Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage

Abstract

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.

Introduction

Chondrocytes in healthy adult articular cartilage have been shown to be differentiated cells, expressing a stable phenotype. The expression pattern of extracellular matrix molecules involves collagens, proteoglycans, and non-collagen (glyco) proteins (reviewed in Kuettner, 1994). The major matrix components, collagen type II and aggrecan, are responsible for the mechanical strength and the hydro-elastic properties of cartilage. Other quantitatively minor matrix components like collagen type XI and IX or decorin, biglycan, and fibronectin help to stabilize the collagenous network and support the macromolecular framework (reviewed in Bruckner and van der Rest, 1994, Kuettner, 1994, Buckwalter and Mankin, 1997). Proteins with a restricted distribution within the cellular environment, as shown for cartilage oligomeric matrix protein, tenascin, fibronectin, or collagen type VI, may function in cell–matrix interactions.

During the disease process of osteoarthritis (OA), a metabolic activation of chondrocytes is accompanied by phenotypical changes in the synthesis of extracellular matrix components. The expression of collagens is increased, as shown for type II and VI (Lippiello et al., 1977, Mankin et al., 1981, Pullig et al., 1999), and the synthesis of non-collagenous matrix molecules, like small proteoglycans, fibronectin, cartilage oligomeric protein and tenascin, is also activated (Chevalier et al., 1994, Cs-Szabo et al., 1997). The enhanced expression of type X collagen and alkaline phosphatase, components that mark chondrocyte differentiation and hypertrophy, are characteristic phenomena of the late stages of osteoarthritis (Rees and Ali, 1988, von der Mark et al., 1992).

For hypertrophic chondrocytes in osteoarthritic cartilage, parallels can be found with the expression pattern of hypertrophic chondrocytes of the epiphyseal growth plate cartilage, where chondrocytes are arranged in zones according to morphological and phenotypical criteria. Hypertrophic chondrocytes near the mineralized bone matrix of the epiphyseal growth plate have a large cell volume and a high metabolic activity (Breur et al., 1991). They not only express matrix molecules which are typical for chondrocytes of the resting zone of the epiphyseal growth plate and chondrocytes of adult cartilage, like the cartilage collagens and proteoglycans (Tacchetti et al., 1987, Gerstenfeld and Landis, 1991), but they also share characteristics with the osteogenic cells of bone. In vivo and in vitro studies have described the expression of alkaline phosphatase, bone sialoprotein, osteocalcin, osteonectin and also osteopontin (OPN) in the hypertrophic chondrocytes of the growth plate (Strauss et al., 1990, Bianco et al., 1991, Gerstenfeld and Landis, 1991, McKee et al., 1992, Lian et al., 1993, Nakase et al., 1994, Lebeau et al., 1995, Neugebauer et al., 1995, Sommer et al., 1996). Up to now, there have been no data on whether OPN can also be expressed by differentiating adult chondrocytes in vivo.

OPN is a highly phosphorylated and sulfated glycoprotein, with a molecular weight of approximately 32 kDa. Variable sizes up to 70 kDa have been reported. It is expressed in bone forming cells, as well as in mesenchymal cells from the uterus, placenta, kidney, and nervous system (reviewed in Denhardt and Noda, 1998). The expression of OPN was also shown in activated macrophages and lymphocytes (Patarca et al., 1989).

The detailed biological function of osteopontin is not understood; however, data based on the localization, structure and in vivo effects in bone suggest that OPN mediates the attachment of osteoblasts to the bone matrix. These considerations are based mainly on the RGDS sequence (Arg–Gly–Asp–Ser) in OPN, that is known to be a potential cell-binding site (Oldberg et al., 1986, Ruoslahti and Pierschbacher, 1987). Species overlapping analysis of the RGDS mRNA coding sequence revealed a high conservation, implying that OPN has an important function in vivo. The integrins αvβ1, αvβ3, αvβ5, α9β1, α8β1 and α4β have also been shown to interact with OPN (Lian et al., 1993, Smith et al., 1996, Bayless et al., 1998, Denda et al., 1998). Non-integrin mediated binding to OPN was also shown for CD44, the receptor molecule for hyaluronic acid (Weber et al., 1996). OPN also interacts with extracellular matrix components like type I collagen, osteocalcin, or fibronectin (Nemir et al., 1989, Singh et al., 1990, Chen et al., 1992, Ritter et al., 1992).

Expression of OPN in cartilaginous tissue is limited to chondrocytes, which differentiate to form hypertrophic chondrocytes in the epiphyseal growth plate, or to chondrocytic cells in a pathologic environment such as a fracture callus (Yoon et al., 1987, Mark et al., 1988, Ikeda et al., 1992, McKee et al., 1992, Roach, 1992, Lian et al., 1993, Hirakawa et al., 1994, Nakase et al., 1994, Sommer et al., 1996).

This study describes the expression and localization of OPN in osteoarthritic cartilage, to confirm that adult human articular chondrocytes may undergo further differentiation in vivo, and have similarities to the hypertrophic chondrocytes of the epiphyseal growth plate. OPN expression in osteoarthritic cartilage correlates with the morphological signs of matrix degeneration. Detailed information about the stage-specific expression of OPN in OA could serve as a useful tool to mark the osteoarthritic disease process and to further elucidate the pathways involved in the progression of the disease.