Mutation in the Heparan Sulfate Biosynthesis Enzyme EXT1 Influences Growth Factor Signaling and Fibroblast Interactions with the Extracellular Matrix

Abstract

Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including members of the fibroblast growth factor (FGF) and platelet-derived growth factor families. We previously reported the structure of HS synthesized by embryonic fibroblasts from mice with a gene trap mutation of Ext1 that encodes a glycosyltransferase involved in HS chain elongation. The gene trap mutation results in low expression of Ext1, and, as a consequence, HS chain length is substantially reduced. In the present study, Ext1 mutant and wild-type mouse embryonic fibroblasts were analyzed for the functional consequences of the Ext1 mutation for growth factor signaling and interaction with the extracellular matrix. Here, we show that the phosphorylation of ERK1/2 in response to FGF2 stimulation was markedly decreased in the Ext1 mutant fibroblasts, whereas neither PDGF-BB nor FGF10 signaling was significantly affected. Furthermore, Ext1 mutants displayed reduced ability to attach to collagen I and to contract collagen lattices, even though no differences in the expression of collagen-binding integrins were observed. Reintroduction of Ext1in the Ext1 mutant fibroblasts rescued HS chain length, FGF2 signaling, and the ability of the fibroblasts to contract collagen. These data suggest that the length of the HS chains is a critical determinant of HS-protein interactions and emphasize the essential role of EXT1 in providing specific binding sites for growth factors and extracellular matrix proteins.

Introduction

Heparan sulfate proteoglycans (HSPGs)², composed of a core protein and one or more negatively charged heparan sulfate chains, are ubiquitous components of the extracellular matrix and cell surfaces. As co-receptors for numerous growth factors and cytokines, HS proteoglycans modify cell behavior and regulate cell growth and thus play vital roles in various biological processes, such as cell proliferation and tissue morphogenesis (reviewed in Ref. 1). The ability of HS to exert its function is embedded in the fine structure of HS chains that seems to be tightly regulated between cell types (2), during development (3, 4), aging (5), and in certain pathological conditions (6–8).

The biosynthesis of HS is a complex process that occurs in the Golgi network (reviewed in Refs. 9–11). Chain elongation is initiated by the formation of a tetrasaccharide linkage region composed of glucuronic acid-galactose-galactose-xylose with xylose covalently bound to a serine residue in the proteoglycan core protein. After the addition of a single N-acetylglucosamine (GlcNAc) residue, elongation proceeds by alternating addition of GlcNAc and glucuronic acid (GlcA) to the nonreducing end of the growing polymer. As the chain polymerizes, it undergoes a series of partial modifications, initiated by removal of the N-acetyl group from selected GlcNAc residues, and substitution of the generated free amino groups with sulfate groups. Subsequent modification reactions occur primarily in these N-sulfated regions. They include partial C5-epimerization of GlcA to iduronic acid and addition of sulfate groups at different positions.

The elongation of HS chains is mediated by the action of two members of the exostosin protein family, EXT1 and EXT2 (12, 13). Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human autosomal dominant genetic disorder, which manifests with short stature and bony capped outgrowths, predominantly originating from the epiphyseal plates at the distal ends of long bones (14). The mechanism by which exostoses develops is poorly understood, but mutations in either EXT1 or EXT2 and the resulting reduction or absence of HS in the exostosis cartilage cap has been implicated in disturbed signaling responses in exostoses chondrocytes (15, 16). Mice carrying a hypomorphic mutation in Ext1 generated by gene trapping (Ext1^Gt/Gt) (17) die by embryonic day 14.5 (18). In contrast, Ext1-deficient mice generated by gene targeting lack HS, fail to gastrulate, and die at around embryonic day 8.5 (19). The longer survival time of the Ext1^Gt/Gt mice may be explained by the fact that they still produce small amounts of EXT1, which results in synthesis of short but apparently normally sulfated HS chains. The embryonic lethal phenotype of Ext1^Gt/Gt may reflect the inability of the mutant HS to mediate some but not all HS-dependent steps in embryogenesis. The mutant chains could either be too short to bind the protein ligand, or the chains may not be able to properly present the growth factor to its high affinity kinase receptor (20).

Several growth factors, including the fibroblast growth factors (FGFs), depend on HSPGs for cell-signaling activity (21). FGFs comprise a family of 23 structurally related members and are essential for normal embryonic development. FGFs act through tyrosine kinase receptors (FGFRs) on a wide range of cell types, such as fibroblasts, endothelial cells, and other cell types. The formation of FGF·HSPG·FGFR ternary complexes at the cell surface leads to activation and phosphorylation of the receptor tyrosine kinase that triggers various intracellular signaling cascades, including the MAPK pathway (22–24). Studies of ternary complex formation at different stages of mouse embryo development suggest that different FGF-FGFR pairs require distinct HS structures for complex formation (25). The binding of FGF2 to HS is the best characterized of the different members of the FGF family. FGF2 binds to HS on the cell surface, and this binding facilitates and stabilizes FGF2 binding to its cell surface receptor (26). HS also mediates binding to various extracellular matrix proteins, including fibronectin, laminin, and different types of collagen including the fibrillar collagen type I. HS interaction with collagen I promote cell attachment (27) and angiogenesis (28). In this study, we investigated how the Ext1 gene trap mutation affects fibroblast growth factor signaling and interaction with collagen I.

EXPERIMENTAL PROCEDURES

Cell Culture

Sv40-immortalized mouse embryonic cell lines derived from wild-type and Ext1 gene-trapped (Ext1^{Gt(pGT2TMpfs)064Wcs}, Ext1^Gt/Gt; Ref. 17) embryonic day 11.5 mouse embryos were prepared as described previously (20). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin (PAA Laboratories). For re-expression of Ext1 in Ext1^Gt/Gt fibroblasts, a full-length mouse Ext1 cDNA cloned into the pBudCE4.1 vector (29) was transfected into Ext1^Gt/Gt fibroblasts using the Nucleofector electroporation kit for adherent cells (Amaxa). Cell clones stably expressing the Ext1 expression constructs were selected using 0.5 mg/ml of Zeocin (Invitrogen). Three individual cell clones (referred to as cl.1.4, cl.4.7, and cl.4.8) stably expressing Ext1 were identified by flow cytometry after staining with the 10E4 antibody and were subsequently used in further experiments.

Isolation of Metabolically Labeled HS

Ext1 gene trap (Ext1^Gt/Gt) and wild-type immortalized embryonic fibroblasts were cultured as described above. 80% confluent cells were labeled for 24 h with 200 μCi/ml Na₂³⁵SO₄, or 50 μCi/ml [³H]glucosamine. The medium was collected and saved, and the cells were washed twice with phosphate-buffered saline (PBS), treated with 1 mg/ml trypsin (Sigma) for 10 min at 37 °C and were then centrifuged at 16,000 × g for 1 min. The cell pellets were saved, and the supernatants were treated with 0.5 m NaOH at 4 °C overnight to release O-linked saccharides from the protein core. After neutralization samples were diluted with H₂O and thereafter applied onto anion-exchange DEAE-Sephacel (Amersham Biosciences) columns. Columns were washed in: 0.15 m NaCl, 50 mm Tris, pH 7.2, 0.1% Triton X-100; 0.15 m NaCl, 50 mm Na-acetate, pH 4.0; 0.15 m NaCl, 50 mm Tris, pH 7.2, 0.1% Triton X-100; and H₂O; and finally, glycosaminoglycans were eluted with 2 m NaCl and desalted using a PD-10 column (GE Healthcare). Galactosaminoglycans were digested with chondroitinase ABC (Seikagaku Corporation) and HS chains analyzed by gel chromatography.

Flow Cytometry

Ext1^Gt/Gt and wild-type fibroblasts were harvested using trypsin/EDTA and washed twice in cold PBS. An aliquot of the cells was resuspended in 1 ml serum-free DMEM containing 20 milliunits of heparitinase I and II (Seikagaku Corporation) and incubated at room temperature for 2 h. After washing with PBS, heparitinase-treated and -untreated cells were incubated with the primary monoclonal anti-HS antibody 10E4 (1:50) or 3G10 (1:50) (both from Seikagaku Corporation) at 4 °C for 30 min. After washing with PBS three times, the cells were incubated with the secondary antibodies: allophycocyanin-conjugated goat anti-mouse IgG for 10E4 and fluorescein isothiocyanate-conjugated goat anti-mouse IgG and IgM (both from Jackson ImmunoResearch Laboratories, Inc.) for 3G10. Wild-type cells incubated with the secondary antibodies alone served as negative controls. For analysis of integrin expression, antibodies against rat/mouse α1 (CD49a, clone Ha31/8), rat α2 (CD49b, clone Ha1/29), and mouse β₁ (CD29, clone 9EG7) were used (BD Biosciences). For analysis of integrin α11, a rabbit polyclonal antibody was used as described (30). Secondary antibodies were: Cy3-conjugated goat anti-hamster IgG for α1 and α2, DyeLight^TM488-conjugated goat anti-rat IgG for β₁ and Cy3-conjugated goat anti-rabbit for α11, all from Jackson ImmunoResearch Laboratories, Inc. Fluorescence was measured using a FACSCalibur or FACSAria flow cytometer (BD Biosciences) and analyzed with the FlowJo software (Tree Star, Inc.).

Analysis of Growth Factor Signaling

Subconfluent cell cultures grown in 12-well plates were washed with serum-deprived DMEM and starved for 18 h in the same medium. Growth factors (FGF2, PDGF-BB, and FGF10; all from R&D Systems) were added at different concentrations (ranging from 0.2–100 ng/ml). After incubation for 10 min at 37 °C, the medium was discarded, and the cells were solubilized by addition of 200 μl of boiling nonreducing SDS sample buffer. Equal amounts of cell extracts were separated on two separate SDS-10% polyacrylamide gels under nonreducing conditions and electrotransferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in 5% skimmed milk in Tris-buffered saline, 0.5% Tween 20 for 1 h at room temperature. One of the membranes was incubated with mouse anti phospho-ERK (1:2,000, Cell Signaling Technology, Inc.) recognizing the activated, phosphorylated kinase, and the other membrane was incubated with mouse anti-total ERK-2 (1:5,000, Santa Cruz) recognizing both the unphosphorylated and phosphorylated kinase. The membranes were developed using ECL reagent (Pierce), and the bands were visualized and quantified with the BioImager (Bio-Rad) using Quantity One software (Bio-Rad).

Glycosyl Transferase Activity Assay

GlcNAc and GlcA transferase activities were measured by incubating enzyme protein preparations with ¹⁴C-labeled UDP sugars (62.5 μCi/μmol; prepared by mixing radiolabeled and unlabeled UDP sugars) and oligosaccharide acceptors as described (31). Protein concentrations were determined using the BCA protein assay (Pierce).

Cell Attachment

Cells were trypsinized, washed three times in Puck's saline containing Mg²⁺ and Ca²⁺, seeded at a concentration of 10⁵ cells/well in 48-well plates precoated overnight with varying concentrations of collagen I. The cells were allowed to attach for 55 min at 37 °C. The plates were then washed three times in Puck's saline. The cells were fixed with 96% EtOH and stained with 0.1% crystal violet for 30 min. After washing under running tap water, cells were lysed by incubation for 30 min with 100 μl of 1% Triton X-100/well. Cell attachment was measured colorimetrically in a Versamax microplate reader (Molecular Devices) at 595 nm. In heparin blocking experiments, wells were precoated with 5 μg/ml of collagen, and the cells were incubated with heparin (50 μg/ml) on ice for 15 min before plating; cell attachment to collagen was analyzed as described above.

Cell Proliferation Assay

Cell proliferation was determined by [³H]thymidine incorporation. 50,000 cells/well were seeded in a 24-well plate. After 24 h in culture, cells were subject to starvation for 24 h in serum-free DMEM. Then, FGF2 or FGF10 was added to cells at a final concentration of 10 ng/ml for another 24 h. Cells incubated with serum-free DMEM alone served as a negative control. During the last 4 h of growth factor stimulation, [³H]thymidine (1 μCi/ml; specific activity, 6.7 Ci/mmol; Perkin Elmer Life Sciences) was added to the cells. Cells were harvested by trypsinization and washed three times in cold PBS. After precipitation with 5% trichloroacetic acid, the cell pellets were dissolved in 0.5 m NaOH containing 0.5% SDS. The amount of incorporated [³H]thymidine was determined by scintillation counting.

Collagen Gel Contraction Assay

Wild-type and Ext1^Gt/Gt fibroblasts were trypsinized, washed, and resuspended in serum-free DMEM containing 2 mm glutamine and antibiotics. The cell suspension was mixed with a collagen I solution (1.2 mg/ml in DMEM with 20 μm Hepes, pH 8.0) to a final concentration of 10⁵ cells/ml. 100 μl aliquots of the resulting solution were transferred to a 96-well plate precoated with 2% bovine serum albumin and incubated at 37 °C for ∼1 h to allow collagen polymerization. Collagen gels were detached by the addition of 100 μl DMEM or 100 μl DMEM containing 10 ng/ml of FGF2. Gels were further incubated at 37 °C, and gel diameters were measured microscopically at successive time points up to 10 h. In a separate series of experiments, 100 μl DMEM or DMEM containing 50 μg/ml heparin or a 5 μg/ml of a blocking antibody (Ha2/5, BD Biosciences) directed against the integrin β1-subunit, were added to the gels at the time of detachment, and the gels were further incubated at 37 °C; gel diameters were measured as above.

RESULTS

FGF2- but Not PDGF-BB- or FGF10-induced Activation of ERK1/2 Is Reduced in Ext1^Gt/Gt Fibroblasts

Previously, we have shown that the HS chains produced by Ext1^Gt/Gt mouse embryonic fibroblasts are shorter than those of the wild-type fibroblasts (20). HS acts as an important co-receptor for several growth factors and to determine whether the reduction in chain length on the Ext1^Gt/Gt cells had a biological effect, we examined the ability of wild-type and mutant cells to respond to various growth factors. FGF2 activated both wild-type and Ext1^Gt/Gt mutant cells in a dose-dependent manner, as determined by the levels of phosphorylated p44/42 (ERK1/2) (Fig. 1A). Comparisons at the highest levels of phosphorylation in the wild-type cells, showed that the relative values of pERK1/2 were decreased by ∼75% in the Ext1^Gt/Gt mutant cells. In contrast, Ext1^Gt/Gt mutant and wild-type cells showed no striking difference in FGF10- or PDGF-BB-stimulated ERK1/2 phosphorylation (Fig. 1, B and C), even though some variations in the response to high concentrations of PDGF-BB (100 ng/ml) were observed in the mutant cells between different experiments.

FIGURE 1.

Signaling response of wild-type and Ext1^Gt/Gt fibroblasts after stimulation with different growth factors. Ext1^Gt/Gt and wild-type cells mutant cells were rinsed in serum-free medium and starved in serum-free medium for 18 h and then stimulated with FGF2 (A), PDGF-BB (B), or FGF10 (C) at the indicated concentrations. After 10 min, cells were lysed, and the cell lysates were subjected to SDS-PAGE followed by transfer to nitrocellulose membranes and immunoblotting with phosphospecific (p) ERK and total ERK antibodies. The left panels show the relative intensity of immunoreactive bands of phosphorylated versus total ERK. The right panels show corresponding Western blot analyses. Compared with wild-type, the mutated cells displayed a strongly reduced phosphorylation of ERK in response to FGF2, whereas phosphorylation of ERK mediated by PDGF-BB or FGF10 was similar in both cell types. The figures show representative results from one of three independent experiments. Wild-type (wt) cells (black bars); Ext1^Gt/Gt cells (white bars).

Reduced Binding of HS-specific Antibodies to Ext1^Gt/Gt Fibroblasts

To directly test whether the gene trap mutation in Ext1 alters ligand binding to cell surface HS, we used flow cytometry to quantify the binding of the HepSS1 and 10E4, two antibodies that recognize sulfation patterns within HS chains (32, 33). Both 10E4 and HepSS1 antibodies showed a markedly reduced binding to Ext1^Gt/Gt cells relative to wild-type cells. The median channel fluorescence (MCF) values after staining with 10E4 were 270 for the mutant and 731 for the wild-type cells (Fig. 2A). In contrast, no significant difference in the binding of the 3G10 antibody could be observed between the wild-type and mutant cells after digestion with a combination of heparitinase I and II (Fig. 2C), MCF_Ext1^Gt/Gt = 433 and MCF_wild-type = 448. The 3G10 antibody does not react with intact HS but recognizes the unsaturated hexuronic acid-containing oligosaccharide stubs that remain associated with core proteins after heparitinase digestion (32). Therefore, 3G10 serves as a marker of the amount of HS chains without providing information about their structure. Thus, our results indicate that the wild-type and the mutant cells have similar numbers of HS chains exposed at the cell surface but that the HS chains on the mutant cells contain less binding epitopes for the HepSS1 and 10E4 antibodies.

FIGURE 2.

Ext1^Gt/Gt fibroblasts have reduced amounts of cell surface HS. Ext1^Gt/Gt and wild-type cells were examined for cell surface expression of HS by flow cytometry using the 10E4 and 3G10 antibodies. Compared with wild-type cells, the mutant cells showed a strongly reduced binding to 10E4 (A) but similar binding to 3G10 before (B) and after (C) heparitinase (Hep) digestion, indicating that the wild-type and mutant fibroblasts have similar numbers of HS chains. The histograms show the intensity of the fluorescence (% of max) and are representative of three independent experiments. Ext1^Gt/Gt cells (black line); wild-type cells (filled black profile); control (using secondary antibody only) (filled gray profile). The binding profiles of the HepSS1 antibody to cell surface HS (data not shown) were similar to the binding profiles shown for 10E4 in A.

Attachment to Collagen and Collagen Gel Contraction Is Attenuated in Ext1^Gt/Gt Fibroblasts

Normal tissue repair involves migration of fibroblasts into the wound followed by tissue remodeling and repair. Cell surface HSPGs are important in mediating the adhesion to the extracellular matrix. To analyze the impact of the Ext1 mutation on cell-matrix interactions, we compared the attachment of wild-type and mutant fibroblasts to collagen I. At low collagen concentrations, the attachment of Ext1^Gt/Gt mutant cells to collagen was reduced ∼20–30% as compared with wild-type cells. At higher collagen concentrations, there was an increased attachment of wild-type cells, which was not observed to the same extent for Ext1^Gt/Gt fibroblasts (Fig. 3).

FIGURE 3.

Ext1^Gt/Gt fibroblasts exhibit reduced attachment to collagen I. Ext1^Gt/Gt and wild-type fibroblasts were allowed to adhere to different concentrations of immobilized collagen and the number of adherent cells determined as described under “Experimental Procedures.” The absorbance reflects the number of attached cells. The numbers of attached Ext1^Gt/Gt fibroblasts (white bars) were reduced compared with wild-type cells (black bars), both at high and low collagen concentrations. The graph shows one of five representative experiments, and each experimental condition was measured in duplicate.

Because the attachment of Ext1^Gt/Gt cells to collagen was reduced, we next investigated the contractile capacity of wild-type and Ext1^Gt/Gt cells to determine whether the mutation in Ext1 influenced cell-mediated remodeling of three-dimensional collagen matrices. Wild-type and Ext1^Gt/Gt mutant fibroblasts were embedded in collagen gels, and the diameter of the contracted collagen gel was compared with the initial gel diameter. Wild-type cells contracted the collagen gel within hours and reached a maximum contraction toward the end of the observation period at 10 h (Fig. 4A). In contrast, Ext1^Gt/Gt fibroblasts showed a considerable decrease in both the initial and overall resultant contraction of collagen matrix as compared with wild-type controls (Fig. 4A). Wild-type cells contracted the gels to ∼35% of the initial size, whereas the corresponding value for the mutant cells was ∼75%. Addition of FGF2 stimulated the contraction of collagen gels by both wild-type and Ext1^Gt/Gt cells (Fig. 4B). However, although gel contraction by Ext1^Gt/Gt cells was increased by the presence of FGF2, it was not comparable with that of wild-type cells.

FIGURE 4.

Ext1^Gt/Gt fibroblasts show impaired ability to contract collagen gels. Wild-type and Ext1^Gt/Gt fibroblasts were embedded in type I collagen as described under “Experimental Procedures.” Contraction of collagen gels was monitored at the indicated time points, in the absence (DMEM) or presence of FGF2 (10 ng/ml). A, Ext1^Gt/Gt fibroblasts displayed a reduced capacity to contract floating collagen gels, as compared with wild-type fibroblasts. The decrease was evident after 2 h and remained consistent throughout the measurements. Re-expression of Ext1 by the mutant cells increased contractility to near wild-type levels (shown for clone 4.7). B, addition of FGF2 caused a moderate increase in contractility for all three cell types. The degree of contraction is presented as the gel area in percentage of the original area ± S.D. (n = 12). wt, wild-type; Gt/Gt, Ext1^Gt/Gt; cl. 4.7, clone 4.7 (Ext1^Gt/Gt+Ext1).

Because integrins are the major collagen receptors on mouse fibroblasts, we analyzed the expression levels of the collagen binding integrins α1, α2, α11, and β1 by flow cytometry (Fig. 5). Except for a slight tendency to increased expression of α1 in the mutant cells, there were no significant differences in integrin expression between wild-type and Ext1^Gt/Gt cells. To further examine the effect of the Ext1 mutation on the ability of the mouse embryonic fibroblasts to interact with collagen I, we analyzed the effect of heparin on cell attachment to collagen I and on cell-mediated collagen contraction. In cell attachment assays, heparin reduced the attachment of wild-type and Ext1^Gt/Gt cells to collagen I by 16 and 32%, respectively (Fig. 6A). Heparin also reduced the contractile capacity of both the wild-type (Fig. 6B) and the Ext1 mutant cells (Fig. 6C), and, similar to the attachment to collagen, the reduction was more pronounced in the Ext1^Gt/Gt cells. Finally, incubation with an antibody to β1 (Ha2/5) inhibited collagen contraction completely. Similar results were obtained with the 9EG7 antibody (data not shown).

FIGURE 5.

Integrin expression by wild-type and Ext1^Gt/Gt fibroblasts. Ext1^Gt/Gt and wild-type cells were examined for expression of collagen binding integrins (α1, α2, α11, and β1) by flow cytometry using integrin-specific antibodies as described under “Experimental Procedures.” The expression levels of these integrins were similar for both cell types. Black line, α1, α2, α11, or β1; filled gray profile, control (using secondary antibody only).

FIGURE 6.

Heparin reduces the interaction of both wild-type and Ext1^Gt/Gt fibroblasts with collagen. A, wild-type and Ext1^Gt/Gt fibroblasts were seeded on plates containing type I collagen and allowed to attach for 1 h. The absorbance at 595 nm reflects the number of attached cells (mean ± S.D., n = 6). The addition of heparin (50 μg/ml) reduced the attachment of both wild-type and Ext1^Gt/Gt cells. Wild-type (B) and Ext1^Gt/Gt (C) fibroblasts were embedded in type I collagen and time-dependent contraction was measured in the absence (DMEM) or presence of heparin (50 μg/ml) or anti β1-integrin blocking antibody (Ha2/5, 5 μg/ml) Contraction of collagen gels was monitored at the indicated time points. For both cell types, heparin significantly reduced fibroblast-mediated collagen contraction and the anti β1-integrin almost completely prevented collagen gel contraction. The degree of contraction is presented as the gel area in percentage of the original area ± S.D. (n = 8). no coll/no hep, control without collagen or heparin.

Cell Proliferation Induced by FGF2 and FGF10 Is Reduced in Ext1^Gt/Gt Fibroblasts

As ERK phosphorylation induced by transient stimulation with FGF2 was reduced in Ext1^Gt/Gt cells as compared with wild-type cells, we wanted to investigate whether there was a functional effect on mitogenic activity following sustained growth factor stimulation. Addition of FGF2 stimulated proliferation of both cell types (Fig. 7). Despite the significantly weaker signaling response of the mutant compared with the wild-type cells after stimulation with FGF2 (Fig. 1A), the proliferation of Ext1^Gt/Gt cells after FGF2 stimulation was ∼65% of the wild-type cells. FGF10 induced a more modest proliferation response of wild-type cells and had virtually no effect on the Ext1^Gt/Gt cells (Fig. 7).

FIGURE 7.

Ext1^Gt/Gt cells show a reduced proliferation rate after growth factor stimulation. Wild-type and Ext1^Gt/Gt fibroblasts were cultured in 24-well plates in DMEM supplemented with fetal calf serum. After 24 h, the medium was changed to starvation medium (without serum), FGF2 or FGF 10 were added to some wells; and cells were maintained for another 24 h with the addition of [³H]thymidine during the last 4 h. [³H]thymidine incorporation was determined as described under “Experimental Procedures”. The graph is depicted as mean values ± S.D., n = 4. Wild-type cells (black bars); Ext1^Gt/Gt cells (white bars). no stim, control without added growth factor.

Re-expression of Ext1 in Ext1^Gt/Gt Fibroblasts Increases HS Chain Length and Rescues HS-dependent Cell Matrix and Growth Factor Interactions

The polymerization of HS is generated by alternating additions of GlcNAc and GlcA. To corroborate that the changes in HS chain length correlated with GlcNAc and GlcA transferase activities, we assayed the in vitro glycosyltransferase activities in cellular extracts. As shown in Fig. 8, A and B, the in vitro enzyme activities of Ext1^Gt/Gt cells were dramatically reduced. To confirm that this and the above-described results were specifically caused by the mutation in the Ext1 gene, Ext1^Gt/Gt fibroblasts were stably transfected with Ext1, generating cellular clones expressing Ext1^Gt/Gt+Ext1. Transfection of Ext1 into Ext1^Gt/Gt fibroblasts restored enzyme activities to approximately the same levels as in the wild-type cells (Fig. 8, A and B). Re-expression of EXT1 also restored HS chain length to that of wild type cells (Fig. 8C). The longer HS chains also resulted in more binding epitopes for the 10E4 antibody (Fig. 8D) and an increased FGF2 signaling response (Fig. 8E). In addition, collagen gel contraction by Ext1^Gt/Gt+Ext1 fibroblasts was similar to that of the wild-type cells (Fig. 4, A and B).

FIGURE 8.

Reintroduction of Ext1 in Ext1^Gt/Gt fibroblasts rescues HS chain length and HS-protein interactions. Stable transfection of Ext1^Gt/Gt cells with Ext1 (clones 1.4, 4.7, and 4.8) resulted in increased GlcNAc and GlcA transferase activities (A and B); increased HS chain length (shown for clone 1.4, triangles) (C) as compared with the same cells transfected with the empty vector alone (open circles). Superimposed is the chromatogram showing ³⁵S-labeled HS from wild-type cells (filled circles). The retarded components eluting at ∼20–23 ml, corresponds to material that is degraded by chondroitinase ABC. The labeled HS was isolated from the cell surface/extracellular matrix. (D) intermediate binding of the 10E4 antibody to cell surface HS (clones 1.4, 4.7, and 4.8) as compared with wild-type and mutant fibroblasts; E) increased intracellular signaling by FGF2. The band intensity of phosphorylated versus total ERK was quantified and presented in the bar graph as relative activation of the receptor. The figure shows one representative experiment of three independent experiments. Black bars, wild-type cells; white bars, Ext1^Gt/Gt cells; gray bars, clone 4.8; cl., clone.

Taken together, we have demonstrated that reduction in EXT1 enzyme activity in mouse embryonic fibroblasts results in decreased MAPK signaling induced by FGF2 but not FGF10 or PDGF-BB and a lower proliferation rate. Another consequence of reduced EXT1 expression is a decreased interaction with collagen I, resulting in impaired cell adhesion and ability of the cells to reorganize collagen gels.

DISCUSSION

In this study, we examined how EXT1 levels affect fibroblast growth factor signaling and interaction with the extracellular matrix. Several signaling molecules, including members of the FGF, Wingless/Wnt, and Hh families, require HSPG at the cell surface for optimal signaling activity. Whereas biochemical studies, using structurally defined HS oligosaccharides in cell culture systems and in vitro binding assays, have provided data concerning the minimal growth factor binding epitope on HS, the influence of HS chain length on intact cell-associated proteoglycans for growth factor binding is not much studied. We have previously observed that HS chain length is substantially reduced in Ext1^Gt/Gt mutant fibroblasts, without significantly affecting the sulfation (20). Our present results demonstrate that the reduced HS chain length has a dramatic effect on fibroblast response to FGF2 and interaction with the extracellular matrix. The interaction of FGF2 with HS has received much attention because of the essential role of HS for FGF2-induced biological cellular responses. A range of size-defined heparin oligosaccharide has been tested in different studies for their ability to bind to FGF2 and to form FGF2-HS-FGFR ternary complexes. These studies show that the N-and O-sulfate groups play essential roles for the FGF2-HS interaction and suggest that although tetra-, penta-, and hexasaccharides are able to bind FGF2, at least an HS octasaccharide with sufficient sulfation is required for binding of HS to both FGF2 and its receptor (34–37). We observed that FGF2-induced MAPK signaling and proliferation was clearly reduced in the Ext1^Gt/Gt fibroblasts indicating that the short HS chains synthesized by Ext1^Gt/Gt fibroblasts have a decreased capacity to respond to FGF2. However, although the FGF2-mediated ERK phosphorylation was reduced in the mutant cells, it was not completely abolished, which could explain their proliferation response during sustained FGF2 stimulation. The shorter chain length may also influence the charge distribution of Ext1^Gt/Gt HS chains and thus the ability to form stable FGF2·HSPG·FGFR ternary complexes when transiently stimulated with the growth factor. It is therefore possible that control over chain length is one of the regulatory factors in HS-dependent signaling. In contrast to our results, FGF2 signaling is not significantly disturbed in Ext1^Gt/Gt mice (18). One possible explanation may be that in the previously mentioned study, Ext1^Gt/Gt limb explants were utilized at a time point (embryonic day 15.5), when the phenotype seems to be somewhat stabilized and less severe. Another explanation could be that a higher concentration of FGF2 was used in that study. Clearly, a more detailed understanding of the role of HS chain length in HS-dependent signaling event is required to understand the overall interactions between HS and protein ligands.

The lack of effect of the Ext1 mutation on FGF10-mediated signaling was surprising because FGF10 is known to require HS for mitogenic activity (38). Our results indicate that the Ext1^Gt/Gt HS contains the sulfation pattern required for FGF10 binding and signal transduction. Yet, FGF10 did not significantly affect the proliferation rate of Ext1^Gt/Gt fibroblasts. This suggests, that even though the initial MAPK signaling induced by FGF10 (seen as elevated levels of phosphorylated ERK1/2) was not affected by the reduced chain length, the sustained signaling, possibly involving other pathways, was affected by the Ext1 mutation.

Furthermore, PDGF-BB stimulated ERK1/2 phosphorylation was unaffected by the mutation in Ext1. PDGFs are dimeric molecules of disulfide-bonded A and B polypeptides that occur as homo- and heterodimers that signal via two different receptors, PDGFRα and PDGFRβ (39). In agreement with our data, previous reports in the literature indicate that PDGF-BB efficiently stimulated activation of PDGFRβ in the absence of HS and that there is no absolute requirement for HS in PDGF-BB-induced PDGFα receptor activation (26, 39).

Collagens are abundant constituents of the extracellular matrix, where they provide important functions as protective support during tensile force and as scaffolds to help cells organizing the matrix. In a recent study, utilizing dermal fibroblasts from patients with systemic sclerosis, it was shown that HS-dependent ERK signaling contributes to the increased contractility seen in the lesions developing during this condition (40). This suggests that the reduced contractility of the Ext1^Gt/Gt fibroblasts observed in the present study may rely on a reduced signaling response to FGF2; however, this remains to be confirmed. FGF2-induced contraction of floating collagen lattices was previously shown to involve phosphatidylinositol 3-kinase, Rac, Rho, and also Rho kinase (41), implying that the intracellular signaling mechanisms that drive collagen gel contraction are complex and that several different signaling pathways act in parallel. HS binds to collagen I via a single binding site near the NH₂ terminus on the collagen monomer (42). The corresponding sequence on the HS chain is less characterized, but our results from the cell attachment assay and also the collagen gel contraction analysis suggest that this domain is absent or less prevalent in the HS chains expressed by Ext1^Gt/Gt cells. Delayed collagen gel contraction was recently reported for fibroblasts deficient in the major cell surface HSPG syndecan-4 (43), supporting the role for HS in remodeling of collagen gels. Previous work has shown that the expression of syndecan-4 is not decreased in Ext1^Gt/Gt mutant cells (44), and also that although these cells are able to adhere to fibronectin, they are unable to form focal adhesion (45). The ability of fibroblasts to contract free-floating collagen gels rely on functional β1-integrins (46, 47) and HS proteoglycans could possibly act both as primary receptors for collagen or as co-receptors in the interaction between collagen and integrins (42). In the present study, heparin reduced the contractile capacity of the mouse embryonic fibroblasts, emphasizing the important role of HS in this context. Recently, cell surface HS chains on syndecan-1 were shown to be essential for α2β1-mediated adhesion of Chinese hamster ovary cells to collagen and for α2β1-mediated cell spreading and actin organization on collagen (48). One of the most prevalently expressed integrins in cultured immortalized mouse embryonic fibroblasts has previously been implicated to be α11β1 (30), and this was also the case in the present study. However, any potential cooperation between HS and α11β1 remains to be established.

It has been shown that there is a reduction, but not a total loss, of HS in the chondrocytes of exostoses, suggesting that exostoses formation is a result of reduced HS (49). An intriguing issue is if hereditary multiple exostoses-associated mutations in either EXT1 or EXT2 result in reduction of HS in affected growth plates, leading to shorter HS chains. This, in turn, could result in growth factor signaling defects in a subset of chondrocytes, as our results suggest that EXT1-dependent HS chain elongation makes a significant contribution to the specificity of HS-protein interactions.