGFRIII antibody and resolved by 6% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected using the streptavidin-horseradish peroxidase and enhanced chemiluminescence detection system. The first lane represents the surface-biotinylated cell lysate from control BAECs (Lysate) whereas the rest of the lanes represent immunoprecipitation of the respective cell lysates (Control = control BAEC; Vector = BAEC transfected with vector alone; IIIR OE = BAEC transfected with the TGF type III receptor cDNA and over-expressing the TGF type III receptor) with antibodies to the type III receptor protein. The molecular mass standards (194, 116, and 85 kDa) are indicated in the margin by the arrows.



BAECs Respond to TGF1 by Increased PAI-1 Protein Production and Enzyme Activity but Are Refractory to TGF2

The effects of varying doses (0.5, 5.0, and 25 ng/ml) of TGF1 and TGF2 on PAI-1 protein levels and enzyme activity in BAECs were examined by immunoblotting and reverse zymography. Both PAI-1 protein and activity were differentially regulated by TGF1 and TGF2. As illustrated by the immunoblotting technique in Fig. 4A, these amounts of TGF1 dose-dependently increased steady state PAI-1 protein levels while TGF2 was ineffective. These results were confirmed by reverse zymography as illustrated in Fig. 4B, that is, TGF1 increased steady state PAI-1 activity levels at all concentrations tested, while TGF2 did not.


Figure 4: A, differential effects by TGF1 and TGF2 on PAI-1 in BAECs. BAEC PAI-1 protein levels were determined by immunoblotting. Lane 1 = control; lane 2 = TGF1 (0.5 ng/ml) treatment; lane 3 = TGF1 (5.0 ng/ml) treatment; lane 4 = TGF1 (25.0 ng/ml) treatment; lane 5 = TGF2 (0.5 ng/ml) treatment; lane 6 = TGF2 (5.0 ng/ml) treatment; lane 7 = TGF2 (25.0 ng/ml); molecular mass of the PAI-1 = 47 kDa. B, BAEC PAI-1 activity was determined by reverse zymography.



Expression of the TGF Type III Receptor Protein Increases the Responsiveness to TGF2 by Increased Induction of PAI-1

The data above indicated that TGF1, but not TGF2, could induce PAI-1 protein in BAECs. We then re-examined this issue in BAECs that expressed transfected type III receptor. Cultures transfected with vector plasmid alone responded to TGF1 and TGF2 in a way that was consistent with untransfected cultures, although the vector itself seemed to decrease basal PAI-1 protein levels (top two panels, Fig. 5). In BAECIIIR OE cells, both TGF1 and TGF2 enhanced PAI-1 protein to essentially equal extents (third panel, Fig. 5). In contrast, BAECIIIR AS cells transfected with the type III receptor cDNA in the reverse orientation were unresponsive to both TGF isoforms (bottom panel, Fig. 5). Notably, PAI-1 protein in the untreated BAECIIIR AS cells was the highest among all the cell lines tested.


Figure 5: Expression of TGFIIIR increases the effect of TGF2 on PAI-1 protein in BAECs. Subconfluent cultures of untransfected BAECs (Control), cells transfected with vector alone (Vector), cells overexpressing type III receptor (IIIR OE), and cells expressing an antisense construct of the type III receptor (IIIR AS) were incubated with 0.5 ng/ml of TGF1 or 0.5 ng/ml of TGF2. Equal amounts of protein from detergent-extracted cell lysates were fractionated on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, probed with antibodies to PAI-1 protein, and visualized by enhanced chemiluminescence. Analogous results occurred in two separate experiments.



Expression of Type III Receptor Protein Increases Inhibition of Migration by TGF2 in BAECs

Our laboratory previously reported that TGF1 inhibited the migration rate of BAECs, whereas TGF2 had a very modest effect (32) . Therefore, we repeated migration assays with control and transfected cells to see if there were any differences in the responsiveness to the two TGF isoforms. Similar to the earlier results, TGF1, but not TGF2, inhibited migration in BAECs and in cultures transfected with vector plasmid alone (Fig. 6). However, with BAECIIIR OE cells, both TGF1 and TGF2 equally inhibited the migration rates of these cells while BAECIIIR AS cells were refractory to both TGF isoforms. Previous studies (32) also demonstrated differences in BAEC proliferation responses to TGF1 and TGF2. TGF1 potently reduced proliferation of BAECs, whereas TGF2 had no effect. In agreement with our PAI-1 and migration studies, when we examined the proliferative response in BAECIIIR OE cells, the cells were inhibited by both TGF isoforms (data not shown).


Figure 6: Expression of TGFIIIR increases the inhibitory effect of TGF2 on migration by BAEC. Migration assays were performed with untransfected cells (BAEC), cells transfected with vector alone (BAEC(V)), or cells transfected with TGFIIIR in the forward (BAECIIIR) or reverse orientation (BAECIIIRAS). The cells were incubated with 0.5 ng of TGF1 or 0.5 ng of TGF2 and assayed 6 days after removal of the fences to allow migration to occur. Relative migration was analyzed with the NIH Image Program.



Expression of the Type III Receptor Protein Increases the Affinity of TGF2 for the BAEC Surface via the TGF Type III Receptor

Our results so far indicated that the expression of TGF type III receptor protein in BAECs increased their responsiveness to TGF2 by three separate criteria: proliferation, PAI-1 protein, and migration assays. TGF2 gave comparable results to TGF1. When an equal amount of unlabeled TGF1 was combined with I-TGF1, binding to the TGF type III receptor was not affected (Fig. 7, lane 2) whereas a 10-fold excess of unlabeled TGF1 showed a 70-80% reduction (Fig. 7, lane 3). Similar results occurred with analogous amounts of unlabeled TGF2, although it appeared to have a slightly higher affinity for transfected type III receptor binding sites (Fig. 7, lanes 4 and 5).


Figure 7: TGF type III receptor actively associates with TGF1 and TGF2. Subconfluent BAECIIIR OE cells (lanes 1-5) were incubated with 100 pMI-TGF1, cross-linked with disuccinimidyl suberate and analyzed as in Fig. 1A. During the incubation period with iodinated TGF1, labeling medium included no addition (lane 1), unlabeled TGF1 at 100 pM (lane 2), or 1000 pM (lane 3); or unlabeled TGF2 at 100 pM (lane 4) or 1000 pM (lane 5).




DISCUSSION

BAECs endogenously express both type I and type II receptors for TGF, respond well to TGF1, but are relatively refractory to TGF2. Expression of transfected type III receptor protein allowed BAECs to now respond to TGF2 in terms of PAI-1 protein levels, migration, and proliferation assays. These results indicate that type III receptor protein expression increases the responsiveness to TGF2 in BAECs that do not normally respond to treatment with TGF2. Our binding studies agree well with previous observations made by Wang and co-workers (26) and Lopez-Casillas and co-workers (25) when they transfected type III receptor cDNA into LE myoblast cell lines lacking the TGF type III receptor protein. In contrast, however, BAEC transfectants containing the type III receptor cDNA in the reverse orientation displayed a dramatic reduction in the surface expression of both type I and type II receptors as assessed by radioligand binding and cross-linking, and were refractory to both TGF1 and TGF2. Therefore, our results indicate that complete down-regulation of the TGF type III receptor could alter the surface expression of the TGF type II and type I receptors in a fashion that is not yet well understood.

The expression of the type III receptor protein increases the responsiveness of BAECs to TGF2. TGF2 has a lower affinity for the cell surface type II receptor (K 500 pM) than does TGF1 or TGF3 (K 25-50 pM) (5, 43) . One possible explanation for this effect is direct physical interaction between the type II and type III receptor proteins on the cell surface. Once TGF2 binds to the type III receptor it could then couple more effectively to the type II receptors. Indirect evidence from our studies with iodinated TGF1 indicate that immunoprecipitation of radiolabeled BAECIIIR OE cell lysates with antibodies to the type III receptor did not co-precipitate much ligand-bound type II receptor. However, immunoprecipitation with anti-type II antibodies co-precipitated a significant amount of the type III receptor protein as well as the type I receptor protein. Differences with these two antibodies may reflect the overexpression of the type III receptor protein relative to the type II and type I receptor proteins on the surface of the BAECIIIR OE cells and suggest that a large fraction of the type III receptors in this situation may not associate with the other receptor types. Therefore, we suggest that there may be two kinds of complexes: 1) complexes composed of TGF, type II, and type I receptors and 2) complexes composed of TGF and all three cell surface components. Expression of the type III receptor may specifically promote TGF2 access to type II receptor thus overcoming the low affinity binding of TGF2 to type II receptors. It is also possible that TGF2 binding to the type III receptor elicits a conformation change in the TGF2 molecule that more favorably associates with type II receptor. Unbound TGF1 might approximate such a conformation better than unbound TGF2. Recent studies by Qian and co-workers (44) , using domain swapping experiments suggest that the different potencies of TGF1 and TGF2 map to a 42-amino-acid region that includes the longest -helix in the monomer. The availability of these chimeric TGF molecules will enable us to identify which portion of the molecule is responsible for interacting with the TGF receptors. The recent crystallography studies of TGF2 (45, 46) will also assist in our understanding of the important residues required for interaction with the receptors.

Large vessel endothelial cells such as BAECs and small vessel endothelial cells such as rat fat pad capillary endothelial cells differ in the expression of the TGF type III receptor protein (32) . BAECs lack the type III receptor protein and are refractory to TGF2 whereas rat fat pad capillary endothelial cells contain the type III receptor protein and are inhibited by TGF1 and TGF2 in proliferation studies (32) . Hence the differences in the surface expression of the type III receptor protein in these two populations of endothelial cells may well modulate their responsiveness to TGF2. These data are consistent with the concept that cells regulate their responsiveness to TGF isoforms by modulation of their TGF receptors. Although the type III receptor does not appear to play a direct role in signaling, recent findings from McAllister et al.(47) suggest that defects in a related TGF-binding protein, endoglin, which does not have direct signaling activity as well, is the cause of hereditary hemeorrhagic telangiectasia type I, an autosomal dominant disorder that is characterized by multisystemic vascular dysplasia and recurrent hemeorrhage. Hence although large vessel endothelial cells lack the type III receptor, perhaps the type III receptor plays an important role in early stages of vascular growth in microvessel endothelial cells. Future experiments will be directed toward down-regulating the expression of endogenous TGF type III receptor in cultured microvascular endothelial cells by using the antisense construct to determine its effect in in vitro angiogenesis.


FOOTNOTES

*
This work was supported by the Reed Foundation Fellowship In Vascular Biology (to S. S.), United States Public Health Service Grants RO1HL28373 and PO1 DK38979 (to J. A. M.), American Heart Association Grants 92006500 (to J. A. M.), RO1AR39201 (to M. C.), and BRSG/RR-05358 (to M. C. and T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, 310 Cedar St., New Haven, CT 06510. Tel.: 203-785-2763; Fax: 203-785-7303.

The abbreviations used are: TGF, transforming growth factor; BAEC, bovine aortic endothelial cells; PAI-1, plasminogen activator inhibitor-1.


ACKNOWLEDGEMENTS

We thank Dr. Anne Romanic, Adeline Tucker, and Joanne Lum for technical assistance. We also thank Dr. Lucia Languino for critically reading this manuscript.


REFERENCES
  1. Roberts, A. B., and Sporn, M. B.(1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds) pp. 419-472, Springer-Verlag, Berlin
  2. Massague, J.(1990) Annu. Cell Biol. 6, 597-641 [CrossRef]
  3. Lin, H. Y., and Lodish, H. F.(1993) Trends Cell Biol. 3, 14-19 [CrossRef]
  4. Sporn, M. B., and Roberts, A. B.(1992) J. Cell Biol. 119, 1017-1021 [Medline] [Order article via Infotrieve]
  5. Massague, J.(1992) Cell 69, 1067-1070 [Medline] [Order article via Infotrieve]
  6. Miyazono, K., ten Dijke, P., Ichijo, H., and Heldin, C. H.(1994) Adv. Immunol. 55, 181-220 [Medline] [Order article via Infotrieve]
  7. Lin, H. Y., Wang, X.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F.(1992) Cell 68, 775-785 [Medline] [Order article via Infotrieve]
  8. 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]
  9. Tsuchida, K., Lewis, A. K., Mathews, L. S., and Vale, W. W.(1993) Biochem. Biophys. Res. Commun. 191, 790-795 [CrossRef][Medline] [Order article via Infotrieve]
  10. Ebner, R., Chen, R.-H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R.(1993) Science 260, 1344-1348 [Medline] [Order article via Infotrieve]
  11. Franzen, P., ten Dijke, P., Ichijo, H., Schulz, P., Heldin, C.-H., and Miyazono, K.(1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  12. Chinkers, M.(1987) J. Cell. Physiol. 130, 1-5 [Medline] [Order article via Infotrieve]
  13. Boyd, F. T., and Massague, J.(1989) J. Biol. Chem. 264, 2272-2278 [Abstract/Free Full Text]
  14. Laiho, M., Weiss, F. M. B., and Massague, J.(1990) J. Biol. Chem. 265, 18518-18524 [Abstract/Free Full Text]
  15. Laiho, M., Weiss, F. M. B., Boyd, F. T., Ignotz, R. A., and Massague, J.(1991) J. Biol. Chem. 266, 9108-9112 [Abstract/Free Full Text]
  16. Segarini, P. R., Rosen, D. M., and Seyedin, S. M.(1989) Endrocrinology 3, 261-272
  17. Segarini, P. R., Ziman, J. M., Kane, C. J. M., and Dasch, J. R.(1992) J. Biol. Chem. 267, 1048-1053 [Abstract/Free Full Text]
  18. Geiser, A. G., Burmester, J. K., Webbink, R., Roberts, A. B., and Sporn, M. B.(1992) J. Biol. Chem. 267, 2588-2593 [Abstract/Free Full Text]
  19. Inagaki, M., Moustakas, A., Lin, H.-Y., and Carr, B. I.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5359-5363 [Abstract]
  20. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  21. Chen, R. H., Ebner, R., and Derynck, R.(1993) Science 260, 1335-1338 [Medline] [Order article via Infotrieve]
  22. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C. H., and Miyazono, K.(1993) Exp. Cell Res. 207, 681-692
  23. Centrella, M., Kim, J., Pham, T., Casinghino, S., Rosen, V., Wozney, J., and McCarthy, T. L.(1995) Mol. Cell Biol., in press
  24. 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]
  25. 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]
  26. Wang, X.-F., Lin, H.-Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and Weinberg, R. H.(1991) Cell 51, 797-805
  27. Moren, A., Ichijo, H., and Miyazono, K.(1992) Biochem. Biophys. Res. Commun. 189, 356-362 [Medline] [Order article via Infotrieve]
  28. Ohta, M., Greenberger, J. S., Anklesana, R., Bassols, A., and Massague, J.(1987) Nature 329, 539-542 [CrossRef][Medline] [Order article via Infotrieve]
  29. Ottman, O. G., and Pellus, L. M.(1988) J. Immunol. 140, 2661-2665 [Abstract/Free Full Text]
  30. Cheifetz, S., Bellon, T., Cales, C., Vera, S., Bernabeu, C., Massague, J., and Letarte, M.(1992) J. Biol. Chem. 267, 19027-19030 [Abstract/Free Full Text]
  31. Jennings, J. C., Mohan, S., Linkart, T. A., Widstrom, R., and Baylink, D. J.(1988) J. Cell. Physiol. 137, 167-172 [Medline] [Order article via Infotrieve]
  32. Merwin, J. R., Newman, W., Beall, L. D., Tucker, A., and Madri, J. A. (1991) Am. J. Pathol. 138, 37-51 [Abstract]
  33. Lopez-Casillas, F., Wrana, J. L., and Massague, J.(1993) Cell 73, 1435-1444 [Medline] [Order article via Infotrieve]
  34. Moustakas, A., Lin, H.-Y., Henis, Y. I., Plamondon, J., O'Connor-McCourt, M., and Lodish, H. F.(1993) J. Biol. Chem. 268, 22215-22218 [Abstract/Free Full Text]
  35. Madri, J. A., and Furthmayr, H.(1987) Am. J. Pathol. 94, 179-187
  36. Madri, J. A., and Furthmayr, H.(1980) Human Pathol. 11, 353-366 [Medline] [Order article via Infotrieve]
  37. Roll, F. J., Madri, J. A., Albert, J., and Furthmayr, H.(1980) J. Cell Biol. 859, 597-616
  38. Truett, M. A., Blacher, R., Burke, R. L., Caput, D., Chu, C., Dina, D., Hartog, K., Kuo, C. H., Masarz, F. R., Merryweather, J. P., Najarian, R., Pachl, C., Potter, S. J., Puma, J., Quiroga, M., Rall, L. B., Randolph, A., Urdea, M. S., Valenzuela, P., Dahl, H. H., Favalaro, J., Hansen, J., Nordfang, O., and Ezban, M.(1985) DNA 4, 333-349 [Medline] [Order article via Infotrieve]
  39. Centrella, M., McCarthy, T. L., and Canalis, E.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5889-5893 [Abstract]
  40. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  41. Pratt, B. M., Harris, A. S., Morrow, J. S., and Madri, J. A.(1984) Am. J. Pathol. 117, 349-354 [Abstract]
  42. Madri, J. A., Pratt, B. M., and Tucker, A. M.,(1988) J. Cell Biol. 106, 1375-1384 [Abstract]
  43. ten Dijke, P., Iwata, K. K., Goddard, C., Peihler, C., Canalis, E., McCarthy, T. L., and Centralla, M.(1990) Mol. Cell. Biol. 10, 4473-4479 [Medline] [Order article via Infotrieve]
  44. Qian, S. W., Burmester, J. K., Merwin, J. R., Madri, J. A., Sporn, M. B., and Roberts, A. B.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6290-6294 [Abstract]
  45. Daopin, S., Piez, K. A., Ogawa, Y., and Davies, D. R.(1992) Science 257, 369-373 [Medline] [Order article via Infotrieve]
  46. Schlunegger, M. P., and Grutter, M. G.(1992) Nature 358, 430-438 [CrossRef][Medline] [Order article via Infotrieve]
  47. McAllister, K. A., Grog, K. M., Johnson, D. W., Gallione, C. J., Baldwin, M. A., Jackson, C. E., Helmhold, E. A., Markel, D. S., McKinnon, W. C., Murrel, J., McCormick, M. K., Pericak-Guttmache, A. E., Letarte, M., and Marchuk, D. A.(1994) Nature Genet. 8, 345-351 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.