ARTICLE |
Correspondence to: Susan Chubinskaya, Dept. of Biochemistry, Rush Medical College at RushPresbyterianSt. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612.
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Summary |
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This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239250, 2000)
Key Words: osteogenic protein-1, human articular cartilage, in situ hybridization, comparative RT-PCR, immunohistochemistry, Western blotting
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Introduction |
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CARTILAGE HOMEOSTASIS is a well-synchronized balance between anabolic and catabolic processes. Although various inflammatory mediators and proteinases present within the cartilage and the joint as a whole can upset this equilibrium, specific growth factors may well be able to reestablish this balance. It has been shown that bone morphogenetic proteins (BMPs), as members of the human transforming growth factor-ß (TGF-ß) superfamily, induce new cartilage and bone formation in vitro and in vivo (
Bovine osteogenic protein was originally purified from bone matrix as a mixture of homodimers of two members of the BMP family. One component was identified as the protein product of the bovine equivalent of the human osteogenic protein-1 (OP-1; BMP-7) gene and the other was identified as the protein product of the bovine equivalent of the human BMP-2A gene (
Recent studies from our department (
Members of the BMP family have also been identified in different connective tissues, but mostly during the process of development. OP-1 was detected in the ectodermal epithelium of the fetal mouse fore- and hindlimbs, heart, teeth, intestinal epithelium, perichondrium, hypertrophic chondrocytes, and the periosteum/osteoblast layer of developing human bones (
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Materials and Methods |
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Reagents
Human recombinant mature and pro-OP-1, polyclonal antiserum against pro-domain of OP-1 (R2854), and anti-OP-1 monoclonal antibodies (12G3 and 1B12) were provided by Creative BioMolecules and Stryker Biotech (Hopkinton, MA;
Tissue Acquisition
Normal Cartilage.
Full-thickness articular cartilage was dissected within 24 hr of death from the load-bearing regions of femoral condyles of human donors with no history of joint disease. Tissues (from both men and women, ranging from newborn to 84 years of age) were obtained through the Regional Organ Bank of Illinois.
Osteoarthritic Cartilage. Human OA cartilage adjacent to eroded areas was obtained as surgical specimens from patients (5988 years of age, both men and women) undergoing total knee arthroplasty due to advanced OA. Within 3 hr after surgery, full-thickness noncalcified cartilage was dissected from the femoral condyle. Osteophytes were excluded from the studies because this tissue represents a completely different biology compared to primary OA cartilage. Adjacent pieces of the cartilage from both patients and donors were either processed for histology, immunohistochemistry, and in situ hybridization or they were used for total RNA extraction for RT-PCR or for protein extraction for Western blot analyses. Cartilages from four human donors (newborn and adult) were used for isolation and culture of chondrocytes in alginate beads.
Preparation of Chondrocytes and Alginate Beads
Chondrocytes were prepared for culture in alginate beads as previously described (
Histological Processing of Cartilage Tissue
Cartilage from newborn donors and articular cartilage from adult donors (~3 x 3 mm2 pieces) were removed from the joints free from subchondral bone or calcified cartilage and processed for in situ hybridization and immunohistochemistry using RNase-free solutions as previously described (
Processing of Alginate Beads for In situ Hybridization and Histology
The beads were processed as previously described (
Sections of cartilage tissue or alginate bead (6 µm) were utilized for in situ hybridization and immunohistochemistry as described (
Oligonucleotide Probes and PCR Primers for OP-1
The OP-1 sequence has about 30% homology to the sequence of TGF-ß and about 60% homology to BMP-2. Therefore, PCR primers and oligonucleotide probes for in situ hybridization were primarily designed in the most conserved and specific area of the sequence in the nontranslated region of the OP-1. Four antisense and sense oligonucleotide probes and primers were designed on the basis of OP-1 sequence (accession X51801) to fulfill requirements of both in situ hybridization and RT-PCR: (a) 21-mer, antisense, location 18101830, 5'-TTTTCCTTTCGCACAGACACC-3'; (b) 24-mer, antisense, location 14991522, 5'-GGGAAGGTCTCACAAAAGGCAGTT-3'; (c) 20-mer, sense, location 13281347, 5'-TGCCATCTCCGTCCTCTACT-3'; and (d) 23-mer, sense, location 15181540, 5'-TTCCCCTCCCTA-TCCCCAACTTT-3'. The specificity of these probes was compared with sequence data available from PC/GENE on the human DNA database. Sequences were checked using Oligo software program, Version 5 for Windows. All oligonucleotide probes were synthesized and purified by Research Genetics (Huntsville, AL). For in situ hybridization, probes were 3'-end-labeled with 5'-[-thiol-35S]-dCTP (NEN Du Pont; Boston, MA) using terminal deoxynucleotidyl transferase (Promega; Madison, WI). Hybridization was done under conditions of highest stringency.
In Situ Hybridization
In situ hybridization was done according to
In Situ Hybridization Controls
Positive Controls.
Human chondrosarcoma tissue (
Negative Controls.
Sense primers and competitive inhibition control served as negative controls. Competitive inhibition control (cold hybridization) was performed by mixing radiolabeled probe with unlabeled (cold) probe in a ratio of 1:1 and 1:2 (amount of isotope was kept constant) as previously described (
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted directly from cartilage tissue with acidguanidinium thiocyanate as previously described (
Cartilage Extraction
Articular cartilage was briefly washed with 0.9% saline, homogenized, and extracted in 1 M guanidine hydrochloride in the presence of proteinase inhibitors (0.1 M -aminocaproic acid, 0.05 M sodium acetate, 0.005 M EDTA, 0.005 M benzamidine chloride, 0.01 M N-ethylmaleimide, 0.0005 M phenymethanesulfonyl fluoride) at pH 7.4. Samples were extracted for 3 hr on ice and the extracts were dialyzed against water for 48 hr at 28C, lyophilized, and solubilized in sample buffer [0.01 M Tris-HCl, pH 6.5, 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.016% bromphenol blue, 8 M urea, 0.2 M sodium chloride, no dithioerythritol (DTT), pH 6.8].Protein concentrations were determined by a colorimetric assay using BSA as a standard (Pierce; Rockford, IL).
Western Blot Analyses
Aliquots of samples (30 µg of protein/lane) were analyzed by electrophoresis and immunoblotting without prior digestion. Samples were heated in boiling water for 5 min and loaded onto a 12% SDS-polyacrylamide gel (PAGE) in a buffer containing 0.2 M glycine, 25 mM Tris, and 3.5 mM SDS. Proteins were transferred from SDS-PAGE gels onto nitrocellulose transfer membranes overnight at 20 V, 48C in a buffer containing 12 mM Tris (pH 7.4), 6 mM Na-acetate, and 0.3 mM EDTA at pH 7.4. Immunoblot analyses were done either with polyclonal antiserum against pro-domain OP-1 (pro-domain primary antibody; R 2854) or with two anti-OP-1 (mature-OP-1) monoclonal antibodies, 12G3 and 1B12 (
Immunohistochemistry
For immunohistochemistry, cartilage sections (6 µm) were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Before incubation with primary antibodies, tissue sections were digested with keratanase (0.01 U/ml), keratanase II (0.0001 U/ml), and chondroitinase ABC (0.01 U/ml) in 100 mM Tris/50 mM Na-acetate buffer (pH 6.5) at 37C for 90 min to increase the penetration of antibodies into cartilage. Primary antibodies were applied at a 1:100 dilution. Secondary antibodies, peroxidase-labeled goat anti-mouse or anti-rabbit immunoglobulin G (Organon Teknika-Cappel), or biotinylated antibodies from ImmunoPure ABC Alkaline Phosphatase Mouse or Rabbit IgG Staining Kits (Pierce) were used at the same, 1:100 dilution. For controls, the primary antibody was replaced with either normal serum, secondary antibody alone, or primary antibody preabsorbed with OP-1. The colored reaction product was developed either with diaminobenzidine/H2O2 (Sigma) or with alkaline phosphatase-based 1 step-NBT/BCIP substrate (ImmunoPure ABC kit; Pierce). To inhibit endogenous alkaline phosphatase activity, ImmunoPure Phosphatase Suppressor (levamisole) was added to NBT/BCIP substrate in 1:100 dilution.
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Results |
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Expression of OP-1 mRNA
We investigated the expression of OP-1 mRNA in human articular cartilage using comparative RT-PCR and in situ hybridization. By RT-PCR mRNA bands for OP-1 in newborn, normal adult, and OA cartilages were detected (Figure 1A and Figure 1B). PCR bands with specific OP-1 primers revealed the same molecular size products (313 bp) as expected (Figure 1A). When densities of OP-1 mRNA bands were normalized to densities of the GAPDH bands, the relative levels of OP-1 expression in newborn and normal adult cartilage (Figure 1A, Lanes 27) were almost the same. However, in OA cartilage (Figure 1A, Lanes 810), mRNA expression of OP-1 appeared to be upregulated by at least twofold (p<0.01; Figure 1B). Actual values of the PCR bands are represented in Table 1. Sequence analyses of the final PCR products from one normal adult cartilage and one OA cartilage showed 100% homology with human OP-1 mRNA (accession number X51801;
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Sequencing of the PCR products served as a positive control for in situ hybridization because the same PCR primers were applied for both methods. Sense primers and the inhibition control were used as negative controls. Sense oligonucleotide probes (or upstream primers) revealed no specific binding to the chondrocytes (not shown). Addition of unlabeled probe to the radiolabeled oligonucleotide probe at a ratio of 1:1 was able to inhibit specific binding of the probe to the mRNA within the chondrocytes (Figure 2A and Figure 2B), thus demonstrating the high specificity of the probes.
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As a first step with in situ hybridization, the mRNA expression of OP-1 was tested in isolated chondrocytes cultured in alginate beads. These cells showed strong expression of OP-1 mRNA (Figure 3A). In situ hybridization on cartilage sections from different donors and patients verified that all types of analyzed cartilage were positive for OP-1 message. In newborn cartilage, expression was evident from the upper epiphyses to the hypertrophic area (Figure 3B). In normal adult cartilage, OP-1 expression was primarily found in the superficial and middle layers of the cartilage (Figure 3C), whereas in the deep layer levels of expression were very low or below the detection limit. In OA cartilage, OP-1 message was detected in all cartilage layers, with the strongest expression in chondrocyte clusters (Figure 3D).
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Expression of OP-1 Protein
To determine if OP-1 protein can be detected in human articular cartilage, extracts from normal adult and OA cartilages were analyzed by Western blotting. Human recombinant mature and pro-OP-1 were used as positive controls (Figure 4A and Figure 4B, Lanes 1 and 2). As expected, antibody to pro-domain (R2854) did not recognize mature recombinant OP-1 (Figure 4A, Lane 1) but did recognize the pro-form of the molecule (Figure 4A, Lane 2). Antibodies to mature domain (12G3) detected both mature OP-1 (Figure 4B, Lane 1) and pro-OP-1 (Figure 4B, Lane 2). In extracts from all cartilages, anti-mature and anti-pro-domain antibodies recognized immunoreactive bands. Under nonreduced conditions, pro-domain antibody (Figure 4A) recognized three major bands in the pro-OP-1 control at about 3640 kD, 70 kD, and 110 kD. Two of these immunoreactive bands (about 3640 kD and 70 kD) were also present in the cartilage extracts. In addition, two other major bands at 50 kD and above 220 kD were identified. The band at 70 kD may correspond to a mature dimer associated with one pro-domain, called a hemidimer, and bands between 4555 kD could represent the monomer of the pro- and mature domains. According to
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Immunoblot probed with antibody to mature domain (Figure 4B) also showed strong immune bands in both controls and in the cartilage extracts. In controls, the same major band at about 3640 kD was recognized (Figure 4B, Lanes 1 and 2). Furthermore, two other bands at about 50 and 84 kD were found in the mature OP-1 control. This antibody revealed specific OP-1 bands in the pro-OP-1 preparation at the same molecular weight (about 70 and 110 kD) as anti-pro-domain antibody. Extracts from human cartilages showed the strongest immunoreactive band at about 70 kD with antibody to mature domain. Other bands that could be identified on the Western blot with the same antibody are 3640 kD, above 220 kD, and a few minor bands between 45 and 55 kD (Figure 4B). It is important to note that in extracts from OA cartilages an additional low molecular weight band at about 17 kD was detected (Figure 4B, Lane 5). This band could also be seen in some of the donor tissue with signs of asymptomatic degenerative morphological changes (Figure 4B, Lane 4). A band at about 220 kD probably indicates high molecular weight aggregates of OP-1 with matrix molecules, such as collagens or fibronectin, because high affinity of OP-1 binding to different collagens was previously reported (
Immunohistochemical studies were performed on newborn chondrocytes cultured in alginate beads and on cartilage sections. The specificity of antibodies was demonstrated with an inhibition control when primary antibodies were preabsorbed with human recombinant OP-1. The addition of recombinant mature OP-1 decreased the intensity of anti-mature OP-1 staining (Figure 5A and Figure 5B). The same type of control was used to demonstrate the specificity of pro-OP-1 immunostaining with antibody to the pro-domain (not shown). When immunostaining was carried out in the absence of primary antibody (negative control), no positive staining of the cartilage section was visible (Figure 5C). Chondrocytes from newborn donors cultured in alginate beads showed positive staining with anti-mature OP-1 antibody (Figure 5D). The positive immunostaining of human cartilage with antibodies to mature and pro-domains is shown in Figure 6.
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The most striking differences in the distribution of pro- and mature OP-1 were found by immunostaining of cartilage sections. Furthermore, there were distinct staining patterns among newborn, normal adult, and OA cartilage. In newborn growth plate cartilage, very strong intracellular staining with antibodies to mature domain was evident throughout the tissue in the epiphysis, proliferative, and hypertrophic regions (Figure 6A and Figure 6B). The extracellular matrix showed no staining. On the other hand, both the perichondrial matrix and the cells entrapped within it were intensively stained (Figure 6A). When the same newborn cartilage was introduced to the antibody to pro-domain, the strongest staining was detected in the lower hypertrophic zone of growth plate cartilage (Figure 6C and Figure 6D). In the resting and proliferating zones, the intensity of pro-OP-1 staining was lower than in the hypertrophic region. Pro-OP-1 was mostly localized intracellularly and was almost undetectable within matrix. In normal adult cartilage, mature OP-1 was found predominantly in the vicinity of the chondrocytes (within the cells and in the surrounding pericellular matrix) primarily in the superficial and upper middle layers of the tissue (Figure 6E). Some staining was evident in the extracellular matrix. Deep-layer chondrocytes showed very low or practically no mature OP-1 (Figure 6F). In contrast to the distribution of mature OP-1, the majority of pro-OP-1 was localized in the deep layer of tissue, with decreased amounts of pro-OP-1 in the middle and superficial zones of the cartilage (Figure 6G and Figure 6H). The same trend in layer distribution of mature and pro-forms of OP-1 was described for OA cartilage. In the chondrocytes from the upper (superficial) part of OA cartilage, little anti-mature OP-1 staining appeared to be cell-associated, with low staining of lacunae walls. In addition, some staining was evident in the territorial and interterritorial matrix of the upper and middle layers of OA cartilage (Figure 6I). The deep layer of OA cartilage, as was seen in normal adult cartilage, showed very little or no anti-mature OP-1 staining (Figure 6J). The overall level of anti-mature OP-1 staining in OA cartilage appeared to be less intense than in newborn and normal adult cartilage. However, when the same OA cartilage was stained with the anti-pro-domain antibody, deep-layer chondrocytes showed very strong immunoreactivity. The intensity of staining was gradually decreased from the deep zone to the upper zone (Figure 6K and Figure 6L). Moreover, pro-OP-1 staining was detected in the territorial matrix of the deep and middle layers, and granular staining of pro-OP-1 was observed in the middle and upper (superficial) layers of OA cartilage.
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Discussion |
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By in situ hybridization, RT-PCR, Western blotting, and immunohistochemistry, we have demonstrated for the first time that human adult articular chondrocytes at different ages and different histopathogical stages synthesize OP-1, a bone morphogenetic protein that has been reported to be involved in a variety of physiological and pathophysiological processes (
The majority of OP-1 studies have focused on the exogenous application of OP-1 and the resulting induction of anabolic response in a variety of connective tissues including bone, cartilage, and disks (
Interestingly, we detected two forms of the OP-1 moleculethe inactive pro-form and the active mature formsimultaneously present in human adult cartilage. The distinction between the two forms of the protein was possible because of the availability of two types of antibodies. The polyclonal antibody (R2854) was raised in rabbits against the entire pro-domain of the OP-1 sequence (amino acids 29 to 292;
Very intriguing data obtained by immunohistochemistry was the inverted pattern in the localization of mature and pro-OP-1. Here it is important to mention that adult articular cartilage is a heterogeneous tissue in which the cells and matrix are arranged in layers (
Unlike the protein data, expression of OP-1 mRNA was upregulated in diseased cartilage compared to normal newborn and adult tissues. Moreover, in OA cartilage, chondrocytes of all layers demonstrated positive expression of OP-1, whereas in normal adult cartilage the superficial chondrocytes were primarily positive for OP-1 message. Furthermore, OP-1 expression was the strongest in cell clusters (areas of OA cartilage with some cell division). These data were supported by the results of RT-PCR showing that OP-1 mRNA expression was more than twofold higher in OA cartilage than in normal newborn and adult cartilage.
The observed results indicate, first, that the OP-1 gene is inducible in the deep-layer chondrocytes when the superficial layer is destroyed and, second, that in diseased cartilage chondrocytes attempt to withstand catabolic events by upregulation of mRNA for OP-1. However, this upregulation is limited to the OP-1 message and does not appear to take place at the level of protein translation or activation.
Collectively, the data presented here suggest a role for OP-1 in cartilage homeostasis/protection and/or repair. These data further imply that the regulation of the activity of OP-1 at the level of protein may occur through the control of the enzymatic cleavage of pro-OP-1 into the mature form, which could be a key mechanism governing the function of OP-1 in cartilage pathophysiology.
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Acknowledgments |
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Supported by NIH grant 2P50-AR-39239-11.
We thank Dr A. Hari Reddi for valuable suggestions and discussions, and Drs Klaus Huch, Wolfgang Eger, and Arcady Margulis for providing cartilage samples. We would especially like to thank Dr Carol Muehleman for helpful comments on the manuscript. Collaboration with Allan Valdellon, MD, of the Regional Organ Bank of Illinois and his staff, as well as the surgeons of the Department of Orthopedic Surgery at Rush-PresbyterianSt Luke's Medical Center, is gratefully acknowledged.
Received for publication July 27, 1999; accepted September 24, 1999.
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