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Originally published In Press as doi:10.1074/jbc.M512821200 on February 21, 2006

J. Biol. Chem., Vol. 281, Issue 17, 11506-11514, April 28, 2006
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Cellular Heparan Sulfate Negatively Modulates Transforming Growth Factor-beta1 (TGF-beta1) Responsiveness in Epithelial Cells*

Chun-Lin Chen, Shuan Shian Huang, and Jung San Huang1

From the Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104

Received for publication, November 30, 2005 , and in revised form, January 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-surface proteoglycans have been shown to modulate transforming growth factor (TGF)-beta responsiveness in epithelial cells and other cell types. However, the proteoglycan (heparan sulfate or chondroitin sulfate) involved in modulation of TGF-beta responsiveness and the mechanism by which it modulates TGF-beta responsiveness remain unknown. Here we demonstrate that TGF-beta1 induces transcriptional activation of plasminogen activator inhibitor-1 (PAI-1) and growth inhibition more potently in CHO cell mutants deficient in heparan sulfate (CHO-677 cells) than in wild-type CHO-K1 cells. 125I-TGF-beta1 affinity labeling analysis of cell-surface TGF-beta receptors reveals that CHO-K1 and CHO-677 cells exhibit low (<1) and high (>1) ratios of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I, respectively. Receptor-bound 125I-TGF-beta1 undergoes nystatin-inhibitable rapid degradation in CHO-K1 cells but not in CHO-677 cells. In Mv1Lu cells (which, like CHO-K1 cells, exhibit nystatin-inhibitable rapid degradation of receptor-bound 125I-TGF-beta1), treatment with heparitinase or a heparan sulfate biosynthesis inhibitor results in a change from a low (<1) to a high (>1) ratio of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I and enhanced TGF-beta1-induced transcriptional activation of PAI-1. Sucrose density gradient analysis indicates that a significant fraction of TbetaR-I and TbetaR-II is localized in caveolae/lipid-raft fractions in CHO-K1 and Mv1Lu cells whereas the majority of the TGF-beta receptors are localized in non-lipid-raft fractions in CHO-677 cells. These results suggest that heparan sulfate negatively modulates TGF-beta1 responsiveness by decreasing the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I, facilitating caveolae/lipid-raft-mediated endocytosis and rapid degradation of TGF-beta1, thus diminishing non-lipid-raft-mediated endocytosis and signaling of TGF-beta1 in these epithelial cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF-beta2 is a family of 25-kDa disulfide-linked dimeric proteins. It has three members in mammals (TGF-beta1, TGF-beta2, and TGF-beta3), which share ~70% sequence homology (1–3). TGF-beta exhibits bifunctional growth regulation; it inhibits growth of most cell types, including epithelial cells, endothelial cells, and lymphocytes, and stimulates proliferation of mesenchymal cells such as fibroblasts. The growth regulatory activity of TGF-beta has been implicated in many physiological and pathological processes (e.g. embryonic development, morphogenesis, carcinogenesis, autoimmune diseases, and Alzheimer disease) (1–3). One other prominent activity of TGF-beta is transcriptional activation of extracellular matrix synthesis-related genes, which has been implicated in tissue fibrosis. TGF-beta also exhibits chemotactic activity toward monocytes and neutrophils and is involved in the process of inflammation (1–3).

The various biological activities of TGF-beta are mediated by specific cell-surface type I, type II, type III, and type V TGF-beta receptors (TbetaR-I, TbetaR-II, TbetaR-III, and TbetaR-V) (4–7). TbetaR-I and TbetaR-II are Ser/Thr-specific protein kinases believed to be primarily responsible for mediating TGF-beta-induced cellular responses (2, 8–10). TbetaR-III, also known as betaglycan, is a 270–300-kDa proteoglycan-containing membrane glycoprotein (8–10). The proteoglycans contained in TbetaR-III include heparan sulfate and chondroitin sulfate (11). TbetaR-III functions as a co-receptor, which presents TGF-beta ligands to TbetaR-II (12, 13). TbetaR-V is a high molecular weight non-proteoglycan membrane glycoprotein (7, 14, 15). It is not directly involved in mediating TGF-beta-induced transcriptional activation but is required for TGF-beta-induced growth inhibition (16, 17).

Among the TGF-beta receptor types, TbetaR-III (betaglycan) is the most abundant in many cell types. Due to its high density, TbetaR-III is capable of modulating TGF-beta binding to other TGF-beta receptor types and of regulating TGF-beta-induced cellular responses (12, 13). Ectopic expression of wild-type TbetaR-III augments TGF-beta-induced cellular responses in myoblasts (12, 13). On the other hand, in epithelial cells, ectopic expression of wild-type TbetaR-III attenuates cellular responses induced by TGF-beta, whereas expression of TbetaR-III mutants lacking proteoglycans promotes TGF-beta-induced cellular responses (13). The distinct effects of wild-type and mutant TbetaR-III on TGF-beta1-induced cellular responses in these two cell types appear to be due to the different sizes of proteoglycans in the TbetaR-III molecules in these cells (13). The proteoglycan sizes of TbetaR-III in myoblasts are smaller than those in epithelial cells. These observations suggest that TbetaR-III with larger proteoglycans negatively modulates TGF-beta1-induced cellular responses, whereas TbetaR-III lacking proteoglycans or containing smaller proteoglycans enhances TGF-beta1-induced cellular responses. However, which proteoglycan (heparan sulfate or chondroitin sulfate) in TbetaR-III is involved in modulating TGF-beta-induced cellular responses remains unknown.

Proteoglycans are large unbranched carbohydrates comprised of repeating disaccharide units. They are components of the extracellular matrix and the external cell-surface membrane and are also present in secretory granules. They have six forms: heparan sulfate, chondroitin sulfate, heparin, dermatan sulfate, keratan sulfate, and hyaluronic acid. Proteoglycans have been shown to regulate cell growth, differentiation, and migration (18, 19). Since TGF-beta is also known to regulate these processes, it would be important to define the molecular mechanisms by which heparan sulfate or chondroitin sulfate (or both) modulate TGF-beta1-induced cellular responses. In this communication, we demonstrate that deficiency or enzymic removal of heparan sulfate in epithelial cells increases the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I, attenuates degradation of TGF-beta1, and augments TGF-beta1-induced cellular responses.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Na125I (17 Ci/mg), [{alpha}-32P]ATP, and [methyl-3H]thymidine (67 Ci/mmol) were purchased from ICN Radiochemicals (Irvine, CA). Molecular mass protein standards (myosin, 205 kDa; beta-galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serum albumin, 66 kDa), nystatin, SDS, Dulbecco's modified Eagle's medium (DMEM), DMEM-F-12 medium, heparitinase, phenylmethanesulfonyl fluoride, bovine serum albumin, trichloroacetic acid, and MES, peroxidase-conjugated anti-rabbit IgG, and disuccinimidyl suberate (DSS) were obtained from Sigma. The prestained protein ladder (64, 49, 37, 26, and 20 kDa) was obtained from Invitrogen. TGF-beta1 was purchased from Austral Biologicals (San Ramon, CA). The ECL system and rabbit polyclonal antibodies to TbetaR-I, TbetaR-II, and TbetaR-III were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Wild-type Chinese hamster ovary (CHO) cells (CHO-K1 cells) and mutant CHO cells (CHO-677 cells) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM-F-12 medium containing 50 µg/ml of streptomycin and 10% fetal calf serum. CHO-677 cells (pgsD 677 cells) specifically lacked heparan sulfate (20). The mutation in CHO-677 cells affected both GlcNAc and GlcA transferase activity required for heparan sulfate polymerization (20). Mink lung epithelial cells (Mv1Lu cells) were maintained in DMEM containing 50 µg/ml streptomycin and 10% fetal calf serum.

125I-TGF-beta1 Affinity Labeling of Cell-surface TGF-beta Receptors in CHO and Mv1Lu Cells—CHO cells (CHO-K1 and CHO-677 cells) and Mv1Lu cells were grown on 6-well cluster dishes in DMEM-F-12 medium and DMEM containing 10% fetal calf serum, respectively, as described (16, 17, 20). 125I-TGF-beta1 affinity labeling of cell-surface TGF-beta receptors was performed using the bifunctional cross-linking agent DSS as described previously (11, 14, 16, 17). The affinity-labeled TGF-beta receptors were then analyzed by 5 and 7.5% SDS-PAGE under reducing conditions and autoradiography. In some experiments, the affinity-labeled TGF-beta receptors were immunoprecipitated by specific antibodies to TbetaR-I, TbetaR-II, and TbetaR-III as described previously (17). The immunoprecipitates were then analyzed by 7.5% SDS-PAGE under reducing conditions and autoradiography.

[methyl-3H]Thymidine Incorporation and Northern Blot Analyses—Cells were grown to near confluence on 24-well cluster dishes and then treated with several concentrations of TGF-beta1 at 37 °C for 18 or 2 h for [methyl-3H]thymidine incorporation into cellular DNA or for plasminogen activator inhibitor-1 (PAI-1) expression analysis, respectively. [methyl-3H]Thymidine incorporation into cellular DNA and Northern blot analysis of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in CHO-K1, CHO-677, and Mv1Lu cells were carried out as described previously (16, 17). The relative level of PAI-1 mRNA was estimated based on the ratio of PAI-1 mRNA and G3PDH mRNA intensities on the autoradiogram. The relative intensities of both mRNAs on the autoradiogram were quantitated by a PhosphoImager.

Treatment of Mv1Lu Cells with Heparitinase or a Heparan Sulfate Biosynthesis Inhibitor—Mv1Lu cells were grown near confluence on 6-well cluster dishes, washed twice with serum-free DMEM, and treated with vehicle only, heparitinase (100 milliunits/ml) (11), or a heparan sulfate biosynthesis inhibitor p-nitrophenyl-beta-D-xylopyranoside (3 mM) in DMEM containing 0.1% fetal calf serum (21). After 3 h at 37°C (for heparitinase) and 72 h at 37 °C (for the inhibitor) in serum-free DMEM containing 1% bovine serum albumin, 125I-TGF-beta1 affinity labeling of cell-surface TGF-beta receptors and Northern blot analysis of PAI-1 or G3PDH mRNA expression was performed as described above.

Western Blot Analysis of TbetaR-I and TbetaR-II in CHO Cells—Cell lysates of CHO-K1 and CHO-677 cells (~50 µg of protein) were subjected to 7.5% SDS-PAGE under reducing conditions and then electro-transferred to nitrocellulose membranes. After being incubated with 5% nonfat milk in Tris-buffered saline plus Tween (TBST) (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature, the membranes were further incubated with specific polyclonal antibodies to TbetaR-I and TbetaR-II in TBST/nonfat milk at room temperature for 1 h and washed three times with TBST for 10 min each. Bound antibodies were detected using peroxidase-conjugated anti-rabbit IgG and visualized using the ECL system (Santa Cruz Biotechnology).

Cellular Degradation of Receptor-bound 125I-TGF-beta1 in CHO-K1, CHO-677, and Mv1Lu Cells—Cells grown on 24-well cluster dishes were pretreated with 25 µg/ml nystatin at 37 °C for 1 h in serum-free DMEM-F-12 medium and then incubated with 100 pM 125I-TGF-beta1 in the presence and absence of 100-fold excess unlabeled TGF-beta1. The presence of 100-fold-excess unlabeled TGF-beta1 was used to estimate nonspecific binding. After 2.5 h at 0 °C, cells were washed and warmed to 37 °C in DMEM-F-12 medium (for CHO cells) or DMEM (for Mv1Lu cells) containing 0.2% bovine serum albumin (22). After several time periods in the presence and absence of nystatin (25 µg/ml), the conditioned medium was precipitated with 10% trichloroacetic acid at 4 °C for 0.5 h. The trichloroacetic acid-soluble radioactive material, which contained the degradation products (e.g. amino acids or peptides) of cell-surface bound 125I-TGF-beta1 after internalization and degradation and were released from cells, was counted. The trichloroacetic acid-soluble radioactive material derived from the specific binding of 125I-TGF-beta1 was estimated as the percentage of the total specific binding of 125I-TGF-beta1 determined before incubation at 37 °C.

Sucrose Density Gradient Analysis—Sucrose density gradient analysis was performed at 4 °C as described previously (23). Briefly, cells were grown to near confluence in 100-mm Petri dishes. After washing with ice-cold HEPES buffer (16, 17), cells were scraped into 0.85 ml of 500 mM sodium carbonate, pH 11.0. The cell pellets were homogenized with a tight-fitting Dounce homogenizer followed by three 20-s bursts of ultrasonic disintegrator on ice. The homogenates were adjusted to 45% sucrose using 90% sucrose in MES-buffered saline, pH 6.5 (25 mM MES and 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. Two solutions (1.7 ml each) of 35 and 5% sucrose were laid sequentially on the top of the 45% sucrose solution. After ultracentrifugation at 35,000 rpm with Beckman SW Ti55 rotor for 16–20 h, 10 0.5-ml fractions were collected from the top of the tubes, and a portion of each fraction was analyzed by SDS-PAGE followed by Western blot analysis using specific antibodies to TbetaR-I, TbetaR-II, or caveolin-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deficiency in Heparan Sulfate Augments TGF-beta1 Responsiveness in CHO Cells—CHO wild-type and mutant cells, which are epithelial cells defective in heparan sulfate and chondroitin sulfate synthesis, have provided an excellent system for defining the roles of proteoglycans in cellular responses to stimuli in CHO cells (20, 24). To define the roles of heparan sulfate and chondroitin sulfate in TGF-beta-induced cellular responses (TGF-beta responsiveness) in CHO cells, we determined the effects of increasing concentrations of TGF-beta1 on cell growth (as determined by measurement of DNA synthesis and cell number) and PAI-1 expression in CHO-K1 and CHO-677 cells. CHO-K1 cells are wild-type CHO cells. CHO-677 cells are CHO mutant cells, which are defective in heparan sulfate synthesis but not chondroitin sulfate synthesis (20). As shown in Fig. 1A, TGF-beta1 inhibited DNA synthesis in CHO-677 cells more potently than in CHO-K1 cells. At 25 pM, TGF-beta1 inhibited DNA synthesis by ~60% in CHO-677 cells and by ~40% in CHO-K1 cells (Fig. 1A, panel a). TGF-beta1 also inhibited cell growth more potently in CHO-677 cells than in CHO-K1 cells. TGF-beta1 (100 pM) inhibited cell growth by ~75 and ~25% in CHO-677 and CHO-K1 cells, respectively (Fig. 1A, panel b). CHO-677 cells also responded more strongly to TGF-beta1-induced expression of PAI-1 when compared with wild-type CHO-K1 cells (Fig. 1B, panel a). At 20 pM, TGF-beta1 stimulated PAI-1 expression by ~2.2-fold in CHO-677 cells (Fig. 1B, panel b). In contrast, TGF-beta1 at the same concentration only slightly stimulated PAI-1 expression by 1.2-fold in CHO-K1 cells (Fig. 1B, panel b). These results suggest that the defect in heparan sulfate biosynthesis augments TGF-beta1 responsiveness in CHO cells.


Figure 1
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  		FIGURE 1.
Effects of TGF-beta1 on growth (A) and PAI-1 expression (B) in CHO-K1 and CHO-677 cells. Cells were treated with several concentrations of TGF-beta1 as indicated (for DNA synthesis and PAI-1 expression analyses) or 100 pM TGF-beta1 (for cell number analysis). After 18 h (for DNA synthesis) and 72 h (for counting cell number) (A) or 2 h (for PAI-1 expression) (B) at 37 °C, the cell growth or PAI-1 expression was determined by measurement of [methyl-3H]thymidine incorporation into cellular DNA (DNA synthesis) (A, panel a) and by counting cell number (A, panel b) or by Northern blot analysis (B). The [methyl-3H]thymidine incorporation or cell number in cells treated without TGF-beta1 was taken as 100% (control). In Northern blot analysis, the expression of G3PDH mRNA was used as control (B, panel a). The relative levels of the transcripts were quantitated with a PhosphorImager. The relative amount of the PAI-1 transcript was expressed as the ratio of the relative levels of PAI-1 and G3PDH (B, panel b). The ratio in cells treated without TGF-beta1 was taken as 1.0. Values in the bar charts are mean ± S.D. (n = 4). *, p < 0.05 or 0.001, TGF-beta1-treated cultures versus cultures treated with vehicle alone (control).

 
Deficiency in Heparan Sulfate Increases the Ratio of TGF-beta1 Binding to TbetaR-II and TbetaR-I in CHO Cells—125I-TGF-beta1 affinity labeling of cells (125I-TGF-beta1 binding and cross-linking with the bifunctional reagent DSS) followed by SDS-PAGE has been commonly used for determining 125I-TGF-beta1 binding to individual cell-surface TGF-beta receptor types (11, 14, 17). To determine the effect of the deficiency in heparan sulfate on 125I-TGF-beta1 binding to TGF-beta receptors in CHO cells, CHO-K1 and CHO-677 cells were affinity-labeled with 125I-TGF-beta1 (100 pM) and analyzed by 7.5% SDS-PAGE under reducing conditions and autoradiography. As shown in Fig. 2A, in CHO-677 cells, 125I-TGF-beta1 affinity-labeled TbetaR-III exhibited a distinct band with molecular mass of ~120 kDa (TbetaR-III*), representing the 125I-TGF-beta1 affinity-labeled core protein of TbetaR-III (Fig. 2A, panel a, lane 3). In CHO-K1 cells, 125I-TGF-beta1 affinity-labeled TbetaR-III (~270–300 kDa) migrated at the top of the separating gel on 7.5% SDS-PAGE (Fig. 2A, panel a, lane 2). 125I-TGF-beta1 affinity-labeled TbetaR-I and TbetaR-II migrated as distinct bands with molecular masses of 68 and 88 kDa, respectively, on SDS-PAGE in CHO cell lines including CHO-LRP-1– cells are CHO mutant cells lacking TbetaR-V (22) (Fig. 2A, lane 1). The lack of TbetaR-V appeared to affect 125I-TGF-beta1 binding to other TGF-beta receptor types in CHO cells. Quantative analysis of 125I-TGF-beta1 affinity labeling of TGF-beta receptors in these CHO cells revealed that CHO-677 cells exhibited an increase of TGF-beta1 binding to TbetaR-II and a decrease of 125I-TGF-beta1 binding to TbetaR-I when compared with CHO-K1 cells (Fig. 2A, panel b). The ratio (~1.4) of 125I-TGF-beta1 affinity-labeled TbetaR-II and TbetaR-I appeared to be higher in CHO-677 cells than that (0.6) in wild-type cells (CHO-K1 cells) (Fig. 2A, panel c). This result suggests that the deficiency in heparan sulfate alters binding of TGF-beta1 to TbetaR-I and TbetaR-II. To further define this, CHO-677 and CHO-K1 cells were incubated with increasing concentrations of 125I-TGF-beta1; 125I-TGF-beta1 affinity labeling of TGF-beta receptors in these cells was then performed and analyzed by 5% (Fig. 2B) and 7.5% (Fig. 2C). SDS-PAGE under reducing conditions and autoradiography. As shown in Fig. 2, B and C, 125I-TGF-beta1 bound to TbetaR-I, TbetaR-II, and TbetaR-III or TbetaR-III* in a concentration-dependent manner in CHO-K1 cells (lanes 1–5) and CHO-677 cells (lanes 6–10). The half-maximum concentration (~50 pM) of 125I-TGF-beta1 for binding to the core protein (~120 kDa) of TbetaR-III (TbetaRIII*) in CHO-677 cells was similar to that of 125I-TGF-beta1 for binding to TbetaR-III (~270–300 kDa) in CHO-K1 cells (Fig. 2B, panels a and b). CHO-K1 and CHO-677 cells also did not exhibit significant differences in the half-maximum concentrations (~50 pM) of 125I-TGF-beta1 for binding to TbetaR-I and TbetaR-II (Fig. 2C, panels b and c). This suggests that the affinities of TGF-beta1 for binding to TbetaR-I and TbetaR-II are similar in CHO-K1 and CHO-677 cells. However, the total binding of 125I-TGF-beta1 to TbetaR-II (determined as the 125I-TGF-beta1 affinity labeled TbetaR-II) increased in CHO-677 cells as compared with that in CHO-K1 cells (Fig. 2C, panel a, lanes 6–10 versus lanes 1–5 and Fig. 2C, panel c versus panel b). The binding of 125I-TGF-beta1 to TbetaR-I (125I-TGF-beta1 affinity-labeled TbetaR-I) decreased concomitantly in CHO-677 cells as compared with that in CHO-K1 cells (Fig. 2B, panel a, and Fig. 2C, panel a, lanes 6–10 versus lanes 1–5 and Fig. 2C, panel c versus panel b). At 100 pM 125I-TGF-beta1, the ratio of 125I-TGF-beta1 affinity-labeled TbetaR-II and TbetaR-I in CHO-677 cells was estimated to be ~ 2 (Fig. 2C, panel d). In contrast, at the same concentration of 125I-TGF-beta1, the ratio of 125I-TGF-beta1 affinity-labeled TbetaR-II and TbetaR-I was estimated to be 0.7 in CHO-K1 cells (Fig. 2C, panel d). CHO-677 and CHO-K1 cells exhibited similar ratios of TbetaR-II and TbetaR-I protein levels as determined by Western blot analysis (Fig. 3, lane 2 versus lane 1). The multiple bands of TbetaR-II shown in Fig. 3 were also demonstrated previously (25). These results suggest that the deficiency in heparan sulfate alters the total binding of TGF-beta1 to TbetaR-I and TbetaR-II without significantly affecting the affinities of TGF-beta1 binding to TbetaR-I and TbetaR-II and their protein expression in these CHO cells. The similar half-maximum concentrations of 125I-TGF-beta1 for binding to TbetaR-III and TbetaR-III* (Fig. 2B, panel b) in CHO-K1 and CHO-677 cells suggest that the binding affinities of 125I-TGF-beta1 for TbetaR-III and TbetaR-III* are similar in these cells. Since TbetaR-III is known to present the ligand TGF-beta to TbetaR-II and then TbetaR-I, and since TbetaR-III is also known to form hetero-oligomeric complexes with TbetaR-I and TbetaR-II in the presence of TGF-beta (11, 26, 27), the lack of heparan sulfate in TbetaR-III* (in CHO-677 cells) may affect the formation of the hetero-oligomeric complexes of TbetaR-III/TbetaR-II/TbetaR-I, which contain different percentages of TbetaR-I and TbetaR-II (28). To test this possibility, we performed immunoprecipitation using specific antibodies to TbetaR-I, TbetaR-II, and TbetaR-III following 125I-TGF-beta1 affinity labeling of CHO-677 and CHO-K1 cells. As shown in Fig. 4, CHO-677 cells exhibited more 125I-TGF-beta1 affinity-labeled TbetaR-II in the TGF-beta receptor complexes when compared with CHO-K1 cells (lanes 2, 4, and 6 versus lanes 1, 3, and 5).


Figure 2
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  		FIGURE 2.
125I-TGF-beta1 affinity labeling of TGF-beta receptors in CHO-K1 and CHO-677 cells. Cell-surface TGF-beta receptors in CHO-K1 cells, CHO-677 cells, and CHO-LRP-1– cells (which are CHO mutant cells lacking TbetaR-V) were affinity-labeled with 125I-TGF-beta1 (100 pM)(A) or increasing concentrations of 125I-TGF-beta1, as indicated (B and C) by cross-linking with DSS after incubation of cells with 125I-TGF-beta1. The cell lysates of 125I-TGF-beta1 affinity-labeled cells were analyzed by 7.5% (A and C) and 5% (B) SDS-PAGE under reducing conditions and autoradiography. The brackets indicate the locations of the 125I-TGF-beta1·TbetaR-I, 125I-TGF-beta·TbetaR-II, 125I-TGF-beta1·TbetaR-III*, and 125I-TGF-beta1·TbetaR-III complexes. The relative intensities of these complexes were quantified with a PhosphorImager (A, panel b; B, panel b; C, panel b; and C, panel c) and plotted against the concentrations of 125I-TGF-beta1 used for affinity labeling (B, panel b; C, panel b; and C, panel c). The ratios of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I in cells incubated with TGF-beta1 (100 pM) (A) or increasing concentrations of 125I-TGF-beta1 (B and C) were estimated and are shown in A, panel c, and C, panel d, respectively.

 


Figure 3
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  		FIGURE 3.
Western blot analysis of TbetaR-I and TbetaR-II in CHO-K1 and CHO-677 cells. Cells were grown to confluence on 24-well cluster dishes in DMEM-F-12 medium. The cell lysates (200 µg of protein) were analyzed by Western blot analysis. The brackets indicates the locations of TbetaR-I, TbetaR-II, and beta-actin.

 
Enzymic Removal of Heparan Sulfate Increases the Ratio of TGF-beta1 Binding to TbetaR-II and TbetaR-I and Augments TGF-beta-induced PAI-1 Expression in Mv1Lu Cells—To see if absent or decreased heparan sulfate alters TGF-beta1 binding to TbetaR-I and TbetaR-II and resultant TGF-beta responsiveness in other epithelial cell systems, mink lung epithelial cells (Mv1Lu cells) were treated with heparitinase for 3 h at 37°C or with a heparan sulfate biosynthesis inhibitor p-nitrophenyl-beta-D-xylopyranoside for 72 h at 37 °C and then affinity-labeled with 125I-TGF-beta1 at 0 °C. The 125I-TGF-beta1 affinity-labeled TGF-beta receptors were analyzed by 7.5% (Fig. 5A) and 5% (Fig. 5B) SDS-PAGE under reducing conditions and autoradiography. Heparitinase or p-nitrophenyl-beta-D-xylopyranoside treatment of the cells appeared to convert ~270–350-kDa TbetaR-III to fast-migrating forms (Fig. 5B, lanes 2 and 3 versus lane 1). Accordingly, heparitinase or p-nitrophenyl-beta-D-xylopyranoside treatment resulted in the increase of 125I-TGF-beta1 binding to TbetaR-II (Fig. 5, A–C) and TbetaR-V (Fig. 5B, lanes 2 and 3 versus lane 1). The ratios of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I in cells treated with vehicle only, heparitinase and p-nitrophenyl-beta-D-xylopyranoside were estimated to be ~0.6, ~1.6, and ~1.1, respectively (Fig. 5D). Heparitinase or p-nitrophenyl-beta-D-xylopyranoside treatment did not affect the protein levels of TbetaR-I and TbetaR-II in Mv1Lu cells as determined by Western blot analysis (data not shown). These results suggest that enzymic removal or synthesis inhibition of heparan sulfate increases the ratio of 125I-TGF-beta1 affinity-labeled TbetaR-II and TbetaR-I in Mv1Lu cells as observed in CHO-677 cells (versus CHO-K1 cells). To see whether heparitinase treatment affects TGF-beta1-induced cellular responses in Mv1Lu cells, the cells were treated with increasing concentrations of TGF-beta1 following treatment with heparitinase. The PAI-1 expression was then determined by Northern blot analysis. As shown in Fig. 6, A and B, heparitinase treatment augmented TGF-beta1-induced expression of PAI-1 in Mv1Lu cells. The half-maximum concentration (ED50) of TGF-beta1 to induce PAI-1 expression are similar (~4–5 pM) in cells treated with and without heparitinase. This is consistent with the observation that the deficiency of heparan sulfate causes increase and decrease of TGF-beta1 binding to TbetaR-II and TbetaR-I, respectively, without altering the affinities of TGF-beta1 binding to TbetaR-II and TbetaR-I in CHO-677 cells. These results support the hypothesis that the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I positively correlates with the magnitude of TGF-beta1-induced cellular responses (28).


Figure 4
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  		FIGURE 4.
Immunoprecipitation of 125I-TGF-beta1 affinity-labeled TGF-beta receptor complexes in CHO-K1 and CHO-677 cells. Cells were affinity-labeled with 100 pM 125I-TGF-beta1 in the presence of DSS. The 125I-TGF-beta1 affinity-labeled cell lysates were subjected to immunoprecipitation with specific antibodies to TbetaR-I, TbetaR-II, and TbetaR-III ({alpha}-TbetaR-I, {alpha}-TbetaR-II, and {alpha}-TbetaR-III, respectively). The immunoprecipitates were then analyzed by 7.5% SDS-PAGE under reducing conditions and autoradiography. The brackets indicate the locations of 125I-TGF-beta1·TbetaR-I, 125I-TGF-beta1·TbetaR-II, and 125I-TGF-beta1·TbetaR-III* complexes. The 125I-TGF-beta1·TbetaR-III complex migrated at the top of the separating gel.

 


Figure 5
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  		FIGURE 5.
Effect of treatment with heparintinase or a heparan sulfate synthesis inhibitor on 125I-TGF-beta1 binding to TGF-beta receptors in Mv1Lu cells. Cells were treated with heparitinase at 37 °C for 3 h or with a heparan sulfate synthesis inhibitor p-nitrophenyl-beta-D-xylopyranoside (inhibitor) at 37 °C for 72 h. After treatment, cell-surface TGF-beta receptors were affinity-labeled with 125I-TGF-beta1 (100 pM) in the presence of DSS. 125I-TGF-beta1 affinity-labeled TGF-beta receptors were then analyzed by 7.5 (A) and 5% (B) SDS-PAGE under reducing conditions and autoradiography. The brackets indicate the locations of 125I-TGF-beta1 affinity-labeled TbetaR-I, TbetaR-II, TbetaR-III*, TbetaR-III, and TbetaR-V. The relative intensities of 125I-TGF-beta1·TbetaR-I and 125I-TGF-beta1·TbetaR-II complexes were quantified with a PhosphorImager (C). The ratios of 125I-TGF-beta1·TbetaR-II and 125I-TGF-beta1·TbetaR-I complexes were estimated in cells treated with vehicle only, heparitinase, or p-nitrophenyl-beta-D-xylopyranoside (D).

 
Cells Lacking Heparan Sulfate or Treated with Heparitinase Exhibit Decreased Cellular Degradation of TGF-beta1—TGF-beta receptor-mediated signaling is known to occur in endosomes (29–31). The TGF-beta receptor complex with a higher ratio of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I (e.g. in CHO-677 cells and Mv1Lu cells treated with heparitinase) is hypothesized to undergo clathrin-mediated endocytosis and transduces signaling in endosomes (28–31). On the other hand, the TGF-beta receptor complex with a low ratio (<1) of 125I-TGF-beta1 binding to TbetaR-II and TbetaR-I (e.g. in CHO-K1 and Mv1Lu cells) is hypothesized to undergo rapid degradation via caveolae/lipid-raft-mediated endocytosis (28–31). To test this hypothesis, we examined the cellular degradation of 125I-TGF-beta1 bound to cell-surface receptors in CHO-677 cells, CHO-K1 and Mv1Lu cells. As shown in Fig. 7, 125I-TGF-beta1 bound to cell-surface TGF-beta receptors underwent rapid degradation, as determined by measurement of specific trichloroacetic acid-soluble radioactive material in the conditioned medium, in wild-type CHO cells (Fig. 7A) as compared with CHO-677 cells (Fig. 7B). The cellular degradation of 125I-TGF-beta1 bound to cell-surface TGF-beta receptors appeared to be inhibited by nystatin in CHO-K1 and Mv1Lu cells but not in CHO-677 cells (Fig. 7, A and C versus B). Nystatin is a cholesterol-binding compound and has been used to inhibit caveolae/lipid-raft-mediated endocytosis/degradation (29–32). This result suggests that caveolae/lipid-raft-mediated endocytosis is involved in rapid degradation of TGF-beta receptor complexes in CHO-K1 and Mv1Lu cells, whereas clathrin-mediated endocytosis/signaling/degradation, which is not inhibited by nystatin, mainly occurs in CHO-677 cells. To test this, we performed sucrose density gradient analysis of TbetaR-I and TbetaR-II in CHO-K1, CHO-677, and Mv1Lu cells. As shown in Fig. 8, a significant fraction of TbetaR-I and TbetaR-II was localized in caveolae/lipid-raft fractions (fractions 4 and 5) in CHO-K1 and Mv1Lu cells. The TbetaR-I localization in Mv1Lu cells was not shown because the antibody to TbetaR-I used in the experiment did not react well with mink TbetaR-I antigen. By contrast, the majority of TbetaR-I and TbetaR-II were localized in non-lipid-raft fractions (fractions 7 and 8) in CHO-677 cells. These results are consistent with the notion that caveolae/lipid-raft-mediated endocytosis facilitates rapid degradation of TGF-beta and attenuates TGF-beta responsiveness (28–31).


Figure 6
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  		FIGURE 6.
Effect of treatment with heparitinase on TGF-beta1-induced PAI-1 expression in Mv1Lu cells. Cells were treated with heparitinase as described above. After the treatment, cells were further treated with several concentrations of 125I-TGF-beta1 (0, 5, 10, and 20 pM). After 3 h at 37°C, the PAI-1 expression was determined by Northern blot analysis (A). The expression of G3PDH was used as the control. The relative amount of the PAI-1 transcript was estimated based on the ratio of the relative intensities of PAI-1: G3PDH and was taken as 1.0 in cells treated with vehicle only (B).

 


Figure 7
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  		FIGURE 7.
Cellular degradation of receptor-bound 125I-TGF-beta1 in CHO-K1 (A), CHO-677 (B), and Mv1Lu (C) cells. Cells were pretreated with nystatin (25 µg/ml) at 37 °C for 1 h and then incubated with 125I-TGF-beta1 (100 pM) in the presence and absence of 100-fold excess unlabeled TGF-beta1 (for estimating nonspecific binding of 125I-TGF-beta1). After 2.5 h at 0 °C, cells were washed and warmed to 37 °C. After several time periods (0.5, 1, and 1.5 h) in the presence or absence of nystatin (25 µg/ml), 10% trichloroacetic acid-soluble radioactive material (derived from the specific binding of 125I-TGF-beta1) in the medium was determined and expressed as the percentage of the total specific binding of 125I-TGF-beta1 at the cell surface determined before incubation at 37 °C. Values in the bar charts are mean ± S.D. (n = 4). *, p < 0.001, nystatin-treated cultures versus vehicle-treated cultures.

 


Figure 8
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  		FIGURE 8.
Sucrose density gradient analysis of TbetaR-I and TbetaR-II in CHO-K1, CHO-677, and Mv1Lu cells. Cells were subjected to sucrose density gradient ultracentrifugation as described (24). The fractions were analyzed by Western blot analysis using antibodies to TbetaR-I, TbetaR-II, and caveolin-1. The arrows indicate the locations of TbetaR-I, TbetaR-II, and caveolin-1. Fractions 4 and 5 were caveolar/lipid-raft fractions whereas fractions 7 and 8 were non-lipid-raft (or clathrin) fractions.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Members of the TGF-beta family are among the most potent growth factors or cytokines known. They are active at subpicomolar concentrations. TGF-beta is regulated at several levels including: 1) transcription (1–3, 10), 2) activation of the latent form of TGF-beta (33, 34), 3) endocytosis of TGF-beta receptor complexes (28–31), and 4) post-TGF-beta receptor signaling (35–38). Among these, the molecular mechanisms underlying the regulation of endocytosis and signaling of TGF-beta receptor complexes have been less studied until recently. TGF-beta-induced signaling has recently been shown to occur in endosomes (29–31). Potassium chloride depletion, which inhibits clathrin-mediated endocytosis, has been shown to attenuate TGF-beta receptor internalization and TGF-beta-induced cellular responses in Mv1Lu cells (29–31). TGF-beta receptors at the cell surface undergo ligand-independent internalization and recycling via clathrin-coated and caveolin-positive vesicles (28–31). In the absence of ligand, TGF-beta receptors undergo constitutive internalization and recycling (30). Following ligand binding, ligand-activated TGF-beta receptor complexes are internalized into endosomes (where they mediate signaling) and caveolin-positive vesicles (where they are subjected to rapid degradation). The biochemical properties of TGF-beta receptor complexes designated for endosomes and caveosomes (caveolin-1-positive vesicles) are unknown (29–31). Here we demonstrate that cells defective in biosynthesis of heparan sulfate or treated with a heparan sulfate biosynthesis inhibitor respond more strongly to TGF-beta1-induced cellular responses than wild-type cells or untreated cells. These cells exhibit a higher ratio (>1) of TGF-beta1 binding to TbetaR-II and TbetaR-I and a decrease of degradation of TGF-beta1 bound to TGF-beta receptors as compared with those observed in wild-type or untreated cells. These results support the hypothesis that the ratio of TbetaR-II and TbetaR-I in the TGF-beta receptor complex provides a signal for determining TGF-beta responsiveness (28).

A model is proposed to demonstrate how the formation of distinct TGF-beta receptor complexes at the cell surface determine the cellular response to TGF-beta1. In this model (Fig. 9) modified from the models reported previously (28, 29), TbetaR-III presents TGF-beta1 to TbetaR-II at the cell surface. TbetaR-I is recruited to form TbetaR-III·TbetaR-II·TbetaR-I ternary complexes that have two forms: I and II. Complex I, which contains more TbetaR-II than TbetaR-I (as determined by 125I-TGF-beta1 affinity labeling), undergoes clathrin-mediated endocytosis and transduces signaling in endosomes. Complex II, which contains more TbetaR-I than TbetaR-II, undergoes caveolae/lipid-raft-mediated endocytosis and rapid degradation. The formation of these complexes can be regulated by the following: 1) the proteoglycan moiety of TbetaR-III: larger proteoglycan moieties in TbetaR-III facilitate the formation of Complex II (13), and no proteoglycan or a small proteoglycan moiety in TbetaR-III facilitates the formation of Complex I (13); 2) altered expression of endoglin: endoglin is a TGF-beta-binding, proteoglycan-containing membrane protein and shares with TbetaR-III a limited amino acid sequence homology (39), and the increased expression of endoglin facilitates the formation of Complex II, leading to the attenuation of TGF-beta-induced cellular responses (40); and 3) altered expression of TbetaR-I or TbetaR-II. Increased expression of TbetaR-II or TbetaR-I facilitates the formation of Complex I or II and enhances or attenuates TGF-beta1-induced cellular responses, respectively (41–43). Blobe et al. (12) reported that stable transfection of L6 myoblasts with TbetaR-III cDNA yields a higher ratio (>1) of TGF-beta binding to TbetaR-II and TbetaR-I and enhances TGF-beta responsiveness. Eickelberg et al. (13) demonstrated that, in LLC-PK (which are epithelial cells, unlike L6 myoblasts), overexpression of TbetaR-III produces a lower ratio (<1) of TGF-beta binding to TbetaR-II and TbetaR-I and attenuates TGF-beta responsiveness. Since TbetaR-III in LLC-PK1 cells exhibits higher molecular weight glycosaminoglycan chains than that in L6 cells, they suggested that the sizes of glycosaminoglycan chains in the TbetaR-III molecule affect the ratio of TGF-beta binding to TbetaR-II and TbetaR-I and thus affect TGF-beta responsiveness. However, which proteoglycan (heparan sulfate or chondroitin sulfate) is involved in modulating TGF-beta responsiveness has not previously been determined. Here we demonstrate that epithelial cells deficient in heparan sulfate or treated with heparitinase or a heparan sulfate biosynthesis inhibitor exhibit a higher ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I, decreased degradation of TGF-beta1, and enhanced TGF-beta1 responsiveness. This suggests that heparan sulfate negatively modulates TGF-beta1 responsiveness by facilitating formation of Complex II in epithelial cells.


Figure 9
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  		FIGURE 9.
Heparan sulfate modulation of TbetaR-I·TbetaR-II complex (Complex I and Complex II) formation in epithelial cells. According to the dominance hypothesis (28), there are two major TbetaR-I·TbetaR-II complexes (Complex I and Complex II) present on the cell surface. Complex I contains more TbetaR-II than TbetaR-I, whereas TbetaR-II is less than TbetaR-I in Complex II. The ratio of TbetaR-I and TbetaR-II in the complexes can be determined by 125I-TGF-beta affinity labeling (as shown on the top of the figure). Complex I preferentially undergoes clathrin-mediated endocytosis of TGF-beta, resulting in promoting signaling (e.g. Smad protein phosphorylation) and cellular responsiveness. Complex II mainly undergoes caveolae/lipid-raft-mediated endocytosis, resulting in rapid degradation of TGF-beta and less cellular responsiveness. In epithelial cells expressing heparan sulfate (HS) (e.g. CHO-K1 and Mv1Lu cells), the majority of the TbetaR-I/TbetaR-II complexes are present as Complex II, which exhibits nystatin-inhibitable endocytosis and degradation of TGF-beta. In epithelial cells lacking heparan sulfate (e.g. CHO-677 cells and Mv1Lu cells treated with heparitinase), the TbetaR-I·TbetaR-II complexes mostly exist as Complex I, which exhibits nystatin-uninhibitable endocytosis, signaling, and degradation of TGF-beta.

 
In murine embryonic fibroblasts, the TbetaR-III gene ablation does not significantly affect the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I in TbetaR-III-null murine embryonic fibroblasts (44), which is >1.0.4 This suggests that the formation of Complex I (with a high ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I) is a default process and that the formation of Complex II (e.g. induced by heparan sulfate) is a regulatory event. TGF-beta is known to stimulate the expression of proteoglycans, which may play a feedback regulatory role in TGF-beta actions (45, 46). Certain carcinoma cells may acquire resistance to TGF-beta growth suppression by up-regulation of heparan sulfate expression (47–49).


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-977-9250; Fax: 314-977-9205; E-mail: huangjs{at}slu.edu.

2 The abbreviations used are: TGF, transforming growth factor; MES, [2-(N-morpholine)ethanesulfonic acid); CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; DSS, disuccinimidyl suberate; PAI-1, plasminogen activator inhibitor-1; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. Back

4 C.-L. Chen, S. S. Huang, and J. S. Huang, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. William S. Sly and Jeffrey H. Grubb for providing CHO-K1 and CHO-677 cells, Dr. Frank E. Johnson for critical review of the manuscript, and John McAlpin for typing the manuscript.



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 DISCUSSION
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