Attachment of articular cartilage chondrocytes to the tissue form of type VI collagen

ELSEVIER

f ic~a Acta etBiochi Blbphysica

Biochimica et Biophysica Acta 1249 (1995) 180-188

Attachment of articular cartilage chondrocytes to the tissue form of type VI collagen Jose Marcelino, Cahir A. McDevitt

*

Section of Musculoskeletal Biology, Department of Biomedical Engineering, The Cleveland Clinic Foundation Research Institute, Cleveland, Oil 44195, USA

Received 29 December 1994; accepted 3 February 1995

Abstract Type VI collagen is composed of a short triple helix rich in RGD sequences with globular domains at each extremity of the helix. Disulfide-bonded tetramers of the monomeric molecule associate non-covalently to form networks of microfibrils in connective tissues, including cartilage. The disulfide-bonded tetramer can be extracted with 6 M guanidine HCI and purified without pepsin digestion and is referred to here as the tissue form of type VI collagen. Type VI collagen in mature articular cartilage appears to be concentrated pericellularly. We undertook a systematic investigation using solid phase assays to establish the nature of the attachment of bovine articular cartilage chondrocytes to the intact, tissue form of bovine type VI collagen. The tissue form of type VI collagen was extracted from bovine meniscus cartilage with 6 M guanidine HC1 and purified by polyethylene glycol precipitation. When equal molar quantities were coated on microwells, the tissue form of type VI collagen attached more cells than the pepsin-digested form of the molecule that lacked the globular domains. The attachment to the intact, tissue form was dose-dependent and saturable and was not inhibited by heparin or type II collagen. A linear GRGDSP peptide failed to inhibit attachment of the chondrocytes to the intact, tissue or pepsin-digested forms of type VI collagen, but totally inhibited the interaction when the intact molecule was reduced and alkylated. In contrast, a cyclic C *GRGDSPC * peptide inhibited attachment to the tissue form of type VI collagen, but not to fibronectin. The attachment had a metal ion dependence that could be satisfied by MnC12, slightly less by MgCI 2, but not at all by CaC12. A direct interaction between the tissue form of type VI collagen and a chondrocyte cell surface receptor or receptors is a structural feature of the pericellular matrix in cartilage. Keywords: Type VI collagen; Chondrocyte; RGD; Cartilage; Cell attachment; Integrin

1. Introduction Mature articular cartilage is comprised of relatively few chondrocytes embedded in an extracellular fibrillar meshwork of type II collagen, with smaller amounts of type IX and type XI collagens [1]. Proteoglycan aggregates, small proteoglycans and a range of glycoproteins are the other major macromolecules in the tissue [2-5]. A distinct subset of the matrix glycoproteins is the family of multi-domain connecting proteins [6], that include type VI collagen [7,8], fibronectin [9], thrombospondin [10] and cartilage oligomeric matrix protein (COMP) [11]. The primary function of the chondrocyte is to maintain the structural integrity of its surrounding extracellular matrix. The pericellular microenvironment of the chondrocyte

* Corresponding author. Fax: + 1 (216) 4449198. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4838(95)00026-7

must play a critical role in the cell-matrix interactions that enable the cell to maintain homeostasis in the normal tissue and to initiate repair processes in damaged tissues. Type VI collagen is a significant constituent of this pericellular microenvironment as demonstrated by immunolocalization [7,12], immunoelectron microscopy [ 13] and the isolation of chondrocytes with their surrounding matrix ( ' c h o n d r o n s ' ) after tissue homogenization [14]. Type VI collagen is also present in the interterritorial matrix. The type VI collagen molecule is composed of three genetically distinct a-chains that form a short triple helix (about 105 nm) with non-helical extensions that appear as large globular domains in the electron microscope [15]. Distinctive features of the molecule are the presence of repeats in the globular domains that have homology with the collagen binding domain of von Willebrand factor and the presence of at least 11 R G D sequences in the triple helical portion of the molecule [16]. Digestion of type VI

J, Marcelino, C.A. McDeL, itt / Biochimica et Biophysica Acta 1249 (1995) 180-188

collagen with pepsin removes most of the globular domains, yielding three fragments of molecular weights with approx. 70 kDa, 55 kDa and 40 kDa respectively that represent primarily the a-chains of the triple helical portion of the molecule [17,18]. Type VI collagen molecules form disulfide-bonded tetramers a n d / o r dimers. Tetramers are considered to bind non-covalently to one another to form branched microfibrils that course throughout cartilage [13] and other connective tissues [19]. The interactions of the type VI collagen microfibrils with cells and extracellular matrix molecules are probably very important in the macromolecular organization in the tissue. Experimental canine [20] and human [21] osteoarthritic cartilages are enriched in type VI collagen, implying a role for this protein in the disease process. The disulfide-bonded tetramer a n d / o r dimer can be extracted from cartilage with 6 M guanidine HCI. The type VI collagen isolated from these extracts is referred to as the tissue form of type VI collagen in this study, to distinguish it from the pepsin-digested forms of the protein. Carter was the first to observe that a preparation of type VI collagen ('GP 140 glycoprotein') could attach human lung fibroblasts in vitro [22]. Subsequent studies showed that type VI collagen isola~Eedfrom pepsin digests, and that therefore lacked the globular domains, could attach a range of fibroblast and tumor cell lines [23,24], embryo arterial smooth muscle cells [25] and neural crest cells [26]. Corneal fibroblasts [27] and the neural crest cells [26] could attach to the intact, tissue form of type VI collagen. Loeser reported that bovine chondrocytes attached to pepsin-digested human type VI collagen and bovine fibronectin, and that the interaction with both proteins could be inhibited by a cyclic RGD-containing peptide [28,29]. A systematic study of the capacity of the intact, tissue form of type VI collagen to attach chondrocytes has not been reported. The presence of type VI collagen in the pericellular domain of articular chondrocytes, the involvement of the protein in osteoarthritis, the presence of relatively large amounts of RGD sequences in the molecule and the demonstration that the protein functioned as a cell attachment factor for other cells ]prompted us to evaluate whether the tissue form of type VI collagen was a cell adhesion protein for articular cartilage chondrocytes. Moreover, we wished to establish whether any interaction between chondrocytes and the tissue form of type VI collagen was RGD and metal ion dependent, and whether there was any difference in attachment properties between the tissue and pepsin-digested forms of the protein.

181

charcoal adsorption. Bicinchoninic acid (BCA) kit for protein assays and Iodo-Beads ® were purchased from Pierce, Rockford, IL. Carrier free Na125I (0.1 mCi//zi) and anti-keratan sulfate monoclonal antibody (5D4) were purchased from ICN Biomedicals, Irvine, CA. Goat anti-type II collagen polyclonal antibody was purchased from Southern Biotechnology Assoc., Birmingham, AL. Linear synthetic peptides (GRGDSP and GRGESP) and cell culture reagents were purchased from Gibco/Life Technologies (Grand Island, NY). Poly-Prep chromatography column was purchased from Bio-Rad, Richmond, CA. Sephadex G-50 (superfine) was from Wallac, Gaithersburg, MD. Synthetic cyclic peptide C* GRGDSPC*, where C ' C * refers to a disulfide-bond, and the scrambled version, C* SRPGGPC*, were kindly donated by the Chemical Sciences Department, Tanabe Research Laboratories, San Diego, CA. Heparin, polyethylene glycol (MW 3350) and pepsin (EC 3.4.23.1) were purchased from Sigma, St. Louis, MO. Pepstatin was from Bachem California, Torrance, CA.

2.2. Isolation of intact, tissue form of type VI collagen The tissue form of type VI collagen was purified as previously described with minor modifications [30]. Bovine meniscus cartilage was ground in liquid nitrogen and extracted for 48 h at 4° C with 6 M guanidine HCI, 0.05 M sodium acetate (pH 6.2), containing the following proteinase inhibitors: 0.01 M EDTA; 0.1 M e-amino-n-caproic acid; 0.005 M benzamidine HCI; 0.001 M N-ethylmaleimide; 0.001 M phenylmethylsulfonyl fluoride (hereafter referred to as 'proteinase inhibitor solution'). The extract was diluted to 4 M guanidine HCI with the proteinase inhibitor solution followed by the 1:1 (v:v) addition of 60% polyethylene glycol in 2.04 M guanidine HCI, 0.2 M CsCI, 0.2% octyl /3-glucoside (pH 6.2). The precipitate that formed overnight at 4° C was washed with cold distilled water, recentrifuged, and dissolved in 4 M guanidine HCI containing proteinase inhibitors. The sample went through two more cycles of polyethylene glycol precipitation and was then dissolved in 4 M guanidine HCI, 25 mM Tris (pH 7.3). The concentration of protein was measured by the bicinchoninic assay with bovine serum albumin (BSA) as standard [31]. A conversion factor was empirically determined to convert concentration units of BSA to those of bovine type VI collagen. The final concentration of type VI collagen was usually = 10 mg/ml.

2.3. Isolation of type H collagen and fibronectin 2. Materials and methods

2.1. Materials Ultrapure guanidine HC1 was purchased from United States Biochemical, Cleveland, OH and further purified by

Type II collagen was purified from pepsin digests of bovine articular cartilage by three cycles of 0.9 M NaC1 precipitation in 0.5 M acetic acid [32]. Fibronectin was purified to electrophoretic homogeneity from bovine plasma by gelatin and heparin-agarose affinity chromatog-

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raphy [33]. This preparation of fibronectin did not cross react with the anti-type VI antibody (5D3). 2.4. lodination of type VI collagen 125I-Labelled type VI collagen was employed to establish the amount of protein coating the microwells. Tissue form and pepsin-digested type VI collagen were iodinated in the presence of Iodo Beads ® using the method recommended by the manufacturer. Carrier free Na125I (0.5 mCi) was incubated with 200 /xg of type VI collagen in 150 /xl of 4 M guanidine HCI, 0.1 M Na2HPO 4 (pH 6.5; reaction buffer) for 15 min at room temperature. The solution was aspirated and the Iodo Beads ® were washed once with reaction buffer and then pooled with the labelled sample. The sample was then chromatographed in a Sephadex G-50 column (Bio-Rad Poly-Prep column, Vt = 6.4 ml) that was equilibrated with the reaction buffer. To determine the amount of type VI collagen binding to the well, radiolabelled protein was mixed with unlabelled type VI collagen (approx. 1.8. 105 cpm/0.5 /xg per 100 /xl per well) and then coated in TBS (0.15 M NaC1, 20 mM Tris, pH 7.5) overnight at 4 ° C. The coating solution was aspirated and the wells were washed three times with TBS. The aspirate and washes for each well were pooled, which constitute the unbound material. The bound material was recovered by incubating the wells with 1 M NaOH for 1 h at 22 ° C. The bound and unbound samples were counted using a LKB-Wallac CliniGamma counter. 2.5. Preparation of reduced and alkylated type VI collagen Type VI collagen (approx. 10 m g / m l in 4 M guanidine HC1, 25 mM Tris, pH 7.3) was reduced with 0.08 M dithiothreitol at 37°C for 30 min and alkylated with 0.12 M iodoacetic acid at 37°C for an additional 30 min. This sample was then dialyzed extensively against water, lyophilized and then reconstituted with 4 M guanidine HC1, 25 mM Tris (pH 7.3). 2.6. Preparation of pepsin digested type VI collagen A solution of the tissue form of type VI collagen (approx. 10 m g / m l in 4 M guanidine HCI, 25 mM Tris, pH 7.3) was dialyzed against 0.5 M acetic acid (pH 2.4) for 24 h at 4 ° C. The dialyzed sample was then incubated with pepsin at an enzyme:substrate ratio of 1:40 (w:w) for 60 h at 4° C. The digestion was terminated by adding pepstatin (10 mM, final concentration). The digested material was precipitated with 2 M NaC1 overnight at 4° C and then centrifuged. The precipitate was dissolved in 4 M guanidine HC1, 25 mM Tris (pH 7.3). 2.7. Cell culture Chondrocytes were isolated from 7-12 month old bovine articular cartilage essentially by the method of

Green [34]. Briefly, the articular cartilage was carefully shaved from the knee joint, minced and then sequentially digested with testicular hyaluronidase (0.05%), trypsin (0.2%) and collagenase (0.2%). Chondrocytes were cultured at high density (4. 106 cells/100 mm dish) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 /xg/ml), amphotericin B (0.25 /xg/ml) and sodium ascorbate (50 /zg/ml). The medium (10 ml/culture) was changed every other day. Cell cultures were maintained at 37 ° C in humidified 95% air, 5% CO 2. The cells were harvested at confluence (5-7 days) with 0.25% trypsin/2.5 mM EDTA. The cells at confluence stained positively for type II collagen and keratan sulfate, consistent with the predominant chondrocyte phenotype noted by Loeser [28] at this time interval.

2.8. Cell attachment and inhibition assays Each assay plate (96-well) was coated with a fixed amount of type VI collagen (0.5 / z g / 1 0 0 / M / w e l l ) in TBS for 16 h at 4 ° C. The wells were blocked with 200 /zl of 0.75% BSA in 0.14 M NaC1, 20 mM Tris (pH 7.5) for I h at room temperature. Primary cultured cells were detached after confluence as described above, washed twice with PBS (0.15 M NaC1, 13 mM Na2HPO4, 2 mM KH2PO4, pH 7.2), resuspended in serum-free DMEM and allowed to recover for 30 min at 37 ° C. For the inhibition experiments, 5 • 104 cells/100/xl per well were mixed with a fixed amount of peptide (GRGDSP or GRGESP, 50 /xg/ml) or varying amounts of peptide (GRGDSP or GRGESP, C* GRGDSPC* or C* SRPGGPC *, 0-500 /zg/ml) in serum-free DMEM. Cells at the same concentration were also mixed with varying concentrations of EDTA (0-2.75 mM), or matrix molecules (type II collagen at 1 m g / m l or heparin at 10 m g / m l ) in serum-free DMEM. The cells and added substance were then added immediately to the coated wells. Cells in serum-free DMEM without any additives that were incubated in type VI collagen-coated wells or in BSA-coated wells as positive and negative controls respectively, for these experiments. After incubation for 2 h at 37 ° C, wells were carefully washed four times with PBS. The relative quantity of attached cells was measured by the bicinchoninic acid protein assay [35]. This assay measures the total amount of cell surface protein on the cells attached to the well. The amount of type VI collagen or BSA coating the wells was below the detection limit for this assay. In pilot experiments, we established a linear relationship between absorbance and number of cells. Cell adhesion experiments were performed several times with different preparations of cells and type VI collagen. The effect of divalent cations on cell attachment was studied with cells that were allowed to recover in serumfree DMEM and then washed twice with PBS and resus-

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t i o n s o f CaC12, MgC12, or MnC12 w e r e a d d e d to the c o a t e d wells a n d i n c u b a t e d for 2 h at 37 ° C. T h e a m o u n t s o f a t t a c h e d cells w e r e a n a l y z e d b y the B C A m e t h o d as described above.

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3. Results 3.1. Electrophoresis

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Fig. 1. Polyacrylamide (3-20%)-agarose (0.4-0%) gradient electrophoresis of type VI collagen preparations. The gel was stained with Coomassie brilliant blue. Lane A, reduced tissue form of type VI collagen showing the typical ladder of bands between 185-240 kDa and a band at around 145 kDa. Lane B, non-reduced tissue form of type VI collagen• Lane C, reduced standards with molecular masses of 200 kDa, 116 kDa, 97 kDa, 66 kDa, and 45 kDa. Lane D, reduced pepsin-digested type VI collagen, prior to salt precipitation, showilag three prominent bands at 65 kDa, 59 kDa, and 47 kDa (arrowheads). Lane E, non-reduced pepsin-digested type VI collagen, prior to salt precipitation, showing an intensely stained band at apparent molecular mass above 400 kDa. Lanes F and G are the reduced and non-reduced pepsin.

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Fig. 3. Effects of increasing GRGDSP peptide concentrations on the attachment of chondrocytes to the intact, tissue form (A), reduced and alkylated tissue form (B), and pepsin-digested tissue form (C) of type VI collagen. Chondrocytes (5. 104/100 /xl per well) were mixed with different concentrations of GRGDSP or GRGESP in serum-free DMEM and added to the coated wells. (A) ( 0 ) RGD (VI): RGD containing linear peptide (GRGDSP); wells were coated with intact, tissue form of type VI collagen. (m) RGE: RGE containing linear peptide (GRGESP); wells were coated with intact, tissue form of type VI collagen. (C)) RGD (FN): RGD containing linear peptide (GRGDSP); wells were coated with fibronectin. (B) Wells coated with reduced and alkylated tissue form of type VI collagen• (C) Wells coated with purified pepsin digest of the tissue form of type VI collagen. Data represent the mean+standard deviation.

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J. Marcelino, C.A. McDevitt / Biochimica et Biophysica Acta 1249 (1995) 180-188

istic 'ladder' of four bands with molecular weights from 185 kDa up to 240 kDa and a strongly staining, slightly broad band at about 145 kDa (Fig. 1, lane A) consistent with previous reports [30,36,37]. No bands were evident in the gel when the tissue form of type VI collagen was run without prior reduction (Fig. 1, lane B), indicating the high degree of purity of the preparation. The reduced pepsin-digest of the tissue form of type VI collagen migrated as three major bands with molecular weights of 65 kDa, 59 kDa and 47.5 kDa respectively, with two faintly staining bands at 127 kDa and 135 kDa (Fig. 1, lane D). Unreduced, the pepsin digested type VI collagen migrated as a broad band with an apparent molecular weight of above 400 kDa, consistent with the structure of a disulfide-bonded dimer or tetramer of the triple helix (Fig. 1, lane E). The preparation of intact, tissue form of type VI collagen appeared pure with the characteristic dumbbell shapes after rotary shadowing in the electron microscope (McDevitt and Dennis unpublished) and did not cross-react with anti-fibronectin antibodies. The preparation interacted with hyaluronan [30] as did native [38], and expressed forms of the protein studied by other investigators [39]. Taken together, these data confirmed that our preparation of type VI collagen was highly purified and had regained its native conformation after dilution with TBS of the 4 M guanidine HC1 solution to 0.002 M. 3.2. Cell attachment to type V/ collagen Chondrocytes in serum-free DMEM readily attached to wells coated with the tissue form of type VI collagen but not to those coated with bovine serum albumin (Fig. 2). The number of cells attaching to the type VI collagen was dose dependent and saturable (Fig. 2). The iodinated tissue and pepsin digested forms, respectively, of type VI collagen permitted us to establish conditions for coating specific quantities of each preparation on the well. Incubating wells with 0.5 /xg of either form of type VI collagen in 100 #1 of TBS (pH 7.5) (see Section 2) resulted in 25 _+ 3% of the incubated protein attaching to the well. Molecular weights of 520 kDa and 172 kDa, based on electrophoretic data, were assigned to the tissue and pepsin digested forms respectively of type VI collagen for coating specific molar quantities on the well. When equal molarities of the tissue and pepsin digested form of type VI collagen were coated on the well, a greater number of cells attached to the tissue form of type VI collagen (Fig. 2). This difference was statistically significant as assessed by Student's t-test: P < 0.01 for 12.5. 103 chondrocytes: P < 0.001 for 25 - 1 0 3 - 2 0 0 • 103 chondrocytes. We explored the possibility that the type VI collagen might be binding to either anchorin CII, a cell surface receptor for type II collagen on chondrocytes [40], or to cell surface heparan sulfate proteoglycan, because of the known binding between type VI collagen and heparin [39] and the presence of a heparan sulfate proteoglycans on the

surface of growth plate chondrocytes [41]. However, preincubation of chondrocytes with heparin or type II collagen solutions had no effect on attachment to type VI collagen (data not shown). 3.3. Effect of RGD-peptide We investigated whether the attachment could be inhibited by synthetic linear or cyclic RGD-containing peptides in competition experiments in which cells were premixed with peptide to mask potential RGD receptors. We first tested the effect of a linear GRGDSP peptide with a linear GRGESP peptide used as negative control. We explored this in two ways: (a) by keeping the quantity of the tissue form of type VI coating the well constant and varying the concentration of the peptide added to the cells: (b) by keeping the concentration of the peptide constant and varying the quantity of type VI collagen coating the wells. Fibronectin, a known RGD-dependent attachment factor [42], was used as a positive control in these experiments. As anticipated, the addition of increasing amounts of the linear peptide GRGDSP to the cells resulted in a progressively lower proportion of the cells attaching to fibronectin (RGD (FN) in Fig. 3A). Mixing cells with GRGESP peptide had no effect on attachment to fibronectin-coated wells (data not shown). In contrast, the addition of increasing amounts of linear GRGDSP (or GRGESP) peptide to the chondrocytes had no effect on the number of cells attaching to the tissue form of type VI collagen (RGD (VI) and RGE (VI) in Fig. 3A). In striking contrast, the attach-

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Concentration of Peptide (p.g/ml) Fig. 4. Effect of cyclic RGD-containing peptide on the attachment of chondrocytes to tissue form of type VI collagen and fibronectin. Intact, tissue form of type VI collagen (0.5/xg/100/.tl per well) and fibronectin (1.0 /zg/100 /xl per well) were both coated in TBS. Chondrocytes (5. 104/100/1,1 per well) with different concentrations o f C * GRGDSPC * or its scrambled sequence C * SRPGGPC * in serum-free DMEM were added to coated wells. Cells were incubated for 2 h at 37 ° C. Attachment to BSA-coated wells was insignificant. Data represent the mean + standard deviation for three wells. (11) cyclic SCR (VI): scrambled sequence cyclic peptide; wells were coated with type VI collagen. ( O ) cyclic RGD (FN): RGD containing cyclic peptide; wells were coated with fibronectin. ( 0 ) cyclic RGD (VI): RGD containing cyclic peptide; wells were coated with type VI collagen.

J. Marcelino, C.A. McDevitt / Biochimica et Biophysica Acta 1249 (1995) 180-188

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Concentration of EDTA (mM) Fig. 5. Effect of EDTA on the attachment of chondrocytes to intact, tissue form of type VI collagen (0.5 ~g/100 /~1 per well) coated in TBS. Chondrocytes (5. 104/100 /xl per well) with EDTA (0-2.75 mM) in serum-free DMEM were added to type VI collagen coated wells. Cells were incubated for 2 h at 37° C Attachment to BSA-coated wells was insignificant. Data represents the mean+standard deviation for four wells.

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ment of chondrocytes to the reduced and alkylated tissue form of type VI collagen was inhibited by the linear GRGDSP, but not the G R G E S P peptide (Fig. 3B). The triple helix containing fragment purified by salt precipitation of the pepsin digested type VI collagen, like the parent molecule, was not inhibited in its attachment properties by the linear G R G D S P peptide (Fig. 3C). Fig. 4 shows the effect of a cyclic C * G R G D S P C *, and a scrambled amino acid sequence of this peptide (C * SRPGGPC * ) that served as a negative control, on the attachment of chondrocytes to the tissue form of type VI collagen and to fibronectin. At 50 / x g / m l or higher, the cyclic RGD-containing peptide, unlike the linear form, inhibited attachment to the intact, tissue form of type VI collagen. At 5 0 / z g / m l , about 93% of the cells attached; at 500 / z g / m l , 53% of the cells attached. No inhibition of attachment was observed with the negative control peptide. Further, the cyclic RGD-containing peptide had no effect on chondrocyte attachment to fibronectin, confirming that the peptide had no effect on cell viability.

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Fig. 6. Effect of specific divalent cations on the attachment of chondrocytes to intact, tissue form of type VI collagen (0.5 /xg/100 /~1 per well) coated in TBS. Chondrocytes (5. 104/100 /xl/well) with CaC12 (0-20 mM) (A) or MgCl 2 (0-0.1 raM) (B) or MnCl 2 (0-1 raM) (C) in HBSS/4.5 g / l glucose/20 mM Tris (pH 7.2) were added into type VI collagen coated wells. (D) Chondrocytes (5 - 104/100 ~l per well) with a fixed concentration (0.05 mM) of MgCl 2 or MnCl 2 and increasing CaC12 concentrations in HBSS/4.5 g / l glucose/20 mM Tris (pH 7.2) were added into type VI collagen coated wells. Cells incubated for 2 h at 37 ° C. Attachment to BSA-coated wells was insignificant. Data represent the mean + standard deviation for four wells.

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J. Marcelino, C.A. McDevitt / Biochimica et Biophysica Acta 1249 (1995) 180-188

cell attachment experiments contained l mM CaC12 and 0.8 mM MgSO 4. The effect on cell attachment of incorporating different concentrations of EDTA into the DMEM is shown in Fig. 5. The number of cells attaching was reduced by about 50% at an EDTA concentration of 2.65 mM and totally abolished at 2.75 mM. The effect of the EDTA was clearly on the cell receptors and not on the type VI collagen, as pre-incubation of the coated wells with 5.0 mM EDTA and the subsequent addition of cells after removal of the chelator had no effect on attachment (data not shown). Cells in HBSS did not attach to the type VI collagen coated wells (Fig. 6A-C; zero concentration of metal ion). No attachment in the HBSS buffer was demonstrable in the presence of CaC12 up to a concentration of 20 mM (Fig. 6A). In contrast, the addition of MgC12 (Fig. 6B) or MnC12 (Fig. 6C) restored attachment of cells, with MnC12 more effective than MgCI 2 in endowing the cells with the capacity to attach. The addition of variable concentrations of CaCI 2 to a fixed concentration of MnC12 or MgCI 2 had no effect on attachment (Fig. 6D), confirming that CaC12 had no antagonistic effect on attachment.

4. Discussion

Our data demonstrate that bovine articular chondrocytes attach to and spread on the intact, tissue form of type VI collagen. This attachment was not inhibited by the synthetic linear GRGDSP peptide, but was significantly inhibited by a cyclic RGD-containing peptide. The interaction has a metal dependency that could be satisfied by manganese, and to a lesser extent by magnesium, but not at all by calcium. The pepsin-digested type VI collagen also promoted attachment of chondrocytes that was not inhibited by linear RGD peptide. In contrast, reduction and alkylation of the tissue form of intact, type VI collagen changed its attachment to an exclusively linear RGD-dependent one. The tissue form of type VI collagen with its globular domains intact was more effective in our study than the pepsin-digested form in attaching chondrocytes. Perris et al. reported that neural crests cells behave similarly, with more attaching to the intact, tissue form of type VI collagen [26]. These observations suggest the presence of multiple and cooperative attachment sites on the type VI collagen molecule, as originally proposed by Perris et al. [26]. Self-association of collagen molecules through their globular ends could provide multiple attachment sites [44]. Our observation that the attachment of articular chondrocytes to the tissue form of type VI collagen is not inhibited by linear RGD synthetic peptide is in accord with previous observations by Perris et al. on neural cells [26] and Doane et al. on corneal fibroblasts [27]. Similarly, the failure of linear RGD peptide to inhibit the attachment of chondrocytes to the pepsin-digested form of type VI colla-

gen agrees with similar observations by Pfaff et al. on a range of tumor cell lines [24], by Doane et ai. on corneal fibroblasts [27], and by Loeser on bovine articular chondrocytes [28,29]. However, a linear form of the RGD peptide was reported to inhibit attachment of a melanoma cell line [24]. It should also be noted that a limited sensitivity of cell attachment to pepsin-digested type VI collagen by linear RGD peptide was noted in other studies [23,25]. Our observation that reduction and alkylation of the native type VI collagen and subsequent unravelling of the a-chains changed the nature of attachment to chondrocytes to an exclusively linear RGD-dependent one, finds broad agreement in the literature [23,24,27]. Collectively, these observations suggest that the RGD sequences in the triple helix of type VI collagen are not readily available to integrins that are inhibited by linear RGD synthetic peptide. The disulfide bridges clearly help to stabilize the triple helix. Indeed, an attachment to type VI collagen that is not inhibited by linear RGD peptide might be considered diagnostic of the presence of a triple helix in this protein. Our observation that a cyclic RGD-containing peptide could inhibit the attachment of chondrocytes to the tissue form of type VI collagen is consistent with and extends the observation by Loeser that the same cyclic peptide could inhibit attachment to the pepsin-digested form of type VI collagen [29]. It is interesting that Cardarelli et al. [45] demonstrated that the same cyclic RGD-containing peptide, but not the linear RGD peptide, could inhibit the attachment of an osteosarcoma cell line MG-63 to type I collagen, apparently by inhibiting an a 2 /31 integrin. In this respect, it should also be noted that the melanoma cell attachment to pepsin-digested type VI collagen that was inhibited by the linear RGD peptide was also inhibited by a cyclic RGD-containing peptide [24]. A cyclic peptide is more restricted in the conformations it can present to an integrin than is a linear peptide. The RGD conformation in the cyclic peptide presumably mimics the conformation of one or more helical RGD sequences in the type VI collagen. Bonaldo et al. [25] has noted that two of the RGD sequences in the a 3 chain of type VI collagen are located in a region where several Gly-X-Y repeats lack a proline in the X or Y position. The triple helix could have less rigidity in this region, with the Arg-Gly-Asp sequence more free to interact with an integrin. Our observation that the cyclic RGD-containing peptide had no demonstrable effect on chondrocyte binding to fibronectin is in accord with previous reports [46,47] but contrasts, however, with that of Loeser who found that the cyclic peptide inhibited the chondrocyte attachment to fibronectin [29]. We have no explanation for this discrepancy. Our inhibition studies with type II collagen and heparin, respectively, demonstrated that the cells did not attach to type VI collagen via anchorin CII or a heparan sulfate proteoglycan on the cell surface. In accordance with Loeser's studies on pepsin-digested

J. Marcelino, C.A. McDeuitt / Biochimica et Biophysica Acta 1249 (1995) 180-188

type VI collagen, our results demonstrate an attachment dependence on manganese or magnesium, but not calcium [29]. Pfaff et al. observed ~Lnidentical metal dependence on tumor cell attachment to type VI collagen [24]. Our data for the intact type VI collagen are consistent with the observations that /31 integrins mediate cellular recognition of the pepsin-derived type VI collagen [24,48-50]. Scanning [51] and transmission electron microscopy [52] of articular cartilage have demonstrated that the pericellular region around the chondrocytes is morphologically distinct from the dense fibrillar weave of the remainder of the extracellular matrix. Poole and his co-workers, in a series of morphological studies, have isolated what are now referred to as chondrons: chondrocytes surrounded by a distinct pericellular matrix [53]. Microfilaments of type VI collagen and proteoglycans are integral components of the pericellular matrix of the chondron [14,54]. Importantly, immunoelectron microscopic studies of chondrocytes grown in agarose revealed that type VI collagen filaments made immediate contact with the chondrocyte plasma membrane as well as connecting with the radial collagen network [14]. Our studies are in striking agreement with these observations of Poole [14] and confirm that type VI collagen interacts specifically with one or more receptors on the chondrocyte surface. The multidomain nature of the type V[ collagen molecule enables it to connect the cell surface to different macromolecules, such as type II collagen [55] or hyaluronan [30,38,39], in this pericellular environment. It may therefore also have a role in organizing other matrix molecules of the capsule. The type VI collagen is now one of a family of proteins that bind to the surface of chondrocytes. These include thrombospondin [56] type II collagen [28,29,57,58], type I collagen [57], fibronectin (this study, [28,29,57,58]), matrix Gla protein, vitronectin and osteopontin [28], and a 36 kDa and 58 kDa protein isolated from cartilage [58]. While our study and the morphological studies of Poole [14,54], taken together, provide direct evidence for a role for type VI collagen in cell attachment in mature articular cartilage, it will be interesting to unravel the roles this and the other adhesion proteins play in the different stages of chondrocyte differentiation and in pathological conditions such as osteoarthritis.

Acknowledgements Support from NIH grant AR39569 is gratefully acknowledged. We thank Nikolaos Sarrimanolis who performed pilot experiments on the attachment of canine chondrocytes to type VI collagen, and Christina Sabo who isolated some of the type VI collagen preparations used. We thank Sue Czabaniuk-Sauer and Judy Christopher for typing assistance, Dr. Eva Engvall, La Jolla Research Foundation, La Jolla, CA, for monoclonal antibodies against type VI collagen and Drs. Dawn Nowlin and

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Thomas Lobl, Chemical Sciences Department, Tanabe Research Laboratories, USA, Inc., San Diego, CA, for the cyclic peptides.

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