ARTHRITIS & RHEUMATISM Vol. 56, No. 11, November 2007, pp 3685–3692 DOI 10.1002/art.22970 © 2007, American College of Rheumatology
Increased Expression of Discoidin Domain Receptor 2 Is Linked to the Degree of Cartilage Damage in Human Knee Joints A Potential Role in Osteoarthritis Pathogenesis Ilse-Gerlinde Sunk,1 Klaus Bobacz,2 Jochen G. Hofstaetter,3 Love Amoyo,3 Afschin Soleiman,3 Josef Smolen,3 Lin Xu,1 and Yefu Li1 collagen breakdown products was elevated as a function of increased DDR-2 expression and cartilage damage. Furthermore, in vitro experiments revealed an upregulation of both DDR-2 and MMP-13 mRNA in human articular chondrocytes after stimulation with type II collagen. Conclusion. Our data indicate that 3 factors, DDR-2 expression, MMP-13 expression, and the degree of cartilage damage, are linked, such that DDR-2 promotes tissue catabolism, and tissue degradation promotes DDR-2 up-regulation and activation. Thus, the perpetuation of DDR-2 expression and activation can be seen as a vicious circle that ultimately leads to cartilage destruction in OA.
Objective. To investigate the relationship between increased discoidin domain receptor 2 (DDR-2) expression and cartilage damage in osteoarthritis (OA). Methods. Full-thickness cartilage tissue samples from 16 human knee joints were obtained and the grade of cartilage damage was evaluated according to the Mankin scale. Expression of DDR-2, matrix metalloproteinase 13 (MMP-13), and MMP-derived type II collagen fragments was visualized immunohistochemically. Moreover, upon stimulation with either type II collagen or gelatin, levels of DDR-2 and MMP-13 messenger RNA (mRNA) in primary human articular chondrocytes were assessed by real-time polymerase chain reaction. Results. Immunohistochemical analysis showed an increase in DDR-2 expression in human articular cartilage, which was correlated with the degree of tissue damage. In parallel, the extent of MMP-13 and type II
Osteoarthritis (OA) is considered to be the most common rheumatic disorder (1). The disease has been associated with a variety of distinct risk factors, but with similar clinical outcomes regardless of the postulated risk factor. Although understanding of the pathogenesis of OA has evolved considerably over the past years, the molecular mechanisms are largely unknown. Nevertheless, alterations that occur during cartilage degeneration, eventually leading to OA, are remarkably similar regardless of the etiology. The pathologic events are characterized by proteoglycan loss, followed by type II collagen degradation, and ultimately by localized or complete destruction of articular cartilage matrix (2). Thus, elucidating a common pathway in OA development leading to cartilage degeneration would provide new insight into the pathogenesis of OA. Our recent findings provide evidence for the existence of such a pathway in articular chondrocytes.
Dr. Bobacz’s work was supported by the Austrian Science Foundation (grant J2528). Dr. Xu’s work was supported by the NIH (grants R01-AR-051989 and P01-AR-050245). Dr. Li’s work was supported by the NIH (grant R01-AR-051989). 1 Ilse-Gerlinde Sunk, MD, Lin Xu, MD, PhD, Yefu Li, MD, PhD: Harvard School of Dental Medicine, Boston, Massachusetts; 2 Klaus Bobacz, MD: Harvard School of Dental Medicine, Boston, Massachusetts, and Medical University of Vienna, Vienna, Austria; 3 Jochen G. Hofstaetter, MD, Love Amoyo, Afschin Soleiman, MD, Josef Smolen, MD: Medical University of Vienna, Vienna, Austria. Drs. Sunk and Bobacz contributed equally to this work. Address correspondence and reprint requests to Lin Xu, MD, PhD, Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (e-mail:
[email protected]); or to Yefu Li, MD, PhD, Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (e-mail: yefu_li@hms. harvard.edu). Submitted for publication March 12, 2007; accepted in revised form July 27, 2007. 3685
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We characterized a molecule that may play an important role in inducing matrix metalloproteinase 13 (MMP-13) synthesis in articular chondrocytes. In fact, we found elevated expression of MMP-13 and MMP-derived type II collagen fragments in murine OA models, namely, type IX collagen–deficient (Col9a1⫺/⫺) mice and type XI collagen-haploinsufficient (heterozygous chondrodysplasia [cho/⫹] mice) (3–6). More importantly, we observed the concomitant up-regulation of a distinct cell membrane collagen receptor, discoidin domain receptor 2 (DDR-2). Additionally, in vitro experiments demonstrated an up-regulation and activation of DDR-2 upon binding to triple-helical type II collagen, culminating in increased synthesis of MMP-13 in primary murine chondrocytes and human chondrocyte cell lines. These findings strongly indicated the involvement of DDR-2 in OA pathogenesis. DDR-2 belongs to a novel family of tyrosine kinase receptors for collagen (7). Those receptors are activated by fibrillar collagens (types I, II, and III); however, DDR-2 binds preferentially to type II collagen (8–10). Although DDR-2 has been shown to be involved in extracellular matrix remodeling during morphogenesis (10), its role in OA pathogenesis has not been widely recognized. Considering this information, we investigated DDR-2 and MMP-13 expression in human articular cartilage/chondrocytes and its relationship to the degree of cartilage damage, hypothesizing that increased DDR-2 expression is related to the severity of tissue destruction. This study is the first to examine the relationship between DDR-2 expression/activation and cartilage degradation in human articular cartilage and primary human articular chondrocytes. MATERIALS AND METHODS Articular cartilage and cell culture. For histologic and immunohistochemical analysis, OA articular cartilage specimens were obtained from the knee joints of 16 subjects (ages 42–81 years, mean age 64.8 years): 7 at the time of endoprosthetic knee replacement due to OA and 9 at autopsy (within 24 hours of death). The removal of the 16 cartilage specimens was in accordance with the ethics committee policies of the Medical University of Vienna. Four tissue punches (5 mm in diameter), including all cartilage layers and the subchondral bone, were obtained from the load-bearing sites of the tibial plateau in each subject. Areas displaying total cartilage loss and/or neocartilage formation were avoided. Additionally, 3 cartilage specimens from subjects who underwent endoprosthetic knee replacement due to OA were obtained for cell culture experiments. All 3 specimens showed late OA changes.
Articular cartilage was carefully, aseptically dissected from the joint surface and finely minced. Chondrocytes were released by overnight digestion in 0.2% collagenase B (Boehringer, Mannheim, Germany) and filtered through a cell strainer (BD Falcon, Lincoln Park, NJ) to remove debris and undissociated cell clusters. The cell filtrate was then centrifuged at 500g for 10 minutes. Pellets were resuspended in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (25 mM HEPES, 4,500 mg/liter glucose, and pyridoxine, without sodium pyruvate; Life Technologies, Gaithersburg, MD) and Ham’s F-12 medium (Life Technologies) containing 10% fetal bovine serum (PAA Laboratories, Linz, Austria) and antibiotics/antimycotics (100 units/ml penicillin G, 100 mg/ml streptomycin, and 0.25 g/ml amphotericin B) (Life Technologies). The chondrocyte number was evaluated after trypan blue staining in a Bu ¨rker-Tu ¨rk chamber. Prior to chondrocyte distribution onto the culture dishes, 6-multiwell plates (Costar, Cambridge, MA) were precoated with either native type II collagen or denatured type II collagen gelatin. Type II collagen from chicken sternal cartilage (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.25% acetic acid at 1 mg/ml and used at a final concentration of 10 g/well; gelatin was obtained by heating the type II collagen solution for 60 minutes at 75°C. The isolated human cells were then grown as monolayer cultures in duplicate at a density of 5 ⫻ 105 cells/well and incubated in serum-free basal medium (11). Cells grown in untreated wells served as controls. After 24 hours, total RNA was isolated using a commercially available kit (RNeasy kit; Qiagen, Valencia, CA). Histologic and immunohistochemical analyses. Tissue punches were fixed in 4.0% paraformaldehyde overnight. Thereafter, samples were decalcified in 14% EDTA (SigmaAldrich) at 4°C (pH adjusted to 7.2 by adding ammonium hydroxide [Sigma-Aldrich]) until the bones were pliable. The 4 tissue punches from each subject were then embedded in 1 paraffin block. Fifty serial sections (5 m thick) of each paraffin block were cut for histologic and immunohistochemical analysis. Every tenth section was collected for Safranin O–fast green staining. Two independent assessors (YL and I-GS) graded the OA damage in each sample, using a modified Mankin score (12,13). For immunohistochemical analysis, successive paraffin sections adjacent to the sections stained with Safranin O were deparaffinized and dehydrated. The tissue sections were blocked for 60 minutes in phosphate buffered saline containing 1.5% goat serum, followed by treatment with chondroitinase ABC (0.25 units/ml; Sigma-Aldrich) and overnight incubation with either a mouse anti-human DDR-2 monoclonal antibody (R&D Systems, Minneapolis, MN), a mouse anti-human MMP-13 monoclonal antibody (Chemicon, Temecula, CA), or a polyclonal antibody, C1,2C (Ibex, Montreal, Quebec, Canada), which recognizes a neopeptide on type II collagen. The monoclonal antibodies were diluted 1:50; the dilution of the polyclonal antibody was 1:200. After rinsing, the sections were incubated with a species-specific anti-IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The bound antibodies were visualized using the appropriate NovaRED substrate kit (Vector, Burlingame, CA). Sections in which the primary antibody was omitted served as negative controls.
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Figure 1. Histologic analysis of full-thickness cartilage specimens using Safranin O–fast green staining. Mankin scores in the 16 samples tested ranged from 1 (low) to 10 (high). Five representative specimens are shown. A and B, Representative samples from the group of specimens with low Mankin scores (ⱕ3.5) (n ⫽ 7), exhibiting a small reduction in staining intensity in the superficial zones. C–E, Representative samples from the group with high Mankin scores (⬎3.5) (n ⫽ 9), exhibiting moderate to severe cartilage degeneration, chondrocyte clusters, and enhanced Safranin O staining around the chondrocyte clusters. (Original magnification ⫻ 50.)
Semiquantitative real-time polymerase chain reaction (PCR). One microgram total RNA from each sample was copied into complementary DNA with oligo(dT) primers using the Superscript first-strand synthesis system (BD Biosciences Clontech, Palo Alto, CA). The conditions and primers for real-time PCR have been described elsewhere (6). Statistical analysis. Correlations between DDR-2 and MMP-13 expression and the degree of cartilage damage were examined using Spearman’s rank correlation coefficient. Student’s t-test was used to determine significant differences between groups. P values less than 0.05 were considered significant.
RESULTS Histologic findings. Cartilage sections from all subjects were examined histologically by Safranin O–fast green staining. To determine the amount of cartilage damage, tissue samples were graded according to the Mankin scoring system. The 16 samples had total Mankin scores ranging from 1 (mild damage) to 10 (severe damage). A cutoff for classifying a sample as showing early OA changes was defined as a Mankin score of ⱕ3.5 (n ⫽ 7). The tissue alterations observed in these samples were minor in nature, comprising a uniform colorization of proteoglycans with only a slight reduction in staining intensity in the superficial zones (Figures 1A and B). In contrast, the evaluation of histologic specimens with
Mankin scores ⬎3.5 (n ⫽ 9) revealed moderate (Figure 1C) to severe (Figures 1D and E) cartilage degeneration, including reduced proteoglycan staining, the occurrence of chondrocyte clusters, enhanced Safranin O staining around the chondrocyte clusters in the middle and deep zones, fibrillations in the superficial zone, and loss of articular cartilage. Increased DDR-2 and MMP-13 expression in damaged cartilage. Immunohistochemical analysis was performed to investigate DDR-2 expression and its relationship to the extent of tissue damage in human knee articular cartilage. In specimens with low Mankin scores (ⱕ3.5), we found few DDR-2–positive cells, restricted to the superficial cartilage layer (Figure 2A). In tissue specimens with high Mankin scores (⬎3.5), however, DDR-2 was detected in the majority of the chondrocytes in the superficial zone; furthermore, with increasing cartilage damage, more cells in the transitional zone expressed DDR-2 (Figures 2B and C). As we have consistently shown before in murine OA models (6), the activation of DDR-2 causes upregulation of MMP-13 and subsequently a breakdown in the type II collagen network. Thus, immunohistochemical analysis was performed to detect MMP-13 expression in the articular cartilage tissue samples. MMP-13
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Figure 2. Discoidin domain receptor 2 (DDR-2), matrix metalloproteinase 13 (MMP-13), and MMP-derived type II collagen degradation fragments (C1,2C) in human articular cartilage sections. A–C, DDR-2 staining in 3 specimens with different Mankin scores. Red/brown staining represents sites of DDR-2 expression. D–F, MMP-13 expression (red/brown staining) in successive sections adjacent to the DDR-2–stained sections. G–I, Immunolocalization of C1,2C. Arrowheads indicate areas of type II collagen degradation. Insets show higher-magnification views of the boxed areas. Negative controls, in which the primary antibody was omitted, were run in parallel. (Original magnification ⫻ 100; ⫻ 200 in insets.)
was detected in the same areas that were positively stained for DDR-2 (Figures 2D–F). As described above for DDR-2, MMP-13 expression increased with the extent of cartilage damage and the Mankin score. As a surrogate marker of type II collagen degradation, cartilage samples were immunostained using a polyclonal antibody against a neoepitope (C1,2C) generated by MMPs, including MMP-13. The areas of type II collagen degradation were consistent with those of DDR-2 and MMP-13 expression. The number of stained C1,2C neoepitopes was elevated in specimens with high Mankin scores (Figures 2H and I), compared with those with low Mankin scores (Figure 2G). These findings in human knee articular cartilage suggest that OA progression might be linked to increased DDR-2 expression and, moreover, to DDR2–governed MMP-13 expression. Link between the number of DDR-2–positive cells and disease severity. Since the Mankin score seemed to be connected to the extent of DDR-2 expression, we performed a semiquantitative analysis by assessing the percentage of DDR-2–positive cells in a cartilage area of a defined size (4 ⫻ 2.5 mm2 per specimen). Parts
from each tissue punch (either the left, right, or middle part, as randomly determined using a computer program) were evaluated. We found a significant correlation between cartilage damage, reflected by the Mankin score, and the amount of positively stained chondrocytes (rs ⫽ 0.89, P ⬍ 0.0002) (Figure 3A). Cartilage specimens with Mankin scores ⱕ3.5 showed an average of 23.1% DDR-2–positive chondrocytes, while samples with Mankin scores ⬎3.5 revealed an average of 46.7% DDR-2– positive chondrocytes (P ⬍ 0.003). Similar to DDR-2, MMP-13 expression was elevated with increasing Mankin scores (rs ⫽ 0.9, P ⬍ 0.0002) (Figure 3B). When we investigated the relationship between DDR-2 and MMP-13 expression, a correlation was apparent (rs ⫽ 0.91, P ⬍ 0.0002). These data implicate a direct correlation between cartilage damage, DDR-2 expression, and MMP-13 expression. Increased DDR-2 and MMP-13 messenger RNA (mRNA) expression in chondrocytes exposed to type II collagen. Chondrocytes were obtained and cultured as described above. After 24 hours, cells were harvested and total RNA was subjected to real-time PCR. Upon contact with type II collagen, chondrocytes showed a
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Figure 3. Percentage of positively stained chondrocytes in relation to the grade of cartilage damage, as assessed by Mankin score. For each specimen (n ⫽ 16), 4 areas (2.5 mm2 each) of localized DDR-2 expression and the congruent areas of MMP-13 expression were evaluated. Values are the mean percentage of positive cells/mm2. A, Increase in the percentage of DDR-2–positive cells with increasing cartilage damage. B, Increase in the percentage of MMP-13–positive cells with increasing cartilage damage. See Figure 2 for definitions.
marked increase in DDR-2 as well as MMP-13 expression compared with control cultures. Denatured type II collagen (gelatin), however, had no stimulating effect on articular chondrocytes with regard to DDR-2 and MMP-13 expression (Figures 4A–D). One of the 3 tissue specimens with the most severe OA changes contained only a few viable cells, and thus PCR analysis showed no detectable differences (data not shown). Nevertheless, the results of the real-time PCR experiments confirmed our previous findings in murine primary rib chondrocytes and a human chondrocyte cell line (6,14). DISCUSSION Our study investigated the linkage between the degree of cartilage damage and the extent of DDR-2
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expression in human articular cartilage. Our results demonstrate increased DDR-2 expression by articular chondrocytes with increased severity of OA changes. The concomitant rise in MMP-13 expression as well as MMP-derived type II collagen cleavage products suggests that OA severity and the extent of DDR-2 expression are crucially linked. During the course of OA development, distinct changes in the articular cartilage occur. Alterations in matrix composition (15,16), reductions in cell numbers (17–19), an increased chondrocyte apoptosis rate (20,21), and a change in growth factor responsiveness (22–24) have been described. Although these phenomena are common, they usually appear as relatively late changes. A very early event in OA pathogenesis, however, is the loss of matrix proteoglycans (25), which are found throughout the extracellular matrix and, maybe more importantly, in the pericellular zones around each chondrocyte (26–29). This pericellular zone is thought to provide mechanical resistance (30–33), it might be important for the assembly of hyaluronan–aggrecan–link protein complexes (34), and its small proteoglycans might have inhibitory/regulatory functions in collagen fibrillogenesis, fibronectin adhesion, and growth factor binding (29,35). Additionally, under physiologic conditions, the high proteoglycan content of the pericellular zone is an effective barrier to the type II collagen in the pericellular capsule and the surrounding matrix. When proteoglycan loss occurs in the early stages of OA, this barrier most likely is destroyed and chondrocytes come into contact with type II collagen and specific receptors become activated. This hypothesis is supported by our recent findings showing that DDR-2, a receptor that preferentially binds type II collagen (8–10), up-regulates MMP-13 upon stimulation with native type II collagen (6). Since this event occurs in the absence of inflammation and without prior collagen damage (14), this process might play a key role in the initiation of type II collagen degradation in early OA. Given the potential importance of DDR-2 in OA, we investigated the relationship between cartilage damage and the extent of DDR-2 expression in human articular cartilage and hypothesized that DDR-2 expression is linked to the grade of cartilage damage such that increased loss of tissue integrity leads to increased DDR-2 expression. Histologic and immunohistochemical analyses indeed revealed a dependence of DDR-2 expression on the grade of cartilage damage. When the percentage of positively stained cells was assessed, a
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Figure 4. Up-regulation of DDR-2 and MMP-13 mRNA in primary human articular chondrocytes. Results from 2 specimens are shown. A and B, DDR-2 expression in human articular chondrocytes upon interaction with native type II collagen or denatured type II collagen (gelatin). C and D, Endogenous MMP-13 expression in human articular chondrocytes after incubation for 24 hours with either native type II collagen or gelatin. Cells cultured in untreated wells served as controls. The scale of the y-axis is different in A and B, as well as in C and D. See Figure 2 for definitions.
significant correlation between the 2 factors was found. This observation is consistent with other studies that showed a connection between cartilage damage/Mankin score and proteoglycan content or chondrocyte gene expression (36–38). Concomitantly with DDR-2 up-regulation, we observed an increase in MMP-13 at the protein level. The collagenase MMP-13 is well known as a major type II collagen cleaving enzyme (39,40). However, its upregulation can be caused by many different factors, such as fibronectin fragments, interleukin-1, and tumor necrosis factor ␣ (41–43). Nevertheless, in early OA, significant tissue degradation and inflammation are absent, so distinct molecules such as DDR-2 might be responsible for the induction of MMP-13 synthesis in articular chondrocytes. In fact, our in vitro experiments in primary human articular chondrocytes also showed an up-regulation of MMP-13 mRNA upon the interaction of DDR-2 with native type II collagen but not with
gelatin, thereby strengthening the above assumption and making our study pathophysiologically more meaningful. These data indicate that not only is the extent of DDR-2 expression linked to the grade of cartilage damage, but the degree of tissue damage itself is also dependent on the extent of DDR-2 expression and concomitant matrix catabolism. The initial event of DDR-2 activation thus seems to induce a steady rise in proteinase synthesis, type II collagen degradation, and again DDR-2 activation, thereby leading to a perpetuation of this mechanism and ultimately to the development and progression of OA. The potential importance of DDR-2 lies in the fact that its activation can be seen as a uniform event in OA development and progression, independent of the disease’s primary cause. In fact, there must be an earlier event that ultimately causes DDR-2 activation. Although initial mechanisms are so far unknown, any disruption of the pericellular microenvironment
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(44,45) might lead to the exposure of type II collagen fibrils to chondrocytes, which would then cause the activation and increased expression of DDR-2, as depicted above. Agents designed to block DDR-2 could be envisioned as potential remedies for OA, since they would be effective before matrix catabolism has reached a point of no return. AUTHOR CONTRIBUTIONS Dr. Li had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Sunk, Bobacz, Xu, Li. Acquisition of data. Sunk, Bobacz, Hofstaetter, Amoyo, Soleiman, Smolen, Xu, Li. Analysis and interpretation of data. Sunk, Bobacz, Xu, Li. Manuscript preparation. Sunk, Bobacz, Li. Statistical analysis. Sunk, Bobacz, Xu, Li.
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