Human Cells Unable to Express Decoron Produced Disorganized Extracellular Matrix Lacking “Shape Modules” (Interfibrillar Proteoglycan Bridges)

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

243, 59 – 66 (1998)

EX984089

Human Cells Unable to Express Decoron Produced Disorganized Extracellular Matrix Lacking “Shape Modules” (Interfibrillar Proteoglycan Bridges) John E. Scott,*,1 Katharine M. Dyne,† Alison M. Thomlinson,* Mark Ritchie,* John Bateman,‡ Giuseppe Cetta,† and M. Valli† *Chemical Morphology, School of Biological Sciences, Chemistry Building, Manchester University, Manchester M13 9PL, United Kingdom; †Dipartimento di Biochimica “A. Castellani,” Pavia University, Via Taramelli 3B, 27100 Pavia, Italy; and ‡Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia

tions are fundamentally important throughout biology. In animals the fibrils are mainly collagenous and the soluble polymers are carbohydrate-rich proteoglycans (PGs). Until recently the functions of the latter were expressed in general terms, e.g., as matrix swellers [1]. The fibrils are rope-like tension-transmitting and resisting elements which determine the maximum size of the tissue. The actual shape of the extracellular matrix is determined by positioning the fibrils in the right places [2]. In many tissues (cornea, etc.) there is no apparent connection between adjacent fibrils. Why then do they stay in place under mechanical stress? An answer emerged when it was discovered that collagen fibrils are linked by anionic glycosaminoglycan chains (AGAGs) of the small PGs, e.g., decoron [3, 4]. The AGAG interfibrillar ties are dermatan or chondroitin sulfates, plus keratan sulfate in the corneal stroma, attached via PG protein moieties to collagen fibrils at specific binding sites. Possible binding sites and their amino acid sequences were proposed [5]. AGAGs probably form antiparallel aggregates, stabilized by hydrophobic and hydrogen bonding [5]. Because the structures are repeated in a regular and specific fashion every 60 – 65 nm along the fibrils, as a series of modules consisting of the combination (collagen fibril 3 PG protein 3 AGAG aggregate 4 PG protein 4 collagen fibril) they are called “shape modules” [6]. Altering the length of the AGAG chains may regulate the distance apart of the collagen fibrils, determining the shape of the tissue. This is a new and specific role for the small PGs. Given the great importance of shape and its maintenance, it is relevant that no example of a malfunction or malformation of the shape module was known. ECMs with apparently normal shape modules were produced under conditions in which collagen fibrils were laid down in the wrong places (liver fibrosis, [7]) or as a result of disturbed fibrillogenesis (dermatospar-

The shapes of extracellular matrices are determined by positioning collagen fibrils in the right places, oriented and maintained viv-a`-vis each other. The fibrils are linked orthogonally by dermatan/chondroitin sulfates or keratan sulfate (in small proteoglycans) attached every ;65 nm via their protein moieties to collagen fibrils at specific binding sites. These regular repeating structures are the “shape modules.” The characteristic arrays of orthogonal interfibrillar bridges were missing and the extracellular matrix was totally disorganized in matrices produced by fibroblasts taken postmortem from skin of an electively aborted fetus which did not express decoron in culture, thus supporting the shape module hypothesis. Biglycon, dermatan sulfate, heparan sulfate, collagen, and hyaluronan were produced by these cells but did not contribute to a normal extracellular matrix. A similar electron histochemical and biochemical survey of extracellular matrices produced by seven normal and eight osteogenesis imperfecta cell lines from donors of different ages and both sexes showed no comparable disruptions of their matrices. This investigation appears to be the first to demonstrate systematically proteoglycan:collagen interactions in matrices produced by cultured human cells. © 1998 Academic Press Key Words: collagen; dermatan sulfate; osteogenesis imperfecta; biglycon.

INTRODUCTION

The extracellular matrices (ECMs)2 of connective tissues are systems of insoluble fibrils and soluble polymers which evolved to take the stresses of movement and the maintenance of shape [1]. These func1 To whom correspondence and reprint requests should be addressed. Fax: (44) 161 275 4598. 2 Abbreviations used: AGAG, anionic glycosaminoglycan; ECM, extracellular matrix; OI, osteogenesis imperfecta; PG, proteoglycan.

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0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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axis, [8]), as long as recognizable collagen fibrils were present. This suggested that significant disturbance of shape module form or biosynthesis was incompatible with organized tissue. An analysis of recorded osteogenesis imperfecta (OI) cases showed none in which the collagen glycine mutations occurred in the putative PG binding site sequence in the collagen fibril d band [9]. Such genetic errors in this sequence might therefore prevent the development of a viable fetus. A decoron3 gene knockout in mouse demonstrated mechanical deficiencies in the skin [10] although the condition was not apparently life threatening (see Discussion). Very recently a disturbance in the production of the second of the two molecules in the shape module, the small PG, was reported [11]. The proband, an electively aborted fetus, did not express decoron mRNA or decoron itself in cultures of skin fibroblasts taken postmortem. By light microscopy the morphology of the cells appeared abnormal. It was of the utmost interest to see whether shape modules would be present in matrices produced by these cells. In fact the characteristic orthogonal arrays of Cupromeronic blue-stained AGAG interfibrillar bridges were missing and the ECM was very disorganized, thus supporting the shape module hypothesis. These preliminary findings were reported [12], but since the proband suffered an OI mutation at glycine 415, it was possible that the picture was due in part at least to that mutation. We have now accumulated the necessary control cases in which identical or nonidentical mutations were found at the same position, together with age-matched and other controls, and we can say that the disorganized ECM was not due to the OI type I collagen mutation but to the lack of decoron. The proband thus represents the first example of a human pathology involving demonstrable changes in the production of shape modules. MATERIALS AND METHODS Fibroblast cell lines are listed in Table 1. Skin biopsies were taken from the forearm (adults) or from sites of incision during surgical or postmortem procedures (children). Normal skin fibroblasts from a 12-week-old fetus {1502}, a 31-month-old male {2127}, and a 26-yearold female {2068} were from the American Type Culture Collection or from the Pavia collection (3- {258} and 4-year-old {335} males). Pathological cells from OI patients included those {251} unable to express decoron [11]. Cells from the parents, {252} and {253}, of {251} were also included. Two OI cell lines, {508} and {353}, that demonstrated mutations at the same glycine (415) as {251} were from Dr. A. Nicholls [13] or the Royal Children’s Hospital, Melbourne, respectively. 3 The terms “decoron” and “biglycon,” used in this paper instead of the older decorin and biglycan, refer specifically to the proteins of these PGs, thus avoiding confusion inherent in the older terms, which referred to the whole molecule, i.e., protein (gene product) plus dermatan sulfate (posttranslational product) [32]. The newer terms are recognized by the IUPAC-IUBMB Nomenclature Committee [33].

TABLE 1 Details of Skin Fibroblasts from Osteogenesis Imperfecta (OI) Patients and Controls

Cell strain

Phenotype

Age at biopsy

Mutated glycine in type I collagen a1 chain, position and product

156 242 251 276 353 383 440 508 252 253 235 236 258 335 1502 2068 2127

OI I OI II OI II/III OI I OI III/IV OI II OI II OI III/V Father of 251 Mother of 251 Control Control Control Control Control Control Control

3 years 16 days Fetus, 24 weeks 29 years 50 years Fetus, 24 weeks Fetus, 23 weeks 19 months

901 Ser 910 Ala 415 Ser 85 Val 415 Cys 994 Asp 319 Val 415 Ser

32 years 32 years 4 years 3 years Fetus, 14 weeks 26 years 2.5 years

Clinical, genetic, and biochemical characteristics of the patients are published ({251} [12, 14], {508} [15], {353} [16]). Plasmids P2 and P16 for identification of human decoron and human biglycon messenger RNA [17] were gifts from Dr. L. Fisher (NIH, Bethesda, MD). Cupromeronic blue and chondroitinases were from Seikagaku (Tokyo, Japan). Carrier-free [35S]sulfate was from NEN Life Science Products (Italy). Other chemicals were best available commercial grade. Cell Culture Cells were grown in Dulbecco’s modified Eagles medium (DMEM) and used between passages 5 and 16. Where possible cells were plated at the same passage number. Biochemical and ultrastructural studies were performed on long-term cultures maintained for up to 3 weeks postconfluency in the presence of 0.25 mM ascorbic acid to enhance collagen matrix formation [18], each series in triplicate. Biochemistry Cells. Before labeling cells were incubated in serum-free DMEM containing ascorbate (100 mg/ml for 4 h), which was replaced with DMEM containing ascorbate and 50 mCi/ml carrier-free [35S]sulfate for 24 h. PGs. PGs were isolated by ion exchange from DEAE–Sephacel columns and treated with chondroitin ABC lyase to remove the AGAG chains [11]. Intact PGs were electrophoresed on 8% SDS– PAGE gels and fluorographed; after chondroitin ABC lyase digestion core proteins were electrophoresed on 10% gels and immunoblotted with antibodies against human decoron [11]. Isolated total RNAs. Isolated total RNAs from confluent cell layers were electrophoresed on 1.5% formaldehyde/agarose gels and analyzed by Northern blotting using 32P-labeled cDNA probes encoding for human bone decoron or biglycon [11]. Collagen. On day 21 washed cell layers were sonicated on ice in 50 mM Tris–HCl, pH 7.5, containing 150 mM NaCl and protease inhibitors. Centrifuged insoluble material was lightly digested with

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MgCl2, pH 5.8, containing 2.5% glutaraldehyde, for 1 h at room temperature [23] in Pavia. After being washed in buffer/MgCl2 and then in 0.5% (w/v) sodium tungstate the petri dishes were freeze dried. Processing for electron microscopy (dehydration, embedding in Spurr resin, sectioning) was completed in Manchester, where ultrathin sections were viewed using either a Philips 400 or a 420 TEM. Staining of ultrathin sections for collagen fibrils and their banding patterns was with uranyl acetate.

RESULTS

Biochemistry

FIG. 1. Electrophoresis of anionic glycosaminoglycans isolated from cultures of the decoronless proband cell line {251} and a normal cell line {1502}. They were run on cellulose acetate in 0.05 M pyridinium formate buffer at pH 3.3 containing 5.0 M urea and 0.2 M guanidinium chloride. The strip was stained with Alcian blue 8G (see text for further details). The markers are labelled on the right: cs, chondroitin sulfate; ds, dermatan sulfate; ha, hyaluronan. AGAGS from (lanes A and B) {251}, (C and D) {1502}, (E) standards of hyaluronan and chondroitin-4-sulfate; (F) standard pigskin dermatan sulfate, (G and H) chondroitin-4-sulfate. AGAGs in lanes A, C, and G were digested with chondroitin AC lyase before electrophoresis. Chondroitin sulfate in lane H was incubated in buffer without enzyme and recovered for electrophoresis. Considerable amounts of AGAG moving at the speeds of hyaluronan and chondroitin sulfate were removed by digestion with the enzyme from A, C, and G, but significant amounts of AGAG moving at the speed of dermatan sulfate remained after digestion in A and C.

pepsin (100 mg/ml in 0.5 M acetic acid, 3 h at 15°C). Collagens were electrophoresed on 6% SDS–PAGE gels containing 0.5 M urea and stained with Coomassie brilliant blue [19]. AGAGs. AGAGs were isolated from total freeze-dried cell cultures by precipitation with cetylpyridinium chloride after digestion with papain at 65°C. They were electrophoresed on cellulose acetate membranes in 0.1 M HCl [20] and stained with Alcian blue [21]. Electrophoresis in 0.05 M pyridinium formate buffer, pH 3.3, containing 5 M urea and 0.2 M guanidinium chloride discriminated between dermatan and chondroitin sulfates. Aliquots digested with chondroitin AC lyase were similarly electrophoresed (Fig. 1). Hydroxyproline. Hydroxyproline was measured by a scaled-down version of Woessner’s [21] method after hydrolysis of the total freezedried cell culture in 5 M HCl and deacidification using methyl didodecylamine [21]. UV absorption. UV absorption at 260 nm was determined on papain digests before precipitation with cetylpyridinium as a rough estimate of total nucleic acids. One unit was defined as an optical density of 1.0 in a 1-ml sample in a 1-cm light path. Ultrastructure Cell cultures at various times after plating were stained for sulfated AGAGs using 0.05% (w/v) aqueous Cupromeronic blue in 0.1 M

Hydroxyproline levels. Hydroxyproline levels as a ratio to 260-nm absorption (mg hyp/absorbance at 260 nm in a 1-cm cell/ml solution, see Materials and Methods) were similar in most cell cultures grown for comparable times, although absolute amounts of hydroxyproline per culture vessel varied considerably and increased with increasing time in culture. Five OI cultures ({156}, {242}, {383}, {440}, and {508}) grown in duplicate for 16 days averaged 0.39 (0.08 – 0.74). Three normal cultures ({236}, {258}, and {335}) similarly grown at the same time averaged 0.27 (0.23– 0.32). {251} grown for 12, 13, or 20 days gave ratios of 0.37, 0.26, and 0.66, respectively. {1502}, a control, gave 0.74 from a 20-day culture. Type I collagen. Type I collagen (including crosslinked dimers) was identified in substantial amounts in cultures of control and pathological cell lines. Mutant trimers were present in matrix from {251} and {508} cells (Fig. 2). AGAGs. AGAGs contained considerable amounts of sulfated material traveling slightly more slowly than a chondroitin sulfate standard on electrophoresis in 0.1 M HCl. This strongly suggests that all the AGAGs were sulfated at less than 1.0 sulfate ester group per disaccharide unit [20]. Although significant amounts of this material were digested by chondroitin AC lyase a relatively large amount of undigested material that migrated in the chondroitin/dermatan sulfate position remained. Some of this material moved to the dermatan sulfate position in pyridinium formate buffer (Fig. 1). Hyaluronan, identified by its low mobility in both buffers and its digestibility by AC lyase, was present in considerable amounts (Fig. 1). Heparan sulfate, identified by its electrophoretic mobility, which is less than chondroitin sulfate but more than hyaluronan in both buffers, and resistance to chondroitin ABC lyase, was also present, in smaller amounts than the other AGAGs. Proteoglycans. Decoron was present in all cell cultures except {251}. Biglycon was present in all cell cultures. mRNA. Decoron mRNA was expressed in all cell cultures except that of {251}. Biglycon messenger was present in all cell cultures.

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staining material, possibly collagenous, conUO21 2 taining parallel fine elements without banding, frequently in sheaf-like groups (Fig. 3). Proteoglycan. Cupromeronic blue-stained filaments were always present, sometimes in great profusion (Fig. 3), but not necessarily in ordered arrangements. {251} ECM showed considerable amounts of disordered PG, associated with UO21 staining material, as did many 2 other cell cultures. Cell membranes frequently contained Cupromeronic blue-stained filaments of size and shape typical of PG AGAGs. Orthogonal PG arrays. Orthogonal PG arrays (shape modules) bridging the collagen fibrils were present in all cultured ECMs except for {251} and {253} (Fig. 3). DISCUSSION

FIG. 2. SDS–PAGE (6% gel) of pepsin-extracted collagens from long-term cultures in the presence of ascobic acid. Lane 1, control; lane 2, {252}; and lane 3, {508}. All fibroblasts deposited collagen containing substantial amounts of b components. Type I collagen a chains are indicated on the left. Abnormally migrating mutant a chains are arrowed (right).

Ultrastructure Collagen fibrils. Collagen fibrils were present in the ECMs of all normal and most OI cell cultures. Widths varied from 15 to 45 nm. Banding Dperiodicity was usually apparent but an a– e banding pattern was seldom clear. Two of three cultures of {251} grown on separate occasions showed no collagen fibrils, nor did those of the mother ({253}) of {251} although the father ({252}), who was a carrier of the OI 415 mutation showed a few fibrils, consistently present on three separate occasions. {251} and {253} showed the sparsest matrices of the 18 cell lines examined. There were considerable amounts of

Investigation of PG-collagen-rich ultrastructure in tissue culture promises answers to problems of ECM temporal and spatial organization during development. Such investigations were rare or nonexistent for technical and theoretical reasons. The invention of reagents to demonstrate PG AGAGs by electron microscopy [24] and of methodology for their application to thin and fragile tissue culture matrices were keys to the technical problems. The recognition of patterns of normal PG– collagen interactions, e.g., shape modules [2, 6, 13], provided criteria on which to judge cultured ECMs. The present investigation appears to be the first systematic examination of PG– collagen interactions in cultured ECMs. ECMs produced by cultured cells from patients with inborn errors were compared with ECMs produced by normal and control cells. Specifically, the aim was to compare ECMs from a decoron-poor cell line with those from control cell lines, to see if the hypothesis of the ECM-organizing function of decoron, in giving shape modules, is valid. Because the decoron-deficient cell line also had an OI mutation, controls had to cope with the effect of that mutation at glycine 415 in the type I collagen a1 chain. In fact we found two OI controls ({508} and {353}), each with a different mutation at glycine 415, one of which ({508}, Gly 3 Ser) was identical with that in the decoron-deficient proband.

FIG. 3. Matrices produced in culture by normal {335} (a and b) and pathological decoronless {251} cells (c and d). All are stained with Cupromeronic blue [22] (0.05% in 0.025 M NaAc buffer, pH 5.8, containing 0.1 M MgCl2) to demonstrate PG AGAGs (arrowed) which are present in highly regular arrays of filaments orthogonal to the collagen fibrils in (a) and (b). Collagen fibrils, which are unstained in (a), where they appear as lighter thin bands against the darker plastic, are oriented in parallel vis-a`-vis each other. PG AGAG filaments (the shape modules) bridge across several fibrils at a time. In (b) collagen fibrils are stained with UO21 2 and interfibrillar AGAG bridges are seen. This type of ordered picture was observable in all normal cell culture ECMs and in many of the OI cell culture ECMs (Results). In (c) the PG AGAG filaments appear in a disordered distribution with collagen fibrils not clearly discernible. After staining with UO21 2 , fibrils (presumable collagenous) are more clearly visible in (d) compared with (c) and some of the PG AGAGs may be associated with these fibrils. This picture shows the most ordered PG– collagen structures observed in over 130 pictures from four independent {251} cultures grown at different times. In general no ordered PG– collagen interactions were seen in ECMs in cultures produced by {251} cells. Bar, 200 nm.

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Significantly both gave good ECMs with recognizable collagen fibrils and shape modules, in contrast to {251}, which produced decoronless ECM with no shape modules. Certain other clear results were obtained: 1. Three of seven OI cell lines produced good ECMs and although matrices from three more ({276}, {353}, and {440}) contained fewer collagen fibrils and orthogonal interfibrillar AGAG filaments, their ECMs were nevertheless qualitatively normal. All of them expressed decoron. Thus OI cells with mutations at various points along the collagen a1 chain retain an ability to produce organized ECMs with shape modules in tissue culture. None of these mutations were in the putative PG binding regions of the collagen fibril (cf. [9]). 2. All cell lines made comparable amounts of hydroxyproline per unit of nucleic acid UV absorption over similar periods of time but not all converted this into recognizable collagen fibrils, periodically banded. 3. Several cell lines, {251, 156, 335, 383, 2127, and 1502} inter alia, produced AGAG with the properties of dermatan sulfate. This implies that a deficiency of decoron in {251} was not associated with a qualitative block in producing dermatan sulfate. Since they all produced biglycon, biglycon can be produced when decoron is absent, as in {251}. 4. Cells from infants in the 2- to 4-year age group— even the OI patients {156} and {508}—produced the most prolific matrices. Other parameters, e.g., number of passages, length of time in culture, and type of surface (plastic, glass, Thermanox) on which cells grew, appeared to be of lesser importance and no clear trends were observed in either biochemical or ultrastructural criteria. Most skin biopsies were taken from forearms (adults) or upper thighs (children). 5. Interestingly, cells from the mother {253} of the decoronless proband produced sparse matrices with low or negligible incidence of interfibrillar AGAG bridges in several cultures at different times. She expressed decoron mRNA [11]. Paradoxically, since he was a mosaic carrying the OI gene that affected the proband ({251}), the father’s ECMs were comparable to many controls and OI patients. He expressed decoron mRNA. It is not known why {251} cells are unable to express decoron messenger. It is intended to immortalize them to facilitate further studies. The clear differences between decoronless {251} and all the other cell lines provide good support for the hypothesis that decoron acts as a tissue organizer [3– 6]. Following up the discovery that decoron and other members of the leucine-rich repeat proteins (e.g., proteokeratan sulfates) were symmetrical horseshoes, it was suggested that their properties would help to

organize collagen fibril formation as well as fibril–fibril orientation in shape modules [5]. Our findings that the matrices from cells producing decoron also contained recognizable collagen fibrils and shape modules is compatible with these ideas. The converse, that a decoronless cell line did not produce either shape modules or significant numbers of collagen fibrils, strongly supports the hypothesis. It appears from this one cell line {251} that biglycon cannot substitute for decoron in either of the two proposed functions of decoron at this stage in culture development. How important is the absence of decoron from the ECM? A decoron knockout mouse developed to a complete animal, although with fragile skin [10]. There were still interfibrillar bridges in the affected skin, whereas human cells {251} produced a decoronless matrix without shape modules (this paper). In this context there are two binding sites for proteodermatan sulfates along the collagen fibril, in the d and e bands, respectively [2, 4], and it is not clear which of them (either or both) is vacant in the absence of decoron. In normal skin and tendon the e band is much less frequently occupied by proteodermatan sulfate than the d band. It is possible that an as yet unrecognized minor proteodermatan sulfate specific to the e band may be produced with or without decoron. The properties of the two sets of proteodermatan sulfate AGAGs differed in corneal stroma [25]. The decoronless proband produced dermatan sulfate (which may have been destined exclusively for biglycon). However, mouse knockout models may not faithfully reproduce human ECM pathology. This was clearly demonstrated by studies on a type X knockout mouse, in which the total absence of type X collagen, a major structural component of the growth plate cartilage, led to little [26] or no pathology [27], whereas a reduction of 50% type X collagen (haploinsufficiency) in human growth plate results in Schmid metaphyseal chondrodysplasia [28, 29]. Furthermore a malfunction affecting a generally occurring ECM component may nevertheless be tissue specific, as with procollagen processing in dermatosparaxis, in which normal ultrastructure is found in tendons but totally abnormal fibrillogenesis occurs in skin [8]. The fibrils of the skin of the decoron-knockout mouse [10] were very reminiscent of those in old tendon [30], in which the ratio of proteodermatan sulfate to collagen is low compared with young tissue, with irregular cross-sectional appearance of fibrils. It was suggested that one function of proteodermatan sulfate was to inhibit fusion of collagen fibrils to form large and irregular fibrils [31]. With the proviso that tissue culture conclusions may not apply unmodified to complete animals, we believe that this is the first human pathology in which the

DECORON-LESS HUMAN FIBROBLAST MATRIX LACKS SHAPE MODULES

absence of shape module-forming function has been demonstrated. Tissues from the aborted fetus were not available to us.

15.

This work was supported by the Medical Research Council, London; MURST and CNR Italy; and the National Health and Medical Research Council of Australia.

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