ROLE OF GLYCOSAMINOGLYCANS AND COLLAGEN IN THE … · ECM may contain glycosaminoglycans (GAGs) such as heparan sulphate, chon-droitin sulphate and hyaluronic acid, as well as glycoproteins

y. Cell Sd. 67, 189-202 (1984) 189Printed in Great Britain © The Company of Biologists Limited 1984

ROLE OF GLYCOSAMINOGLYCANS AND COLLAGENIN THE DEVELOPMENT OF A FIBRONECTIN-RICHEXTRACELLULAR MATRIX IN CULTUREDEMBRYONIC CORNEAL EPITHELIAL CELLS

D. L. MATTEY AND D. R. GARRODCRC and Wessex Regional Medical Oncology Unit, Centre Block, Southampton GeneralHospital, Southampton S09 4XY, England

SUMMARY

Corneal epithelial cells from 15-day chick embryos produce a fibronectin-rich extracellular matrixwhen cultured on glass, plastic and fibronectin-coated substrata. Cell culture in the presence ofStreptomyces hyaluronidase or chondroitinase ABC resulted in considerable reduction of thematrix; collagenase had a lesser effect but nevertheless also reduced the matrix. In all enzymetreatments the cells attached and spread to form characteristic epithelial cell islands, but the margi-nal cells of these islands showed a marked reduction in the number of lamellipodia and focalcontacts. Also, the immunofluorescent staining pattern for fibronectin was considerably reduced.Control cells cultured on a fibronectin-coated surface were able to reorganize the fibronectin intofibrils, whereas cells cultured in enzymes showed little or no ability to do so. The cellular reorganiza-tion of fibronectin could also be inhibited by the addition of L-azetidine-2-carboxylic acid (LACA),an inhibitor of collagen secretion. Cells plated out in the presence of LACA spread much better oncollagen substrata than on plastic, glass or fibronectin. However, in all cases very little fibronectinmatrix was detectable in the epithelial islands.

The results suggest that components of the extracellular matrix (ECM) such as collagen,hyaluronic acid and chondroitin sulphates are not essential for the initial attachment and spreadingof corneal epithelial cells in culture, but are important in the development of the ECM, and inmaintaining a flattened morphology and spreading behaviour. It is suggested that fibronectin playsan important role in these interactions and that the ability of cells to organize fibronectin into fibrilsis dependent on the presence of other ECM components such as glycosaminoglycans and collagen.

INTRODUCTION

Many of the extracellular matrix (ECM) components of normal tissues aresynthesized and secreted by cells in culture, where they may play a role incell-substratum and cell-cell adhesion. Apart from various types of collagen theECM may contain glycosaminoglycans (GAGs) such as heparan sulphate, chon-droitin sulphate and hyaluronic acid, as well as glycoproteins such as fibronectin andlaminin (Hay, 1981). Recently, it has become evident that interactions occur betweenvarious components of the ECM to produce macromolecular complexes, which maybe important for attachment and spreading of cells in culture. For example, there isstrong evidence to suggest that interactions of fibronectin with GAG-containingcomplexes on the surface of fibroblasts are functionally important in their adhesionto the tissue-culture substratum (Culp, Murray & Rollins, 1979; Laterra, Ansbacher& Culp, 1980; Perkins, Ji & Hynes, 1979; Laterra, Silbert & Culp, 1983). However,

190 D. L. Mattey and D. R. Garrod

Bruns & Gros (1980) reported that enzymes directed against collagen, hyaluronic acidand chondroitin sulphates had no apparent effect on the attachment and spreading ofhuman skin fibroblasts in vitro and suggested that these macromolecules did not playa significant role in the early attachment and spreading of fibroblasts. On the otherhand, an enzyme directed against heparan sulphate has been shown to inhibit spread-ing of fibroblasts in culture (Laterra et al. 1983).

Relatively little attention has been paid to the role of GAGs and fibronectin incell—substratum adhesion of epithelial cells even though these ECM components areimportant constituents of basement membranes in vivo. The embryonic chick cornealepithelium provides a useful model for studies on epithelial cell-substratum adhesionsince a number of the basement membrane components have been identified andshown to be synthesized and secreted in culture. Synthesis and secretion of lamininhave been reported by Sugrue & Hay (1982) and production of at least two collagentypes has been demonstrated by Linsenmayer, Smith & Hay (1977). The cornealepithelium has also been shown to synthesize GAGs such as chondroitin sulphates,heparan sulphate and keratan sulphate, as well as non-sulphated hyaluronate (Hart &Lennarz, 1978). In the preceding paper (Mattey & Garrod, 1984) we showed that15-day embryonic chick corneal epithelial cells synthesize fibronectin in culture.Furthermore, they organize exogenous fibronectin into a fibrillar matrix. Here weinvestigate the effects of enzymes directed against collagen, hyaluronic acid andchondroitin sulphates on the morphology and spreading of cultured corneal epithelialcells, and on the fibronectin matrix.

MATERIALS AND METHODS

MaterialsHighly purified bacterial collagenase was obtained from Sigma (type VII; collagenase activity,

1300units/mg). The preparation was described as substantially free of non-specific protease,clostripain and tryptic activities. Highly purified Streptomyces hyaluronidase (2000 TRU/mg) waspurchased from Miles, and chondroitinase ABC from Sigma. Hyaluronic acid from human umbili-cal cord (grade I) and chondroitin sulphate from shark cartilage (grade III, mixed isomers) wereobtained from Sigma, as was the proline analogue L-azetidine-2-carboxylic acid (LACA). Fibronec-tin was purified from chicken plasma (Billig et al. 1982).

Measurement of proteolytic activityThe proteolytic activity of the enzyme preparations was determined according to the method of

Tomarelli, Charney & Harding (1949) with azoalbumin as a substrate. The assay is capable ofdetecting lOng of trypsin (2Xcrystallized, Sigma).

Cell cultureCorneal epithelial cells from 15-day chick embryos were isolated and cultured as described by

Nicol & Garrod (1979). Enzymes were added to dissociated cells in complete MEM with 10 % foetalcalf serum just before plating out, or 24 h after culturing in normal medium. Enzymes were usedat the following concentrations: collagenase, lOOu./ml; Streptomyces hyaluronidase, 20u./ml;chondroitinase ABC, 1 .u./ml. The medium was replaced after the first 24 h and subsequently every48 h with fresh enzyme-containing medium. Cells were also plated out in the presence of LACA(lOO^g/ml), or in some cases were allowed to attach and spread (24 h) before addition of theinhibitor.

GAGs and collagen in cornea! epithelial cells 191

Control cultures were carried out in which the enzymes were boiled for 15 min before additionto the medium.

Substrata

Cells were cultured on plain glass coverslips or plastic tissue-culture dishes, or on coverslipscoated with plasma fibronectin or rhodamine-labelled plasma fibronectin, prepared as describedpreviously (Mattey & Garrod, 1984). Some cells were cultured on collagen gels prepared from ratskin collagen (see accompanying paper, Mattey & Garrod, 1984).

Immunofluorescent stainingCells were fixed and stained at various times for fibronectin, as before (Mattey & Garrod, 1984).

MicroscopyPhase-contrast microscopy was performed with a Nikon model M inverted phase-contrast

microscope. Fluorescence microscopy and interference reflection microscopy were carried out ona Zeiss photomicroscope III as described (Mattey & Garrod, 1984).

RESULTS

Measurement of enzyme protease activity

Commercial enzyme preparations are often quite variable in the amount of non-specific protease activity present, so we carried out measurements on the enzymepreparations to rule out the possibility that any enzyme effects were due to proteolyticdigestion. The results are presented in Table 1. With twice-crystallized trypsin(Sigma) as a standard (detectable at 0-01 /ig/ml) no proteolytic activity was detectableat the concentrations of enzymes used in the cell cultures. Furthermore, all cellcultures were carried out in the presence of 10% foetal calf serum, which shouldinhibit any undetected proteolytic activity.

Effect of glycosaminoglycan lyases and collagenase on cell attachment and spreadingCells attached and spread on plastic, glass and fibronectin-coated glass in the

presence of collagenase, hyaluronidase and chondroitinase ABC. The use of enzymesindividually or in combination revealed no apparent differences in the ability of cellsto attach to various substrata. In all cases the cells spread to form characteristicepithelial cell islands. However, slight differences were noted in the morphology andinitial spreading behaviour of enzyme-treated cells when compared with controls.

Table 1. Measurement of enzyme activity

Concn of enzyme Protease activity inEnzyme activity in culture medium culture medium

Enzyme (units/mg) (units/ml) (^gtrypsin/ml)

100 <0-01

20 <0-011 < 0 0 1

• Chondroitinase ABC was supplied as a freeze-dried powder with activity of 5 units /ampoule.

Bacterial collagenaseStreptomyces

hyaluronidaseChrondroitinase ABC*

1310

20005 u/amp.

in D. L. Mattey and D. R. Garrod

Figs 1-6

GAGs and collagen in corneal epithelial cells 193

It

Figs 7, 8. Effect of LACA on corneal epithelial cell morphology and spreading. Cellsplated out in LACA-containing medium attach and spread poorly on glass (Fig. 7),although they attach and spread better on collagen gels (Fig. 8). However, there is littleor no production of lamellipodia at the leading free edge. Bars, 200 ism.

Control cultures quickly formed a cohesive sheet of cells in the first 24 h, while islandsof cells incubated in enzymes revealed a more reticulate pattern in their initial forma-tion (Figs 1,2). However, by 48 h there was little difference between the appearanceof enzyme-treated and control cultures. With further time in culture, differencesbecame apparent in the marginal cells of the epithelial islands. In control cultures themarginal cells had flattened lamellipodia at the free leading edge. These were veryobvious in controls after 48 h in culture (Fig. 3). However, the number of lamellipodiawas considerably reduced in the presence of hyaluronidase and chondroitinase ABC(Figs 4, 5), although the effect was less obvious in collagenase-treated cells (Fig. 6).It was usually noticeable between 48 h and 72 h in culture but became more pronouncedwith continued exposure to the enzymes. The edges of islands generally appearedmuch smoother than controls and often took on a scalloped appearance.

When enzymes were added to cells previously cultured in normal medium for 24 h

Fig. 1. Control culture of corneal epithelial cells after 24 h on glass. The cells haveattached and spread to form a cohesive sheet of cells. Bar, 400[lm.

Fig. 2. Cells plated out in the presence of chondroitinase ABC (1 u./ml). The cell islandappears to have a more reticulate appearance than in controls. Bar, 400/tfn.

Fig. 3. Control cells after 96 h have flattened lamellipodia at the free leading edge of thecell island. Figs 3-6: bar, 200^m.Fig. 4. Cells plated out in chondroitinase ABC show very little lamellipodial activity atthe island margins after 96 h in culture.

Fig. 5. Cells cultured in Streptomyces hyaluronidase (20u./ml) again have nolamellipodia at the edges of the epithelial islands after 96 h.

Fig. 6. Collagenase (100 u./ml) has less effect on the marginal cells and a few well spreadlamellipodia are still seen (96 h culture).

194 D. L. Mattev and D. R. Garrod

Fig. 9. Periphery of corneal epithelial cell island (control) after 72 h in culture, viewedusing IRM. Focal contacts are common in the marginal cells of the island (arrows). Figs9-12: bars, lOjim.

Fig. 10. The marginal cells of islands cultured in chondroitinase ABC for 72 h show aconsiderable reduction in focal contacts and possess large grey areas at the leading edge.

Fig. 11. Cells cultured in Streptomyces hyaluronidase (72 h) showing lack of focal con-tacts and large grey areas at the leading free edge. Retraction fibrils are seen on thesubstratum beyond the margin of the cells.

Fig. 12. Collagenase-treated cells still possess well-spread lamellipodia and focal contacts,as well as a few large grey areas.

the effect on cell morphology and spreading was less obvious, although marginal cellsgenerally appeared less well spread after a further 48 h in culture.

Inhibition of collagen secretion by LACA produced dramatic effects on adhesionand spreading. Cell plated out in the presence of LACA spread better on collagensubstrata than on plastic, glass or fibronectin, although the marginal cells were still


poorly spread and possessed few lamellipodia (Figs 7, 8). Islands cultured for 24 h innormal medium and then for 24 h in LACA also showed poor spreading of marginalcells. These effects were more dramatic in cells plated on glass and fibronectin thanon collagen. With further culture on glass and fibronectin, the free edges of islandsbecame highly irregular and fragmented, and the islands became vacuolated.

Effect of enzyme treatment on formation of focal contacts

Interference reflection microscopy (IRM) revealed the presence of numerous focalcontacts in the marginal cells of control cultures. These appeared as fine black streaksbetween 24 and 48 h in culture and became larger and more prominent over the next48 h (Fig. 9). Cells cultured in hyaluronidase or chondroitinase ABC showed amarked reduction in the number of focal contacts. The edges of the cells were usuallysmoother in appearance with large grey areas, and retraction fibrils were sometimesseen attached to the substratum beyond the margin of the cells (Figs 10, 11). Similarresults were obtained on all substrata used. Focal contacts were found quite frequentlyin collagenase-treated cells, although they were less common than in controls (Fig.12). Cells cultured in the presence of LACA also showed a reduction in the numberof focal contacts.

Effect of enzyme treatment on the fibronectin matrix

Cells in normal medium produced an extensive fibronectin matrix as described inthe accompanying paper (Mattey & Garrod, 1984).

Compared with controls there was a marked reduction in the amount of fibronectinstaining in the presence of hyaluronidase and chondroitinase ABC. The cells demon-strated some intracellular and fibrillar staining in the first 48 h in culture, but thefibronectin was never properly organized into a fibrillar network. In most cases thefibronectin matrix appeared disrupted and took the form of short fragments ratherthan long, branching fibrils (Figs 13-16). Regions with virtually no fibronectin stain-ing were common. Collagenase had a less noticeable effect on the fibronectin matrix,although the staining pattern was usually less extensive than in controls (Figs 17, 18).However, inhibition of collagen secretion by LACA resulted in a marked decrease inthe amount of fibronectin staining. Cells plated out in LACA on collagen substrataalso demonstrated very little fibronectin staining (Figs 19, 20). In contrast, LACAhad little or no effect on the fibronectin staining of corneal fibroblasts (Fig. 21).

The addition of enzymes to cells already attached and spread had less effect on thefibronectin matrix, especially if added after 2 or 3 days of culture (Fig. 22).

Effect of enzyme treatment on the reorganization of substratum-bound fibronectin

As reported in the accompanying paper (Mattey & Garrod, 1984) control cellscultured on glass coverslips coated with plasma fibronectin or rhodamine-labelledfibronectin showed considerable reorganization of the fibronectin into a fibrillarpattern. This was particularly noticeable at the island margins (Figs 23, 24). How-ever, in the presence of enzymes the cells showed little or no ability to reorganize the


Figs 13-18


fibronectin coat (Figs 25, 26). Only in collagenase was there some reorganization ofthe coat into a fibrillar form (Figs 27, 28). When collagen secretion was inhibited thecellular reorganization of substratum-bound fibronectin was also inhibited and thefibronectin coat remained largely undisturbed.

Figs 13-20. Phase-contrast (Figs 13, 15, 17, 19) and corresponding fibronectin-stainingpattern (Figs 14, 16, 18, 20) of cells cultured for 96 h in chondroitinase ABC (Figs 13,14), Slreplomyces hyaluronidase (Figs 15, 16), collagenase (Figs 17, 18) and LACA(Figs 19, 20). Compare fibronectin patterns with that shown by control cells after 96hin culture, as shown in the accompanying paper (Mattey & Garrod, 1984; fig. 4).Bars, 20 fjm.

Fig. 21. Fibronectin staining of corneal fibroblasts after 96 h in the presence of LACA.Figs 21-28: bars, 20fim.

Fig. 22. Fibronectin matrix of 96 h culture to which chondroitinase ABC had been added48 h after plating out.


Figs 23-28


Recovery of enzyme-treated cells

The effects of the enzymes could be reversed by replacing enzyme-containingmedium with normal medium. In most cases the cells had formed an extensivefibronectin matrix and displayed the full complement of focal contacts within 48 h ofchanging the medium. The effects of LACA could also be reversed by replacinginhibitor-containing medium with normal medium.

Addition of plasma fibronectin (100-250 fig/m\) during enzyme treatment did notrestore cell morphology or matrix organization. Similarly, addition of hyaluronic acid(0-01—lOmg/ml) or chondroitin sulphates (mixed isomers, 0-1—30mg/ml) after 24or 48 h in culture was unable to promote reversal of the enzyme effects.

DISCUSSION

The results of this study suggest that hyaluronate, chondroitin sulphate andcollagen are not essential in the initial attachment and spreading of corneal epithelialcells. However, they are important for the development and organization of theextensive fibronectin-containing extracellular matrix formed by these cells in culture.Thus this matrix that is detected by staining with anti-fibronectin antibody appearsto be a complex consisting of at least three components, GAGs, collagen and fibronec-tin. (The presence of GAGs in the matrix was suggested by ruthenium red stainingand electron microscopy in the accompanying paper.) Furthermore, enzyme treat-ments, which prevent formation of the matrix, also prevent the development of a fullyspread morphology and the full complement of cell-substratum adhesions or focalcontacts. We should also stress, however, that cell—cell adhesion was apparentlyunaffected by the enzymes and cells were able to form characteristic epithelial islandswith well-formed junctions (desmosomes and intermediate junctions) at theultrastructural level (unpublished observations).

A number of studies have suggested that hyaluronate-chondroitin sulphate com-plexes in substrate-attached material (SAM) are important in facilitating cell move-ment by promoting detachment from the substratum (Rollins & Culp, 1979; Culp etal. 1979; Abatangelo, Cortivo, Martelli & Vecchia, 1982). However, the initial attach-ment and spreading of fibroblasts in vitro are apparently unaffected by hyaluronidaseand chondroitinase ABC (Bruns & Gross, 1980; Laterraef al. 1983). In the case ofcorneal epithelial cells the effects of hyaluronidase and chondroitinase ABC onlybecome manifest after about 36— 48 h in culture, when the epithelial islands are wellformed. According to Heath (1982), active migration of these cells virtually ceasesafter this time. Focal contacts become prominent beneath the lamellipodia of cells atthe edges of islands after 48 h and this corresponds with the appearance of an extensive

Figs 23-28. Phase-contrast (Figs 23, 25, 27) and corresponding fibronectin-stainingpattern (Figs 24, 26, 28) of cells cultured for 96 h on plasma fibronectin-coated coverslipsin normal medium (Figs 23, 24), chondroitinase ABC (Figs 25, 26) and collagenase (Figs27, 28).


fibronectin matrix (Mattey & Garrod, 1984). Inhibition or reduction of matrix forma-tion by GAGases or collagenase results in reduced spreading of lamellipodia and a lossor reduction of focal contacts. This suggests that the development of lamellipodia andfocal contacts in these cells is related to the formation of an extracellular matrix. It issignificant that enzymes added at the time of plating out had more effect than enzymesadded when cells had already attached and spread. This suggests that GAGs andcollagen are not essential to maintain the integrity of the fibronectin matrix once it hasformed, although insoluble ECM and cell surface complexes might be protected to acertain degree from enzymic attack.

Fibronectin has binding sites for heparin, heparan sulphate, hyaluronic acid andcollagen as well as the cell surface (see Yamada, 1981), but how it becomes organizedinto its characteristic fibrillar patterns is not clear at the molecular level. Our worksupports the view that other extracellular matrix components are involved in normalfibronectin fibrillogenesis (Iwanaga, Susuki & Hashimoto, 1978; Jilek & Hormann,1979; Vuento et al. 1980). A number of studies have shown that GAGs enhance thebinding of fibronectin to collagen, and that they form insoluble complexes with thesecomponents (Ruoslahti & Engvall, 1980; Johansson & Hook, 1980). Oldberg &Ruoslahti (1982) have recently demonstrated that chondroitin sulphate proteoglycanis able to co-precipitate with collagen and fibronectin to form macromolecular com-plexes. They suggested that proteoglycan induced self-assembly of insolublecollagen-fibronectin-proteoglycan complexes could, along with collagen fibrilformation, play a central role in the formation of the extracellular matrix. Enzymesdirected against GAGs and collagen might thus be expected to reduce or inhibitformation of the extracellular matrix by preventing co-precipitation with fibronectininto insoluble complexes. McDonald, Kelley & Broekelmann (1982) have recentlyshown that Fab to the gelatin binding domain of fibronectin inhibits both fibronectinand collagen organization in fibroblast extracellular matrix. They suggested thatcollagens and fibronectin interact to form a fibrillar component of the matrix, and thatfibronectin is required for normal collagen organization and deposition by fibroblastsin vitro. Our results suggest that secretion of collagen is necessary for deposition ofa fibronectin matrix in corneal epithelial cells, although we have no information as towhether collagen and fibronectin are secreted sequentially or simultaneously, eitheras individual molecules or as part of a macromolecular complex.

Sugrue & Hay (1981) have shown that laminin, fibronectin and collagen can causedisappearance of blebbing activity and reorganization of the cytoskeleton at the basalcorneal epithelial surface. Collagen and laminin were shown to have a direct effect onthe cell surface, although fibronectin was only effective if the cells were able to carryout protein synthesis and/or collagen secretion (Sugrue & Hay, 1982). This led to thesuggestion that, while collagen and laminin can act directly on the cell surface,fibronectin interacts with the basal surface via an intermediate molecule that is likelyto be collagen. Our results support that theory since reorganization of fibronectin into•fibrils is inhibited when collagen secretion is inhibited by L-azetidine-2-carboxylicacid (LACA). Furthermore, cells spread poorly on a fibronectin-coated substratumif LACA is added at the time of seeding. They spread better on collagen substrata,


although the production of a fibronectin matrix and the formation of well-spreadmarginal cells with flattened lamellipodia is considerably inhibited. These observa-tions further suggest that secretion of cellular collagen is necessary for developmentof a fibronectin matrix, and that such a matrix is necessary for expression of a flattenedmorphology. In all cases where the fibronectin matrix is reduced, as in the presenceof GAGases or LACA, the epithelial islands show a similar morphology with lack offlattened marginal cells, and reduction of focal contacts.

A reduction of cell surface fibronectin is commonly associated with the transformedphenotype in fibroblasts (Hynes, 1973; Vaheri & Ruoslahti, 1974), although it is notclear exactly what causes this loss. Transformed cells are still able to synthesizefibronectin, although they may do so at a slower rate, but they are unable to retain iton their surfaces. Transformed cells are well equipped to hydrolyse matrix com-ponents, such as fibronectin, proteoglycans and collagen, although a number ofstudies have failed to implicate proteolysis directly as the reason for fibronectin lossin transformed cells (Vaheri & Mosher, 1978). Also, there is no evidence that lowerproduction or changes in fibronectin can explain the difference in cell surfacefibronectin. Many transformed and tumorigenic cells do show a reduced synthesis ofother matrix components such as collagen and proteoglycans (Kleinman, Klebe &Martin, 1981; Nigram, Brailovsky & Bonaventure, 1982). Our results emphasize thata loss or reduction of any one of these fibronectin-binding molecules may influence theamount of fibronectin retained at the cell surface.

This work was supported by the Cancer Research Campaign.

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(Received JO August 1983-Accepted 24 November 1983)

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ROLE OF GLYCOSAMINOGLYCANS AND COLLAGEN IN THE … · ECM may contain glycosaminoglycans (GAGs) such as heparan sulphate, chon-droitin sulphate and hyaluronic acid, as well as glycoproteins