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Human Molecular Genetics Advance Access originally published online on December 24, 2007
Human Molecular Genetics 2008 17(7):996-1009; doi:10.1093/hmg/ddm372
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Changes in heparan sulfate are associated with delayed wound repair, altered cell migration, adhesion and contractility in the galactosyltransferase I (ß4GalT-7) deficient form of Ehlers–Danlos syndrome

Martin Götte1,*,{dagger}, Dorothe Spillmann2,{dagger}, George W. Yip3,{dagger}, Elly Versteeg4, Frank G. Echtermeyer5, Toin H. van Kuppevelt4 and Ludwig Kiesel1

1 Department of Gynecology and Obstetrics, University of Münster, Medical Center, Albert-Schweitzer-Str. 33, D-48149 Münster, Germany 2 Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, SE-75123 Uppsala, Sweden 3 Department of Anatomy, National University of Singapore, 117597 Singapore, Singapore 4 Department of Biochemistry, NCMLS, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands 5 Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, D-30625 Hannover, Germany

* To whom correspondence should be addressed at: Department of Gynecology and Obstetrics, University of Münster Medical Center, Research Laboratory Domagkstr. 11, D-48149 Münster, Germany. Tel: +49 2518356117; Fax: +49 2518355928; Email: martingotte{at}uni-muenster.de

Received September 10, 2007; Accepted December 19, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Reduced activity of ß4-galactosyltransferase 7 (ß4GalT-7), an enzyme involved in synthesizing the glycosaminoglycan linkage region of proteoglycans, is associated with the progeroid form of Ehlers–Danlos syndrome (EDS). In the invertebrates Drosophila melanogaster and Caenorhabditis elegans, mutations in ß4GalT-7 affect biosynthesis of heparan sulfate (HS), a modulator of several biological processes relevant to wound repair. We have analyzed structural alterations of HS and their functional consequences in human ß4GalT-7 Arg270Cys mutant EDS and control fibroblasts. HS disaccharide analysis by reversed phase ion-pairing chromatography revealed a reduced sulfation degree of HS paralleled by altered immunostaining patterns for the phage-display anti-HS antibodies HS4E4 and RB4EA12 in ß4GalT-7 mutant fibroblasts. Real-time PCR-analysis of 44 genes involved in glycosaminoglycan biosynthesis indicated that the structural alterations in HS were not caused by differential regulation at the transcriptional level. Scratch wound closure was delayed in ß4GalT-7-deficient cells, which could be mimicked by enzymatic removal of HS in control cells. siRNA-mediated knockdown of ß4GalT-7 expression induced morphological changes in control fibroblasts which suggested altered cell–matrix interactions. Adhesion of ß4GalT-7 deficient cells to fibronectin was increased while actin stress fiber formation was impaired relative to control cells. Also collagen gel contraction was delayed in the ß4GalT-7 mutants which showed a reduced formation of pseudopodia and filopodia, less efficient penetration of the collagen gels and a diminished formation of collagen suprastructures. Our study suggests an HS-dependent basic mechanism behind the altered wound repair phenotype of ß4GalT-7-deficient EDS patients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Glycosaminoglycans (GAGs) are long, unbranched polysaccharides composed of repeating disaccharide units which consist of alternating uronic acids and amino sugars (1,2). Most GAGs are covalently attached to conserved serine residues of core proteins via a defined linkage region of xylose, two galactoses and a glucuronic acid, thus assembling to proteoglycans (PGs). Alternative addition of N-acetylglucosamine or N-acetylgalactosamine to the terminal glucuronic acid of the linkage region leads to the formation of heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS), respectively (1). Post-translational modifications such as epimerization and sulfation result in the formation of diverse motifs in the GAG chains, that allow binding to a large variety of ligands, thus regulating growth factor signaling, cell adhesion, proliferation, differentiation and motility (3–5).

Amino acid exchanges in galactosyltransferase I (β4GalT-7; E.C. 2.4.1.133 [EC] ), the enzyme that catalyzes the transfer of the first galactose to the xylose residue in the linkage region of PGs, have been linked to the pathology of the progeroid form of the Ehlers–Danlos syndrome (EDS) (6,7). EDS is a heterogeneous group of heritable connective tissue disorders characterized by fragile and hyperextensible skin, joint hypermobility and delayed wound healing accompanied by atrophic scarring (8). Apart from mutations in β4GalT7, molecular defects in several extracellular matrix proteins, including collagen I, III and V, collagen-modifying enzymes, thrombospondin and tenascin X are associated with different forms of EDS (9,10). In patients, point mutations which lead to amino acid exchanges that reduce the activity of ß4GalT-7 can lead to an aged appearance, developmental delay, dwarfism, craniofacial disproportion and generalized osteopenia (7,11). In addition, hypermobile joints, hypotonic muscles, defects in wound healing and loose but elastic skin are observed. While it was previously shown that compound heterozygous amino acid exchanges (186D, 206D) in ß4GalT-7 result in an inefficient substitution of the PG decorin with DS chains (6,12), we have recently characterized the molecular phenotype of an Arg270Cys exchange in the C-terminal catalytic region of β4GalT-7, using patient-derived and healthy control skin fibroblasts (13). The patient's cells exhibited reduced galactosyltransferase activity, defective biosynthesis of mature decorin and biglycan and reduced epimerization of CS/DS GAG chains. In addition, morphological alterations and an intracellular accumulation of degradative vacuoles were seen in β4GalT-7Arg270Cys cells. Analysis of endogenous collagen fibrils showed that the β4GalT-7-deficient cell collagen has a different suprastructure, no banded collagen fibrils and an altered ratio of {alpha}(1) to {alpha}(2) chains compared with controls. Moreover, β4GalT-7Arg270Cys cells exhibited reduced cell proliferation rates.

While some of the aspects of the EDS phenotype, in particular the defects in collagen fibrillogenesis which are modulated by decorin and biglycan, can be explained by the aberrant CS/DS substitution of these PGs, other phenotypic aspects may be due to more complex changes. We had previously hypothesized that changes in GAG structure caused by ß4GalT-7 deficiency may have very profound effects on the structure and composition of the extracellular matrix. This would lead to altered ‘inside-out’ signaling in response to the aberrant matrix, and possibly to the formation of alternative glycosylation patterns, which can sustain viability, but not all functions of a healthy individuum (13). Although it is well known that ß4GalT-7 is involved in the synthesis of the linkage tetrasaccharide common to both HS and CS/DS PGs (14–17), the consequences of ß4GalT-7 mutations on HS structure and function in the context of EDS have not been previously addressed in detail. However, changes in HS may very likely contribute to several aspects of the EDS phenotype. For example, the co-receptor role for cell surface HS for growth factor signaling and thus, modulation of cell proliferation, is well established (18,19). HS also mediates binding to ligands in the extracellular matrix and modulates integrin signaling, contributing to the regulation of cell adhesion, focal adhesion formation and cell motility (20–22). Cell surface HS PGs of the syndecan family, the matrix HS PG perlecan and the HS degrading enzyme heparanase modulate wound repair in vivo in an HS-dependent manner (23–27).

In this study, we investigated structural changes in HS and their functional consequences in human ß4GalT-7 mutant and control fibroblasts. We demonstrate that altered HS structure translates into several changes of cellular phenotype, including delayed wound repair in vitro, as well as changes in fibronectin adhesion, actin stress fiber and filopodia formation and collagen gel contraction. Our results suggest that HS structures, along with the previously described changes in CS/DS PG function (11–13,15), make a significant contribution to the wound repair phenotype in EDS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
In vitro wound repair is delayed in ß4GalT-7 deficient fibroblasts
Since EDS is characterized by impaired wound repair, we employed an established in vitro-scratch wound healing assay (28) to test if reduced ß4GalT-7 activity can be linked to this aspect of the complex EDS phenotype. Compared with control cells, wound closure was decreased by 25% in ß4GalT-7-deficient cells (Fig. 1). HS-degradation by heparitinase-treatment resulted in a similar inhibition (20%) of wound closure in control cells, but led to no further decrease in the mutant cells. Treatment of the cell lines with chondroitin-lyase ABC inhibited wound closure in control cells by 25%, and led to a dramatic 74% decrease in ß4GalT-7-deficient cells. These data confirm a role for GAGs in the wound repair process (28) and point at altered GAG function as a possible cause for the wound repair phenotype in the ß4GalT-7-deficient form of EDS.


Figure 1
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  		Figure 1. Delayed in vitro wound closure in ß4GalT-7 deficient fibroblasts. Confluent cultures of control (WT) and ß4GalT-7 deficient fibroblasts were subjected to scratch wounding as described in Materials and Methods. Wound closure was monitored 6 h after wounding and expressed as the percentage of the original wound margin distance (recorded at time point 0). (A) Representative image of the in vitro wound closure assay (untreated control fibroblasts shown); (B) Quantitative analysis of wound closure (n = 3–9). Indicated cultures were additionally treated with heparitinase I (H'ase); or chondroitin lyase ABC (ABC). *P < 0.05, error bars = SEM.

 
Sulfation of HS is decreased in ß4GalT-7-deficient fibroblasts
We had previously observed reduced epimerization of the dematan sulfate chains of decorin and biglycan in ß4GalT-7-deficient fibroblasts (13). Since ß4GalT-7 catalyzes a key reaction in the initiation of both CS/DS and HS, and since compensatory structural changes have been described in animal models of genetically altered HS biosynthesis and degradation (29,30), we aimed at analyzing if ß4GalT-7 deficiency translated into structural differences between control cell and ß4GalT-7 mutant GAGs. Total cellular HS chains were isolated from control and mutant cells and subjected to complete lyase depolymerization using heparin lyase I, II and III (see Materials and Methods). The lyase cleavage products were analyzed using reversed phase ion-pairing chromatography (31). All cleavage products contain an unsaturated hexuronic acid ({Delta}HexA) as consequence of cleavage by the lyases. The ß4GalT-7 mutation resulted in an increase of non-sulfated disaccharides {Delta}HexA-GlcNAc and a parallel decrease of the disulfated 2-O-, N-sulfated and 6-O-, N-sulfated disaccharides {Delta}HexA2S-GlcNS and {Delta}HexA-GlcNS6S while other disaccharide species were not altered significantly (Fig. 2). These changes resulted in an overall lower total sulfation level of the mutant cells due to a decrease of N-sulfation and 2-O-sulfation paralleled by an increase in non-sulfated disaccharide species (Fig. 3). At the same time, cell associated CS disaccharides were not altered significantly both on disaccharide level (data not shown) and overall sulfation level (Fig. 4). To further characterize the changes in the expression of GAG structures between ß4GalT-7 mutant and control cells, we performed immunostainings with single-chain Fv antibodies directed against 10 different GAG epitopes. No antibody reactivity was observed upon staining with a negative control antibody and antibodies directed against heparin or CSE, respectively (Table 2, Fig. 5). Both mutant and control fibroblasts showed similar antibody reactivity for the DS recognizing antibody LKN1, the anti-CS antibody IO3H12 and the anti-HS antibody AO4B08, which presented as a filamentous matrix staining. The HS epitope antibodies HS4C3 and EV3C3, as well as the CS epitope antibody IO3H10 displayed an additional slightly increased intracellular staining of GAG epitopes in ß4GalT-7-deficient cells. Antibody HS4E4, which preferentially recognizes N-sulfated epitopes in HS, showed a clear membranous staining in the control cell line, whereas only a weak intracellular staining was observed in ß4GalT-7-deficient cells (Fig. 5). Moreover, the RB4EA12 HS antibody displayed a strong, more fuzzy staining pattern in the ß4GalT-7-deficient cells relative to controls. This staining pattern may indicate an aberrant distribution of PGs carrying the RB4EA12 HS epitope (Fig. 5).


Figure 2
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  		Figure 2. Composition of HS from control and mutant cells. HS was isolated from control and mutant fibroblasts and products were exhaustively digested with a mixture of heparin lyases (see Materials and Methods). The disaccharide products, containing non-reducing-terminal 4,5-unsaturated hexuronic acid residues ({Delta}HexA) were analyzed by RPIP-HPLC, as described in Materials and Methods. The disaccharide composition is indicated for control fibroblasts (black bar) and mutant cells (white bar) with N-Acetylglucosamine (GlcNAc), N-sulfated glucosamine (GlcNS) and sulfate group at indicated position (S).

 


Figure 3
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  		Figure 3. Overall sulfation content of HS from control and mutant fibroblasts. Overall sulfate contents (Total sulfation), specified according to type of substituent as non-sulfated (0S), total N-sulfated (NS), total 6-O-sulfated (6S) and total 2-O-sulfated disaccharides (2S) were calculated based on results described in Figure 2. Control fibroblasts (black bar) and mutant cells (white bar).

 


Figure 4
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  		Figure 4. Overall sulfation content of CS from control and mutant fibroblasts. Overall sulfate contents (Total sulfation), specified according to type of substituent as total 2-O-sulfated (2S), total 6-O-sulfated (6S) and total 4-O-sulfated disaccharides (4S) were calculated from CS-disaccharide analysis (Materials and Methods).

 


Figure 5
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  		Figure 5. Immunostaining for HS and CS epitopes in ß4GalT-7 deficient and control fibroblasts. Cells were methanol-fixed and stained with scFv antibodies of different GAG epitope specificity. See text and Table 2 for details.

 
Expression analysis of genes involved in GAG biosynthesis and modification in ß4GalT-7 mutant cells
To analyze if the alterations in HS structure in ß4GalT-7 mutant cells were due to transcriptional changes, we performed a quantitative real-time RT–PCR analysis of the expression of 44 gene products involved in GAG biosynthesis, and of 11 HS core proteins including perlecan and all members of the syndecan and glypican families (Table 1). Fifteen genes were found to be either not expressed or only expressed at low levels in human skin fibroblasts. Gene products which were differentially up- or downregulated more than 2-fold between control and mutant fibroblasts in the initial screening were further investigated in multiple biological replicates. Final analysis of the five most strongly regulated candidate genes in four independent control cell lines and in ß4GalT-7 deficient fibroblasts revealed a significant downregulation of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, encoding an enzyme mediating 6-O-sulfation of CS (P < 0.05) (Table 1, Fig. 6). However, no significant changes in the expression of genes encoding HS biosynthetic enzymes were detected.


Figure 6
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  		Figure 6. Quantitative RT-PCR analysis of selected genes involved in GAG biosynthesis. mRNA was isolated from biological replicates of ß4GalT-7 and control cells, and cDNA was analyzed by qRT-PCR for 54 genes as described in Materials and Methods and in Table 1. The expression of five genes differentially regulated in the initial screening was further investigated using ß4GalT-7 mutant and four control cell lines. Apart from a downregulation of GALNAC4S-6ST in ß4GAlT-7 deficient cells, no significant changes in gene expression were noted. *P < 0.05, error bars = SEM.

 


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  		Table 1. qRT-PCR screening for expression of HSPG core proteins and of enzymes involved in HS-/CS- biosynthesis

 


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  		Table 2. scFv GAG epitope antibody specificity and staining results

 
ß4GalT-7-deficient cells display increased adhesion to fibronectin and impaired actin stress fiber formation
PGs and GAGs play a major role as mediators and modulators of cell adhesion to the extracellular matrix (5). We had previously observed morphological alterations in ß4GalT-7 mutant patient fibroblasts which suggested that cell adhesion may be altered in these cells (13). siRNA-mediated knockdown of ß4GalT-7 mRNA expression in control fibroblasts resulted in a similar phenotype (Fig. 7A–D), as a flattened, extensively spread cell morphology was frequently observed after ß4GalT-7 siRNA treatment. Immunostaining for the ß-subunit of the vitronectin adhesion receptor {alpha}Vß5-integrin did not reveal obvious changes in distribution between control and ß4GalT-7 siRNA-treated cells (Fig. 7 A and C). To further test the hypothesis that altered cell adhesion may contribute to the delayed wound repair phenotype of ß4GalT-7 deficient cells, we measured fibroblast adhesion to a fibronectin substratum in vitro. Both ß4GalT-7 deficient and control fibroblasts showed little adhesion to BSA, which served as a negative control (Fig. 7E). In contrast, adhesion to fibronectin was significantly increased for both cell types, and ß4GalT-7 mutant cells showed a 70% increased adhesion to fibronectin relative to control cells (Fig. 7E). Since cell adhesion to fibronectin was increased, but cell migration during wound repair was decreased in GalT-7 deficient cells, we investigated if formation of focal adhesions and actin stress fibers was altered in these cells. Using immunofluorescence microscopy, we could demonstrate that both control and ß4GalT-7 mutant cells formed focal contacts on fibronectin, as indicated by vinculin staining (Fig. 8). However, focal contacts in control cells showed a more polarized, focal distribution, whereas vinculin staining in ß4GalT-7 mutant cells appeared more evenly distributed, covering large areas of the ventral cell body. The phalloidin-stained actin cytoskeleton of fibronectin-attached control fibroblasts was more prominent and showed clearly distinguishable actin stress fibers, whereas the actin filaments in ß4GalT-7 mutant cells appeared more delicate and had a smaller diameter (Fig. 8). Also large cortical actin fibers were only occasionally seen in mutant cells. Similar alterations in actin filament formation were observed after siRNA-mediated knockdown of ß4GalT-7 expression in control skin fibroblasts (Supplementary Material, Fig. S1).


Figure 7
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  		Figure 7. Altered cell morphology of ß4GalT-7 silenced fibroblasts and increased adhesion of ß4GalT-7-mutant cells to fibronectin. (A–D) Control fibroblasts were transfected with siRNA directed against ß4GalT-7 (C and D), or a control siRNA (A and B) and analyzed after 72 h. Knockdown of ß4GalT-7 expression was confirmed by quantitative real-time PCR, resulting in a >90% reduction of ß4GalT-7 expression (P < 0.05, n = 3). Silencing of ß4GalT-7 induced a more spread, flattened morphology (C and D) compared with controls (A and B), as previously described for ß4GalT-7 mutant fibroblasts (13). (B and D, negative control antibody; A and C, ß5-integrin immunostaining). (E) ß4GalT-7 mutant and control cells were allowed to adhere to fibronectin (FN)- and bovine serum albumin (BSA)-coated plates for 1 h as described in Materials and Methods. Then, cells were stained with methylene blue, followed by photometrical quantification of bound dye at 620 nm. *P1 = significantly different from BSA control, P2 = significantly different from control cells (FN). Error bars = SEM.

 


Figure 8
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  		Figure 8. Impaired actin stress fiber formation in ß4GalT-7 mutant fibroblasts. ß4GalT-7 mutant and control fibroblasts were cultured on a fibronectin substratum for 16 h. Actin stress fibers were detected using fluorescently labelled phalloidin, and focal adhesions sites were detected by vinculin immunostaining as described in Materials and Methods. Blue color indicates DAPI nuclear staining.

 
Collagen gel contraction and fibril reorganization is perturbed in ß4GalT-7 deficient fibroblasts
Since immunofluorescence staining results suggested that formation of actin stress fibers and focal contacts was altered in ß4GalT-7 deficient cells, we tested whether these alterations resulted in impaired cellular contractility. For this purpose, control and ß4GalT-7 deficient fibroblasts were embedded in type I collagen gels, and gel contraction was monitored. After 24 h, control fibroblasts had contracted the collagen gel by 86% of its initial size (Fig. 9A). In contrast, ß4GalT-7 mutant fibroblasts were only able to reduce the linear gel dimension by 70%. The ability to contract the collagen gel was significantly reduced in ß4GalT-7 mutant over control cells for all timepoints investigated (1.5, 3, 7 and 24 h). Syndecan-4 is a cell surface HS PG and matrix receptor which is highly expressed by fibroblasts, acting as a modulator of integrin activity, focal adhesion formation and wound repair (20,21,39,40). Using wild-type and syndecan-4 deficient murine fibroblasts (40), we addressed the question if loss of this downstream target of the ß4GalT-7-dependent HS alterations resulted in a comparable collagen gel contraction phenotype. Collagen gel contraction by murine syndecan-4 deficient cells was significantly delayed at early timepoints (1.5–6 h, P < 0.05) compared with wild-type controls (Fig. 9B). However, after 24 h, syndecan-4 deficient cells had caught up with the wild-type fibroblasts, and the difference in collagen gel diameter was no longer statistically significant. Histological investigation of formalin-fixed gels revealed that human control fibroblasts penetrated the gel more deeply and displayed a more even distribution within the gels compared with the ß4GalT-7 mutant cells (Fig. 10A and B). Both control and ß4GalT-7 mutant fibroblasts were able to reorganize collagen into suprastructures, however, these structures were more frequently seen with control cells (Fig. 10C–F). We employed transmission electron microscopy to investigate cell and collagen fibril morphology in more detail. Control fibroblasts extended numerous pseudopodia and filopodia into the gel matrix (Fig. 11), whereas ß4GalT-7 mutant fibroblasts displayed only a few extensions. Moreover, mutant fibroblasts were associated with a much smaller number of collagen fibrils in comparison with control cells, and were less capable of organizing the collagen intro fibrillar suprastructures (Fig. 11).


Figure 9
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  		Figure 9. Impaired collagen gel contraction in ß4GalT-7- and syndecan-4 deficient fibroblasts. (A) Control (white columns) and ß4GalT-7 deficient (grey columns) fibroblasts were embedded in type I collagen gels as described in Materials and Methods. Contraction of collagen gels was monitored at the indicated timepoints. (B) Collagen gel contraction of murine wild-type (white columns) and syndecan-4 knockout (grey columns) 3T3 mouse fibroblasts. *Significantly different from control cells at P < 0.001 (n > 10).

 


Figure 10
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  		Figure 10. Histological staining of collagen gel-embedded fibroblasts. ß4GalT-7 mutant (B,E,F) and control (A,C,D) fibroblasts were embedded in collagen gels as described in Figure 9, and processed for standard histology after 24 h as described in Materials and Methods. (A and B) Hematoxilin/eosin (H&E) staining of collagen gel margins reveals decreased depth of gel penetration by ß4GalT-7 mutant fibroblasts. (C and E) Both control and ß4GalT-7 were able to reorganize the collagen matrix at gel margins (H&E staining). (D and F) Masson Trichrome staining of central gel area. Control fibroblasts show a more regular distribution within the gel and increased suprastructural organization of collagen (arrows). Bar = 50 µm (applies to all figure parts).

 


Figure 11
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  		Figure 11. Transmission electron microscopy of collagen gel embedded control and ß4GalT-7 mutant fibroblasts. Wild-type (A–D) and ß4GalT-7 mutant (E and F) fibroblasts were processed for electron microscopy 24 h after collagen embedding. Wild-type cells extend numerous pseudopodia and filipodia (arrows) into the gel matrix (A and B). Many fibrillar collagen suprastructures (arrows) are seen in the gel adjacent to the fibroblasts (C and D). In contrast, mutant fibroblasts have a reduced number of cellular extensions, and are associated with a much smaller number of collagen fibrils (E and F).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
We have previously shown that a point mutation causing an Arg270Cys substitution in ß4GalT-7 led to a significant decrease in enzymatic activity and to changes in CS/DS chain attachment and epimerization of the PGs decorin and biglycan (13). Since ß4GalT-7 activity is required for the synthesis of both HS and CS PGs (14–17), we aimed at analyzing potential changes in HS structures in ß4GalT-7 mutant fibroblasts, which could add to the complex pathology of EDS. Such changes would have profound effects on the structure and function of the extracellular matrix (13). GAGs could be at an excess under physiological conditions and some alterations in HS structure may still be tolerable and sustain viability of EDS patients, however, in stress situations such as wounding, the defect may become apparent. Disaccharide analysis of ß4GalT-7 mutant cells indicated a reduction in HS sulfation, due to a decrease of the 2-O-, N-sulfated and 6-O-, N-sulfated disaccharides {Delta}HexA2S-GlcNS and {Delta}HexA-GlcNS6S, respectively, and a parallel increase of the non-sulfated disaccharide {Delta}HexA-GlcNAc. Also, the reduced immunoreactivity of ß4GalT-7 deficient fibroblasts for the scAb HS4E4, recognizing N-sulfated HS, indicated a decreased sulfation of HS in these cells. To our knowledge, this is the first description of a structural alteration of HS in EDS.

Although a study by Grobe and Esko (41) had previously demonstrated that expression of NDST genes can be transcriptionally regulated via structured 5'-UTR-sequences which are present in several genes involved in HS biosynthesis, control of gene transcription does not necessarily affect product structure, as seen in the case of NDST-1 and NDST-2-deficient mice (42). In the present study, we could not find any evidence for a differential transcriptional regulation of HS biosynthetic enzyme expression between ß4GalT-7 deficient and control fibroblasts. We can only speculate if compensatory signaling from the differently composed extracellular matrix, or the activation of alternative signaling pathways by structurally altered GAGs at the surface of ß4GalT-7 mutant cells may have contributed to the changes in HS structure of ß4GalT-7 mutant cells. The activity of HS biosynthetic enzymes may furthermore have been regulated at a functional level, e.g. by direct interactions with ß4GalT-7 or its reaction products, or through enhanced turnover. Similar to our study, there are some examples demonstrating that alterations in HS biosynthesis and degradation can lead to secondary changes in HS structure: HS from HS 2-O-sulfotransferase-deficient mice shows decreased 2-O-sulfation, but increased 6-O-sulfation compared with controls (29), whereas transgenic overexpression of the HS degrading enzyme heparanase in mice results in shorter, yet oversulfated HS chains (30). Thus, while there is evidence of secondary alterations in HS in response to indirect primary genetic defects, regulatory mechanisms are still only poorly understood.

Sulfation patterns determine the ligand-binding properties of HS (1,5,43) potentially affecting several cellular functions and biological processes relevant to the pathology of EDS. ß4GalT-7-deficient cells displayed significantly delayed wound closure in an in vitro model, which can be mimicked by enzymatic degradation of HS in control fibroblasts. In contrast, enzymatic degradation of CS by chondroitinase treatment resulted in a profound decrease in wound closure in ß4GalT-7 mutant cells while the same treatment in control fibroblasts lead to a delay in wound repair which was similar to the one caused by HS degradation in mutants, suggesting that CS may at least in part compensate for HS in the mutants. These findings go in line with a previous observation of reduced in vitro wound repair in rabbit palatal fibroblasts subjected to chlorate-induced reduction of GAG sulfation (28).

Increased substrate adhesion and reduced cell migration may have played a major role in delaying wound repair of ß4GalT-7 mutant fibroblasts compared with controls. The sulfation pattern of CS can have a profound effect on cell adhesion, since primarily 4-O-sulfated CS reduced adhesion, whereas 6-O-sulfated CS increases adhesion of palatal fibroblasts to cell culture dishes (28). While chinese hamster ovary (CHO) cells completely devoid of galactosyltransferase I activity attach to fibronectin substrates to the same extent as wild-type CHO cells (44), the ß4GalT-7 mutant human skin fibroblasts displayed residual galactosyltransferase activity (13) and showed an increased adhesion. N-sulfation of HS on cell surface PGs such as syndecan-4 is important for the adhesion of fibroblasts to the HepII domain of fibronectin FN (HepII) (39). It could be envisaged that syndecan-4 and syndecan-1 ectodomain shedding may convert these molecules from attachment sites into competitive inhibitors for HepII-dependent fibronectin binding during wound repair (24,45). Therefore, shed syndecans containing HS of a lower N-sulfation degree may be less efficient inhibitors of fibroblast adhesion to fibronectin. Similar to our previous observations on ß4GalT-7 mutant fibroblasts (13), siRNA-mediated knockdown of ß4GalT-7 gene expression leads to a change in cell morphology (Fig. 7A–D): The widely spread appearance of cells subjected to ß4GalT-7 siRNA treatment suggests altered cell–matrix interactions. Although the cell surface HSPG syndecan-4 can mediate fibroblast adhesion and spreading on HepII fibronectin domains in an HS-dependent manner (39), it was recently shown that syndecan-2 and syndecan-4 ectodomains can also mediate fibroblast adhesion via an HS-independent pathway (46). Shedding of syndecans followed by matrix deposition may induce the required trans conformation of this HS independent adhesion mode in vivo. Of note, syndecan-2-mediated spreading leads to a more rounded morphology, whereas syndecan-4-mediated spreading resulted in a more polygonal appearance (46). Different modes of adhesion may also be active in wild-type fibroblasts and in fibroblasts with reduced ß4GalT-7 expression and activity, since the latter cannot rely on wild-type HS structures, and may thus recruit alternative adhesion domains.

Besides increased adhesion of ß4GalT-7 mutant fibroblasts to fibronectin, also the impaired actin stress fiber formation, the reduced ability to generate collagen contracting forces and the impaired ability to reorganize a collagen matrix into fibrillar suprastructures may contribute to the pathology. Mutant CHO cells devoid of ß4GalT-7 activity fail to form focal adhesion and actin stress fibers (44). Focal adhesion formation depends on the presence of N- and 6-O-sulfated glucosamine in HS, whereas CS is not important for this interaction (39). In this context, focal adhesion formation may be modulated either directly by syndecan-4, or via syndecan-4 interactions with tenascin-C, an inhibitor of focal adhesion formation (21,47). Mahalingam et al. (39) noted impaired focal adhesion formation in EXT1-deficient mouse fibroblasts. Similarly, we found a reduction in N-sulfation of cell-associated HS in ß4GalT-7 mutant cells. While our ß4GalT-7 mutant fibroblasts were able to adhere to fibronectin and to recruit vinculin into adhesion sites, they were unable to bundle filamentous actin into stress fibers. Thus, changes in HS in ß4GalT-7 mutant fibroblasts appear to support some residual cellular functions (fibronectin adhesion), but cannot fully compensate for the structural deficiency caused by the reduced galactosyltransferase I activity (deficiency in actin stress fiber formation). siRNA-mediated silencing of ß4GalT-7 expression resulted in a similar phenotype, independently confirming a role for ß4GalT-7 in this process (Supplementary Material, Fig. S1). Syndecans act in concert with integrins during spreading on different substrates, leading to the activation of Rho-family GTPases (21,22). While activation of Rac1 drives the formation of nascent focal complexes, RhoA activation induces focal adhesion maturation and actin filament bundling (48). At present, we can only speculate if these syndecan-dependent processes are differentially affected by impaired ß4GalT-7 function and the associated changes in HS structure.

To our knowledge, this is the first functional investigation of ß4GalT-7 deficient cells in a three-dimensional extracellular matrix. CS and fibronectin enhance collagen gel contraction by human gingival fibroblasts (49), suggesting that alterations in fibronectin adhesion and GAG biosynthesis in ß4GalT-7 deficient cells may contribute to this phenotype. Reduced actin stress fiber formation on a two- dimensional substratum translated into a decreased ability to generate contractile forces in collagen gels. Compared with controls, ß4GalT-7 deficient cells penetrated collagen gels less efficiently, indicating reduced cellular mobility similar to the wound repair assay. These findings are further supported by the observed reduction in pseudopodia and filopodia formation, and the reduced ability to reorganize the collagen into fibrillar suprastructures. GAGs can play a direct modulatory role on collagen fibrillogenesis (50), and collagen I-embedded fibroblasts have been shown to reorganize the fibrils in an orientation parallel to their filopodia (51). However, the reduced ability to reorganize a fibrillar collagen matrix may at least in part be due to an inefficient substitution of decorin and biglycan with DS chains, as we have previously demonstrated defects in the fibrillogenesis of endogenously synthesized collagen I in ß4GalT-7-deficient fibroblasts (13). This view is further supported by our finding that murine fibroblasts deficient in a major HSPG matrix receptor, syndecan-4, showed a comparably mild, yet significant, delay in collagen gel contraction. This finding is conform with in vivo studies on the role of syndecan-4 in skin wound repair (40), and establishes a role for HSPGs in collagen gel contraction. However, it also demonstrates that deletion of a major downstream HSPG target of ß4GalT-7 is not sufficient to induce the substantial reduction in collagen gel contraction, which may be due to combined changes in HS and CS/DS biosynthesis.

In summary, we have demonstrated that the reduction in galactosyltransferase activity caused by the Arg270Cys substitution in ß4GalT-7 leads to a reduction in the sulfation degree of HS. The structural alterations, which were particularly prominent for cell-associated HS, were paralleled by significantly delayed wound closure in vitro, increased adhesion to fibronectin, a reduced capability to form actin stress fibers, filopodia and collagen gel-contracting forces. An important part of the EDS phenotype is a marked delay in wound repair and a malformation of connective tissues rich in fibrillar collagens (8). In this study, we could replicate this phenotype on the cellular level, and demonstrate for the first time that, in addition to altered CSPG function (13), changes in HS sulfation patterns are associated with the pathobiology of EDS. Thus, targeting and use of structural analogs of HS may be worth considering in the future treatment of EDS symptoms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cell culture
Skin fibroblasts from an EDS patient carrying the C808T mutation in the ß4GalT-7 gene, and skin fibroblasts from a healthy age-matched donor have been previously described (13,52). Cultures of additional control skin fibroblasts from three sex- and age-matched donors (aged 19, 22 and 31 months) with no known defects in carbohydrate metabolism were a generous gift of Dr Thorsten Marquardt, Department of Pediatrics, Münster University Hospital. Syndecan-4 deficient murine dermal fibroblasts were isolated as previously described (40), and immortalized using the 3T3 method (53). Cells were cultured in modified Eagle's minimum essential medium (MEM) with Earle's salts, 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (PAA, Cölbe, Germany).

Cell adhesion assay
Cell adhesion assays were performed using a modification of published procedures (54,55). Plates were coated with either 10 µg/ml BSA or 10 µg/ml human fibronectin (BD Biosciences) in PBS at 4°C. After 24 h, coated 96 well plates were washed once with PBS and blocked with 1% BSA in PBS for 1 h at RT. One day prior to the assay, cells were cultured in DMEM containing antibiotics and 0.5% FCS. Fibroblasts were washed once and gently removed with 2 mM EDTA in PBS washed twice and finally resuspended in DMEM with 0.5% BSA at a concentration of 250 000 cells/ml. Cells (100 µl/well) were transferred to the pretreated 96-well-plate and incubated for 1 h at 37°C, followed by three washes with PBS, and fixation with 3.7% PBS-buffered formalin for 30 min at RT, followed by staining with 1% methylene blue (Sigma) in 0.01 M borate buffer (pH 8.5) for 30 min. The wells were washed four times with borate buffer prior to cell lysis in ethanol/0.1 M HCl (mixed 1:1). The absorption of extracted methylene blue dye at 620 nm was determined photometrically in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

In vitro wound repair assay
In vitro scratch wound healing assays were performed using a modification of published procedures (28). Fibroblasts (100 000/well) were cultured in six well plates for 7 days prior to wounding. The cell monolayers were wounded by scraping with a 100 µl pipet tip, and closing of the scratch wound was monitored by Nomarksi contrast light microscopy. In some cases, cells were treated either with 0.4 mU/ml (1 mU total) heparin lyase I (Sigma) or 12 mU/ml (30 mU total) protease-free chondroitin ABC lyase (Seikagaku, Kogyo, Japan). The migration distance of the cells was documented with a Zeiss Axiophot camera and Zeiss Axiovision software immediately and 6 h after wounding. For statistical analysis, Student's t-test was employed. A P-value < 0.05 was considered statistically significant. Experiments were repeated at least three times (n = 3–9 for the individual treatment groups).

Collagen gel contraction assay
Human skin fibroblasts were trypsinized, resuspended in DMEM containing antibiotics and 20% FCS and adjusted to a concentration of 106 cells/ml. The cell suspension (0.6 ml) was mixed with 1.2 ml of a buffered collagen I solution (2.4 mg/ml in PBS pH 7.4) (Stem Cell Technologies, Vancouver, BC, Canada). The resulting solution (250 µl/well) was poured into 8-well chamber slides (Nunc) and incubated at 37°C in a humid atmosphere containing 5% CO2 for 1.5 h. Collagen gels were released from the chamber slides and individual gels were cultured in 3.5 cm-dishes containing DMEM, antibiotics and 10% FCS. The size of the collagen gels was documented in at least two linear dimensions using a stereo microscope at 8x magnification (Zeiss, Göttingen, Germany) equipped with a Zeiss Axiocam MRc camera and Zeiss Axiovision imaging software. The size of 10–22 individual gels was determined in three independent experiments 1.5, 3, 6, 7 and 24 h after pouring the gels. For histochemical staining, collagen gels were fixed in 10% formalin for 4 h at RT and embedded in paraffin using standard procedures. Sections (3 µm) were cut from the paraffin blocks and placed on poly-L-lysine coated coverslips. Dried coverslips were de-paraffinized, rehydrated and subjected either to routine hematoxilin/eosin (H&E) staining or Masson's Trichrome staining (Sigma) according to the manufacturer's recommendations.

Immunocytochemistry
For phalloidin/vinculin colocalization experiments, 10 000 cells/well were grown for 16 h in 8-well chamber slides (Nunc, Wiesbaden, Germany) that had been precoated with a 10 µg/ml solution of human fibronectin (BD Biosciences, Heidelberg, Germany). Cells were washed once with PBS prior to fixation with 3.7% PBS-buffered formaldehyde (10 minutes, RT). Following two washes with PBS, cells were permeabilized with 0.1 Triton-X100 in PBS for 5 min. After two additional washes with PBS, non-specific antibody binding sites were blocked with PBS containing 1% Aurion BSA-c (DAKO, Glostrup, Denmark). Slides were subsequently incubated with a monoclonal mouse-anti human vinculin antibody (Sigma), diluted 1:300 in PBS/1% BSA, over night at 4°C. Omission of the primary antibody served as a negative control. Following 2 x 5 min washes with PBS, the samples were incubated for 1 h in the dark with ALEXA-Fluor 568-labelled phalloidin (1:1000; Invitrogen, Karlsruhe, Germany) and ALEXA-Fluor-488 donkey-anti-mouse IgG (1:600; Invitrogen) diluted in PBS/1% BSA. Slides were washed three times with PBS, followed by DAPI staining of cell nuclei, and mounted in glycerol. Slides were analyzed with a Leica DMLB fluorescence microscope equipped with a Leica DC300F camera. Images obtained using different fluorescence channels were merged using Adobe Photoshop 6.0 software. Immunostaining of methanol-fixed cells for ß5integrin was performed essentially as described (13) using a monospecific rabbit anti-human ß5integrin antibody (Santa Cruz, 1:50) and the anti-rabbit EnVision system with the AEC+ substrate (DAKO Glostrup, Denmark), followed by Mayer's haemalum counterstaining (Merck, Darmstadt, Germany). Immunostaining with single chain Fv (scFv) anti-GAG antibodies was performed essentially as described (32). Briefly, fibroblasts were seeded onto coverslips in 24-well plates and cultured for two additional days after reaching confluency. Cells were fixed for 10 min in –20°C cold methanol, dried and blocked for 10 min with PBS containing 2% BSA. Cells were subsequently incubated with primary scFv antibodies for 90 min at 22°C (see Table 2 for specificities). Bound antibodies were visualized using anti-VSV antibody P5D4 followed by Alexa-labeled (488) anti-mouse IgG antibodies. Finally, cells were fixed in ethanol, air dried and embedded in Mowiol. As a control, primary antibodies were omitted or substituted by an irrelevant single chain antibody (MPB59). Sections were observed with a Zeiss Axioscope microscope equipped with a CCD camera.

Electron microscopy
Collagen-embedded control and β4GalT-7 mutant fibroblasts were fixed in 2% paraformaldehyde and 3% glutaraldehyde at 4°C, washed, and postfixed for 2 h using 1% osmium tetroxide. The cells were then dehydrated in an ethanol series, transferred to acetone and embedded in araldite. Ultra-thin sections were cut at 80 nm thickness. After staining with lead citrate and uranyl acetate, samples were examined in an FEI EM 208S transmission electron microscope (FEI, Hillsboro, OR, USA).

Quantitative real-time PCR analysis
Reverse transcription was carried out using the Advantage RT-for-PCR-Kit (Clontech, Heidelberg, Germany). Quantitative PCR was performed using the Qiagen QuantiTect SYBR Green PCR kit in a LightCycler (Roche, Indianapolis, IN, USA) as previously described (56,57). The primers used are listed in Supplementary Table S1. Briefly, after an initial activation step of 94°C for 15 min, 45 PCR cycles were carried out under the following conditions: Denaturation at 94°C for 15 s, annealing at 60°C for 25 s and extension at 72°C for 18 s. Specificity of the amplification was verified using melting curve analysis, and the size of the PCR product was checked by electrophoresis on a 2% agarose gel. The 2–{Delta}{Delta}Ct method was used to determine relative gene transcript levels after normalization to GAPDH.

siRNA-mediated knockdown of ß4GalT-7 expression
siRNA-mediated knockdown of ß4GalT-7 expression was performed using siRNA #111770 (Ambion, Cambridgeshire, UK) and a negative control siRNA (#301698 RNAi control kit, Quiagen, Hilden, Germany). Fibroblasts were transfected with 40 nM siRNA using the Dharmafect reagent (Dharmacon Lafayette, CO, USA) according to the manufacturer's instructions. Knockdown of expression was confirmed at the mRNA level by quantitative real-time PCR 72 h after transfection as described earlier, and lead to a >90% decrease in ß4GalT-7 mRNA expression (P < 0.05, n = 3).

Disaccharide analysis of GAGs
For disaccharide analysis confluent monolayers of fibroblasts were washed twice with ice-cold PBS and removed gently from the tissue culture plates with a cell scraper. Cells were pelleted by centrifugation at 100g and washed twice with PBS, followed by lyophilization. GAGs were isolated and digested essentially as described before (31). In short, dried cell pellets were suspended in 0.5 ml of digestion buffer (50 mM Tris/HCl, pH 8, 1 mM CaCl2, 1% Triton X100) and incubated with 0.4 mg protease (Sigma P5147, type XIV) for 16 h at 55°C followed by an additional aliquot of protease for 2 h before heat inactivation. Benzonase treatment (12.5 U in digestion buffer as above complemented with MgCl2 to a final concentration of 2 mM) was performed for 2 h at 37°C as described (31). Samples were cleaned up by passage over DEAE anion exchange columns and digested with 50 mU chondroitinase ABC (Seikagaku) in 50 µl of 40 mM Tris–Acetate pH 8 before repeated cleanup on DEAE columns. A fraction of the sample was analyzed for CS disaccharides while the remaining part was digested with 0.4 mU each of heparin lyase I, II, III (IBEX) in 15 µl of heparin lyase buffer (5 mM Hepes, pH 7.0, 50 mM NaCl, 1 mM CaCl2, 0.7 mg/ml bovine serum albumin) and incubated for 16 h at 37°C. Heat-inactivated digests were dried and resuspended in 45 µl of H2O for analysis by reversed phase-ion pair chromatography (RPIP-HPLC) as described (31).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This study was financially supported by Polysackaridforskning AB (D.S.), the National Medical Research Council, Singapore (G.W.Y.) and Deutsche Forschungsgemeinschaft DFG GO 1392/1-1 and DFG GO 1392/2-1 (M.G.).


   ACKNOWLEDGEMENTS
 
We thank Birgit Pers, Gunilla Pettersson, Yee-Gek Chan, Siew-Hua Choo, Monika Offers and Ruth Goez for excellent technical assistance, Dr Thorsten Marquardt for the generous gift of control skin fibroblast cultures and Drs Lena Kjellén and Carl Heldin for helpful discussions.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger}The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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