EXPRESSION OF CARTILAGE OLIGOMERIC MATRIX PROTEIN IN ARTICULAR CARTILAGE IN RESPONSE TO BONE MORPHOGENETIC PROTEIN-7 AND TUMOUR NECROSIS FACTOR

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1 Volume 25 No. 1 June2011 Peer reviewed ORIGINAL ARTICLE EXPRESSION OF CARTILAGE OLIGOMERIC MATRIX PROTEIN IN ARTICULAR CARTILAGE IN RESPONSE TO BONE MORPHOGENETIC PROTEIN-7 AND TUMOUR NECROSIS FACTOR S CKM Motaung (D.Tech), JJ Pieterse (D.Tech) Department of Biomedical Sciences, Tshwane University of Technology, Faculty of Science, Pretoria, South Africa Corresponding author: Shirley Motaung motaungsckm@tut.ac.za tel: +27 (0) /6265 ABSTRACT Introduction: The extracellular matrix of articular cartilage consists of collagens, proteoglycans and noncollageneous proteins. Cartilage oligomeric matrix protein (COMP) is a non-collagenous protein isolated from articular cartilage. It is localised in articular cartilage and the proliferative and hypertrophic zones of the epiphyseal growth plate. Aim: The aim of the study was to investigate the role of bone morphogenetic proteins (BMP-7) and tumour necrosis factor (TNF-α) on expression of COMP in articular cartilage. Methods: Stifle joints (n=12) from three month-old calves were obtained from a local abattoir. Articular chondrocytes were isolated from three distinct zones of cartilage and cultured as monolayers in a serum-free chemically defined medium. Results: BMP-7 increased COMP accumulation in dose and time dependent manner. TNF-α was however found to significantly reduce COMP accumulation Conclusion: BMP-7 was shown to be important regulators of the COMP gene in different zones of articular cartilage KEYWORDS Articular cartilage, bone morphogenetic proteins (BMP-7), tumour necrosis factor (TNF-α), cartilage oligomeric matrix protein (COMP) INTRODUCTION Articular cartilage consists of non-collagenous matrix proteins [4], which are important for the interaction and assembly of the various macromolecules. These molecules have different functions and interact specifically with other matrix molecules, either contributing to the structural network or interacting directly with the chondrocytes to modulate the phenotype [1]. Other non-collagenous proteins include COMP, fibronectin and tenascin. COMP, also known as TSP-5, a member of the thrombospondin (TSP) gene family [2], has been isolated from bovine, mouse, swarm rat chondrosarcoma and humans. It has a molecular weight of 524 kda [3-5], with subunits of kda [3, 4, 6] disulphide-bonded in a five-armed structure to the central core. The pentameric structure of COMP was confirmed by electron microscopy [3]. This protein comprises a high content Figure 1: The morphology of cartilage (Vinther et al., 2003). of aspartic acid and glutamic acid and is therefore highly negatively charged. The absence of hydroxyproline showed its noncollagenous nature [3, 4]. COMP is localised in the proliferative region and hypertrophic zones of growth plate cartilage as well as in articular cartilage. The function of COMP is not yet known but based on it being a member of the TSP family [2], it has been suggested that this protein plays a role in the preservation of chondrocyte phenotype, cell growth and matrix development [2]. Mutations of the COMP gene are located on chromosome 19 and have been linked to pseudoachondroplasia (PSACH) [7] and multiple epiphyseal dysplasias (MED) [8]. These disorders are characterised by mild to severe short stature and early onset of osteoarthritis. It has also been reported that elevated serum levels of COMP have been found in patients with knee osteoarthritis (OA) [9] and rheumatoid arthritis (RA) [10]. Therefore, it is a potential biomarker for monitoring the progression of cartilage destruction and cartilage damage [11]. Articular cartilage is a heterogeneous avascular, aneural tissue that provides a smooth bearing surface for movement. Cartilage has a defined stratified structure composed of superficial, middle, deep, and calcified zones each with distinct cell densities and phenotypes, molecular architecture, and mechanical properties. The superficial zone is the site of superficial zone protein synthesis that functions as a boundary lubricant [12]. The middle and deep layers abound in collagen II and proteoglycan aggrecan. Each of these layers has specific functions and therefore, replication of these layers may be important in obtaining a functional tissue-engineered construct for tissue regeneration [13-18]. Figure 1 illustrates the general structure of articular cartilage. It is also known that growth factors stimulate the ex ISSN

2 Volume 25 No. 1 June 2011 Figure 2: (A) Dissected bovine stifle (knee joint). (B) Osteochondral plugs were extracted from the lateral femoral condyles. pression of COMP [19-21]. However, the regulation of cartilage oligomeric matrix protein (COMP) synthesis and secretion is critical for the understanding of cartilage homeostasis in health and disease. We hypothesised that chondrocytes from the superficial, middle and deep zones of articular cartilage would respond differentially to BMP-7 and TNF-α. The main objective in this study was to investigate the effects of BMP-7 and TNF-α on COMP, mrna and protein level in cultured cells from superficial, middle and deep zone chondrocytes of bovine articular cartilage. MATERIALS AND METHODS Tissue Acquisition for Cell Culture Bovine stifle (knee joints) n=12 from 3 month old calves were obtained from an abattoir and dissected under aseptic conditions to expose the femoral condyles as shown in figure 2A. Osteochondral plugs were removed from femoral condyles using a 5mm-diameter cork borer (Fisher Scientific, Hampton, NH) as shown in figure 2B. The superficial zone cartilage of the femoral condyles (200μm) was harvested using a dermatome (Integra, Plainsboro, NJ) as shown in figure 3. Figure 3: Chondrocytes harvested from superficial layer of femoral condyle of calves using a dermatome. Monolayer Culture The superficial zone cartilage was digested with 0.2% collagenase-p (Roche Pharmaceuticals, Nutley, NJ) for 3 hours, the middle and the deep zones overnight (16 hours). Chondrocytes were plated as monolayers at a density of cells/well in 12-well culture plates and incubated at 37 o C in a humid atmosphere of 5% carbon dioxide and 95% air. Chondrocytes were cultured overnight in DMEM/F-12 medium containing 1% fetal bovine serum. The following day media was changed to serumfree DMEM/F-12medium with insulin transferrin selenium (ITS) + Premix (BD Bioscience, Bedford, MA). Cells were treated with various morphogens according to the experimental design described below. Monolayer cell cultures were treated for 4 days and 7 days respectively with the following morphogens: BMP-7 (100 ng/ml, 300 ng/ml and 1000 ng/ml) and TNF-α (1.0 ng/ml, and 3.0 ng/ml). These concentrations of morphogens were based on previous experiments [12]. One half of the culture medium was changed on day 4 and replaced with fresh medium supplemented with the same concentration of the different morphogens. Cells and medium were collected on days four and seven for analysis. Real-time reverse transcriptase polymerase chain reaction analysis Total RNA was isolated from cells culture using RNeasy mini kit (Qiagen, Valencia, CA) with on-membrane DNase 1 (Qiagen) digestion to avoid genomic DNA contamination. Total RNA was reverse transcribed into single-strand cdna using superscript First-Strand Synthesis System with Oligo (dt) primers (Invitrogen, Carlsbad, CA). Real-time quantitative PCR was performed in triplicate on cdna with an ABI 7700 Sequence detector and SYBR Green reagents (Applied Biosystems, Foster City, CA) following the recommended protocols. COMP mrna levels were normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH) levels and expressed relative to the control (untreated) culture levels (ΔΔC T methods, Applied Biosystems) [29]. The primers for bovine COMP (forward: 5 -GAGACCGGGCAG- CATAACTG-3 ; reverse: 5-AGGGCCACACTGGAAGGAG-3 ). GAPDH (forward: 5-GCCAAGAGGGTCAT-3, reverse: 5 -GT- GGTTCACGCCCATCACA-3 ) were designed using Primer Express 1.9 software (Applied Biosystems). Enzyme-linked immunosorbent assay (ELISA) analysis of COMP protein levels An ELISA kit was used for quantitative determination of COMP (MD Biosciences, St. Paul, MN) levels in the conditioned cul- ISSN

3 Volume 25 No. 1 June2011 ture media. The assay is based on the competitive inhibition of primary antibody binding to COMP coated plates. Immunolocalisation of COMP Full-thickness articular cartilage was dissected from bovine femoral condyles. Specimens were fixed with 4% paraformaldehyde (Fisher Scientific, Fair Lawn, NJ) and embedded in paraffin wax. Tissue sections (5 µm thick) were prepared and COMP was localized using rabbit anti-comp polyclonal antibody. Sections were deparaffinized, and endogenous peroxidase was blocked with 3% hydrogen peroxide. Sections were then incubated overnight at 4 C with a 1:200 dilution of rabbit anti-comp polyclonal antibody. The sections were then incubated with biotinylated anti-rabbit immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA) for one hour. No primary antibody was applied in the control experiments. Visualisation was achieved using diaminobenzine/peroxidase reaction (ImmPACT diamino benzine peroxidase substrate, Vector Laboratories) resulting in a brown precipitate. Statistical Analysis For all experiments, the sample size was 12, representing 12 different animals. Results are presented as the mean and standard deviations for each experiment. A one way analysis of variance (ANOVA) with Tukey s HSD (Honestly Significant Differences) to account for multiple comparisons was used to determine the effects of BMP-7 and TNF-α on COMP accumulation in different zones of articular cartilage. A significance level of p<0.05 was used to determine the difference between the control and treated cells and the dose-dependence of morphogens within the zone. RESULTS Dose-and Time-Dependent Actions of BMP-7 on COMP mrna and protein levels. As previously mentioned articular chondrocytes from the superficial, middle and deep zones were cultured for four days (figure 4A and figure 4C) and seven days (figure 4B and figure 4D) as monolayers in a serum free chemically defined medium with graded levels of BMP-7 (100 ng/ml; 300 ng/ml and 1000 ng/ml). COMP mrna expression was assessed using real-time reverse transcriptase polymerase chain reaction (figure 4A and figure 4B) and COMP expression in the culture medium was quantified by ELISA (figure 4C and figure 4D). In the superficial zone COMP accumulation was significantly higher in both dose and time-dependent treatment in response to BMP-7. The middle and deep zones showed no time or dose dependence (figure 4A, figure 4B and figure 4C). Interestingly, on day seven BMP-7 stimulates COMP expression significantly in dose dependence treatment in the middle zone (figure 4B) and deep zone (figure 4B and figure 4D). BMP-7 up regulated COMP expression and protein level in the superficial zone, compared to untreated control cells, treatment with 100 ng/ml, 300 ng/ml and 1000 Figure 4: Influence of BMP-7 on COMP expression in the superficial, middle and deep zones of articular cartilage. Articular chondrocytes were cultured for 4 days (Fig. 4A and Fig. 4C) and 7 days (Fig. 4B and Fig. 4D) as monolayers in serum-free chemically defined medium with graded levels of BMP-7 (100 ng/ml; 300 ng/ml and 1000 ng/ml). COMP protein level in culture medium was quantified by ELISA (Fig. 4C and Fig. 4D). COMP mrna expression was assessed using real-time reverse transcriptase polymerase chain reaction (Fig. 4A and Fig. 4B). The COMP mrna expression and protein level between untreated control and treatment groups in different zones were statistically significant. (*p<0.05) ISSN

4 Volume 25 No. 1 June 2011 Figure 5: The effect of TNF-α and BMP-7 on COMP expression in the superficial, middle and deep zones of articular cartilage. Articular chondrocytes were cultured for 4 days (Fig. 5A and Fig. 5C) and 7 days (Fig. 5B and Fig. 5D) as monolayers in serum-free chemically defined medium with graded levels of BMP-7 (300 ng/ml) and TNF-α (1.0 ng/ml, and 3.0 ng/ml). COMP protein level in culture medium was quantified by ELISA (Fig. 5C and Fig. 5D). COMP mrna expression was assessed using real-time reverse transcriptase polymerase chain reaction (Fig. 5A and Fig. 5B). The COMP mrna expression and protein level between untreated control and treatment groups in different zones were statistically significant (*p<0.05). ng/ml BMP-7 for day seven increased COMP mrna and protein level by 10% in the superficial zone (figure 4B and figure 4D). All fold increase was compared to deep zone without treatment. Relative differences in COMP expression and protein levels in different zones of articular cartilage between the control, treatment groups and dose-dependence were statistically significant respectively (*=p<0.05, **= p<0.01). Action of TNF-α alone and in combination with BMP-7 on COMP mrna expression and protein level Compared to untreated controls, chondrocytes treated with TNF-α resulted in down regulation of COMP levels on both days (figure 5A-figure 5D). BMP-7 resulted only in a significant COMP increase when added to TNF-α. Despite this increase, COMP expression in TNF-α treated cells was still below control. Immunolocalisation of COMP in bovine articular cartilage Strong COMP staining was observed in the uppermost surface layer in the superficial zones of articular cartilage (figure 6B), decreased in the middle zones (figure 6B) and decreased even further in the deep zones (figure 6A). The sections where no primary antibody was applied showed no staining thereby demonstrating the specificity and reliability of the method (figure 6C and figure 6D) mrna expression and protein level between untreated control and treatment groups in different zones were statistically significant (*p < 0.05). DISCUSSION The present study investigated the influence of BMP-7 and TNF-α on COMP mrna expression and protein levels from the surface, middle and deep zones of articular cartilage. Articular cartilage has a limited capacity to repair and regenerate itself due to the avascular nature of the tissue [10] and relatively low cell density. The articular cartilage consists of chondrocytes surrounded by vast expanses of extracellular matrix (ECM). The membrane ECM continuum functions at the organ, tissue, and cellular levels. It is well known that genetic factors are key in tissue morphogenesis. The ability of morphogens to up regulate COMP expression may relate to its anabolic effect on ECM proteins. Although it is known that growth factors stimulate the expression of COMP it is not clear if there is a differential response ISSN

5 Volume 25 No. 1 June2011 to the BMP-7 and TNF-α in the different zones of articular cartilage. It was hypothesised that cells from superficial, middle and deep zone chondrocytes of articular cartilage in monolayer cell culture would respond differently to BMP-7 and TNF-α. BMPs have been shown to induce new cartilage and bone formation in vitro and in vivo [22] and regulate cell proliferation and differentiation. Among the morphogens and growth factors investigated in this study, it was found that BMP-7 significantly stimulated the expression of COMP at mrna and protein levels. Treatment with BMP-7 significantly stimulated chondrocytes more in the superficial zone than in the middle and deep zones. This is similar to observations in superficial zone protein accumulation [12, 23] by morphogens. BMP-7 is a key regulatory factor in chondrocyte metabolism and influences matrix production, proliferation and differentiation [24]. It was found that catabolic cytokines, such as TNF-α suppressed COMP levels in monolayer cell cultures which is consistent with other studies on articular cartilage COMP [25, 26]. It is noteworthy that we observed the inhibitory effect of TNF-α to be partially reversed by the addition of BMP-7. Immunohistochemical findings revealed that COMP localisation is strongest in the superficial zone which confirms the observations reported in another study [27, 28]. CONCLUSION AND RECOMMENDATIONS COMP gene responded positively to stimulation of BMP-7 members in different zones of articular cartilage. This study has provided new information about the synthesis and secretion of the COMP gene in different zones of articular cartilage with the treatment of BMP-7. Furthermore, regulation of this COMP gene will be necessary for tissue engineering to recreate the different articular cartilage zones. BMP-7 in the superficial zones does indeed play a role in the homeostasis of articular cartilage. Stimulation of COMP by growth factors morphogens may slow progression of pathological conditions such as osteoarthritis (OA) and rheumatoid arthritis (RA), which will have an effect in the field of rheumatology. ACKNOWLEDGMENT This work was funded by the Lawrence Ellison Endowment as well as the Fulbright Fellowship to Shirley Keolebogile Motaung from the Institute for International Education, South Africa. REFERENCES 1. Dicesare, P., Hauser, N., Lehman, D., Pasumarti, S. & Paulsson, M. Cartilage oligomeric matrix protein (COMP) is an abundant component of tendon. Federation of European Biochemical Societiestt. 1994a; 354: Oldberg, A., Antonsson, P., Lindblom, K. & Heinegard, D. A. Collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin). European Molecular Biology Organization Journal. 1989; 8: Morgelin, M., Paulsson, M., Malmstrom, A. & Heinegard, D. 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