View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector OsteoArthritis and Cartilage (2005) 13, 120e128 ª 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2004.06.015 International Cartilage Repair Society Inhibition of lysyl oxidase activity can delay phenotypic modulation of chondrocytes in two-dimensional culture1 J. Farjanel Ph.D.*,S.Se`ve Ph.D., A. Borel Ph.D., P. Sommer Ph.D. and D. J. S. Hulmes Ph.D. Institut de Biologie et Chimie des Prote´ines, CNRS UMR 5086, Universite´ Claude Bernard Lyon I, IFR 128 BioSciences Lyon-Gerland, 69367 Lyon Cedex 07, France Summary Objective: Chondrocytes frequently de-differentiate in two-dimensional (2D) culture, especially in the presence of serum. To examine the role of lysyl oxidase (LOX) induced cross-linking in this phenomenon, the effect of the specific LOX inhibitor b-aminopropionitrile (BAPN) was studied in 2D chondrocyte culture. Design: Chick embryo sternal chondrocytes (both proliferative and hypertrophic, from caudal and cranial zones, respectively) were cultured in the presence and absence of BAPN. The production and activities of LOX and LOX-like (LOXL) were assessed by enzyme assay and the use of specific antibodies. Seventeen batches of serum of different origin were compared. Chondrocyte phenotype was assessed both morphologically and biochemically, the latter by quantitative analysis of production of radiolabeled cartilage collagens II, IX, X and XI, and the de-differentiation marker collagen I, for up to 4 weeks in culture. Results: LOX and LOXL were identified, by Western blotting and immunofluorescence, and LO activity was measured in the medium, with both proliferative and hypertrophic chondrocytes. Inhibition of LO activity prevented or delayed chondrocyte de-differentiation, as characterized by changes in cell shape and synthesis of the five different collagen types, from the first days of culture for up to 4 weeks, depending on the origin of the serum added to the culture medium. Conclusion: LO activity may be involved in the control of chondrocyte phenotype, in addition to serum factors. Inhibition of LO activity by BAPN may be useful for the maintenance of the chondrocyte phenotype in 2D culture. Specific variations in the relative proportions of collagens II, IX and XI could be involved in the mechanism underlying these observations. ª 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Lysyl oxidase, b-Aminopropionitrile, Collagen, Chondrocyte. Abbreviations: LOX, lysyl oxidase; LOXL, lysyl oxidase-like; BAPN, b-aminopropionitrile. Introduction Accordingly, precise fibrillar forms of collagen, as well as some soluble factors, can induce the phenotypic modulation In normal cartilage, chondrocytes are known to produce of chondrocytes in culture. collagen types II, IX and XI in proportions leading to their 1 The above observations suggest that the physico-chem- association into thin cartilage-specific heterotypic fibrils .A ical properties of the collagenous matrix might influence sub-family of chondrocytes can become hypertrophic and chondrocyte phenotype. To examine this possibility, we progressively switch to production of collagen X, charac- 2 present here the results of experiments carried out in 2D teristic of terminal differentiation . culture designed to test the effects of de novo collagen cross- In two-dimensional (2D) cell culture, de-differentiation of linking on the maintenance of the chondrocyte phenotype. chondrocytes in the presence of serum is a well documented Lysyl oxidases (LOXs) initiate covalent cross-linking in phenomenon. In contrast, in three-dimensional culture, in 3 collagens and elastin (and perhaps other substrates) by agarose gels, the chondrocyte phenotype is maintained . oxidizing certain peptidyl lysine/hydroxylysine residues to Soluble factors present in serum are known to influence 4 peptidyl lysyl/hydroxylysyl aldehydes which then spontane- chondrocyte phenotype . In addition, many effects of ously condense with neighboring amino groups or additional extracellular matrix variations on cell differentiation have 8,9 5,6 peptidyl aldehydes . In cartilage, LO activity is known to been reported . In the case of chondrocytes, for example, promote covalent cross-links in heterotypic collagen II/IX/XI we previously showed, in the absence of serum, that de- fibrils10. We show here that inhibition of LO activity using the differentiation can be induced by culture in three-dimensional 7 specific inhibitor b-aminopropionitrile (BAPN) can prevent or gels of collagen I , while in gels of cartilage collagens II, delay de-differentiation of chondrocytes for up to 4 weeks of IX and XI, the differentiated phenotype is maintained. culture, depending on the origin of the serum present in the culture medium. 1This work was supported by the Centre National de la Recherche Scientifique and by the Universite´ Lyon I. AB and SS were recipients of studentships from the French government. Materials and methods *Address correspondence and reprint requests to: J. Farjanel, Institut de Biologie et Chimie des Prote´ines CNRS UMR 5086, 7 CELL CULTURES passage du Vercors, 69367 Lyon Cedex 07, France. Tel: 33- 472722666; Fax: 33-472722604; E-mail: [email protected] Seventeen-day-old chick embryo chondrocytes, prepared Received 22 June 2004; revision accepted 22 October 2004. by collagenase digestion of caudal and cranial regions of 120 Osteoarthritis and Cartilage Vol. 13, No. 2 121 sternal cartilage, were plated at a density of 2.0 ! 105 cells/ collagen II was observed just below the HMW band (not cm2, unless otherwise mentioned, in 35 mm diameter shown), as previously described11. Amounts of each plastic Petri dishes (P35). The medium was DMEM collagen were then expressed as percentages of total peak supplemented with 25 mg/ml ascorbate, 2 mM glutamine, area. Measured dpm values from pepsinized cell-layer 100 mg/ml streptomycin, 100 units/ml penicillin and 10% extracts and culture media were then used to convert serum (complete medium). Seventeen different batches of percentages of each collagen type into dpm, which were sera from various origins were compared: 12 fetal calf sera then added for each collagen type to determine total (cell (FCS) from Biomedia (B1 and B2), Sigma (S1 to S7), layer C medium) percentages. In this way, results could be Hyclone (H1) and GIBCO/Life Technologies (G1 and G2); compared between collagen types, expressed as percen- two neonatal calf sera (NCS) from Sigma (S8) and GIBCO/ tages (cell layer, medium and total), and between different Life Technologies (G3); two calf sera (CS) from Hyclone experiments. The average variation between duplicates in (H2) and GIBCO/Life Technologies (G4) and one adult each experiment was found to be about 10% of the measured healthy human serum pool (HS). In all, six experiments percentage values shown in Figs. 5e7. were carried out as follows: experiment 1 e FCS B1; It should be noted that, after 14C-proline labeling of experiment 2 e FCS B2; experiment 3 e FCS S1; synthesized collagens, the measured intensities for the experiment 4 e FCS B2, S2, S3; experiment 5 e FCS S2, different collagen types depend on their proline C hydrox- S4, S5, S6, S7 and H1, NCS S8, CS and H2, human adult yproline contents. These proportions are known to be rather serum HS; experiment 6 e FCS S2, S7, G1, G2 and H1, similar among the fibrillar collagens I, II and XI, particularly NCS G3 and CS G4. When FCS was absent, the medium between different a chains of the same collagen type. In was supplemented with 1 mM pyruvate and 1 mM cysteine contrast, due to their smaller triple-helical regions, collagens as protective agents. BAPN was added as indicated at IX and X contain less proline C hydroxyproline than the a concentration of 60 mg/ml. Media including all additives fibrillar collagens12. This was particularly so for collagen IX, (purchased from Sigma) were changed every 2e3 days and where only the HMW fragment was measured, as identified cultures were maintained for up to 4 weeks. by its well characterized differences in migration on SDS- PAGE with and without reduction11, the LMW fragment being negligible in most of the fluorograms. No corrections were COLLAGEN SYNTHESIS made to allow for these differences in proline C hydroxypro- Newly synthesized collagens were metabolically labeled line content, which has no effect when comparisons are made for 24 h with 1 mCi/ml 14C-proline (285 Ci/mmol: Amersham with and without BAPN for each collagen type. International), beginning on days 2, 13, 20 or 27. Culture media were submitted to pepsinization (pepsin from Serva, Heidelberg, FRG) as previously described7 at 0.1 mg/ml for ANTIBODIES 18 h. Cell layers were treated similarly except for pepsin- Two different polyclonal antibodies were used13e15, ization with 0.3 mg/ml pepsin for 48 h. The amounts of total directed against peptides corresponding to residues newly synthesized pepsin-resistant proteins were deter- e 7 228 279 of human preprolysyl oxidase (LOX) and residues mined by liquid scintillation counting as described . 355e416 of human preprolysyl oxidase-like (LOXL). In the Collagen types were analyzed by SDS-polyacrylamide gels e case of LOX, chicken and human forms show 92% (PAGE) on 4.5 15% gradient gels (in non-reducing sequence identity in the region used as antigen. No conditions except when mentioned) followed by fluorogra- sequence