Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon

Abstract

Cartilage superficial zone protein/proteoglycan (SZP) or proteoglycan 4 (PRG4), has been demonstrated to have the potential for several distinct biological functions including cytoprotection, lubrication and matrix binding. In the present study, we have examined both the immunolocalisation and the mRNA expression pattern of PRG4 in tissue harvested from the compressed and tensional regions of young and mature bovine tendons. Immunohistochemical analyses, utilizing monoclonal antibody 3-A-4 which recognizes a conformational-dependent epitope on native PRG4, demonstrated that PRG4 is present predominantly at the surface of fibrocartilaginous regions of tendon, with the intensity of immunoreactivity in this region increasing with age. RT-PCR analyses revealed that the expression of PRG4 mRNA can be modulated by exposure to cytokines and growth factors. In addition, analyses of human pathological tendon revealed that PRG4 may also be expressed as an alternatively spliced form lacking exons which encode part of the N-terminal matrix-binding and cell-proliferative domain; however, it remains to be determined whether such splice variants are a feature of human tendon, regardless of disease state. Taken together, these data indicate that PRG4 may play an important cytoprotective role by preventing cellular adhesion to the tendon surface as well as providing lubrication during normal tendon function, in a manner complimentary to cartilage PRG4. Structural modifications to SZP, together with a reduction in synthesis during tendon inflammation with injury and disease may account for the formation of tendon adhesions and contribute to the overall dysfunction of the tissue.

Introduction

Superficial zone protein/proteoglycan (SZP), recently renamed proteoglycan 4 (PRG4) by the human gene nomenclature committee, was originally isolated and purified from the culture media of explants derived from the superficial zone of bovine articular cartilage (Schumacher et al., 1994). In subsequent studies, SZP (PRG4) and a related homologue, megakaryocyte stimulating factor precursor protein (MSF), first detected in the urine of patients with acute thrombocytopenia undergoing bone marrow transplantation, was cloned and its primary amino acid sequence determined (Merberg et al., 1993, Flannery et al., 1999). The isolation, characterization and mapping of mouse and human PRG4 genes was later described by Ikegawa et al. (2000). SZP/MSF (PRG4) has a molecular mass of approximately 345 kDa and is synthesized by both superficial zone chondrocytes (but not chondrocytes derived from the mid- and deep zones) and some of the synovial cells which border the joint cavity (Schumacher et al., 1999). The core protein of PRG4 is composed of large (76–78 repeats) and small (6–8 repeats) mucin-like O-linked oligosaccharide-rich repeat domains flanked by cysteine-rich N- and C-terminal domains that are homologous to the somatomedin B and haemopexin domains of vitronectin, respectively (Merberg et al., 1993, Flannery et al., 1999). There are also potential sites for heparin binding, N-linked oligosaccharide substitution and glycosaminoglycan (GAG) attachment. A low level of substitution with chondroitin sulfate or keratan sulfate GAGs, in addition to the secretion of PRG4 into the synovial fluid of the joint distinguishes it from matrix-bound proteoglycans, such as aggrecan, which are primarily retained within the tissue (Schumacher et al., 1994, Schumacher et al., 1999). Within tendons, increased concentrations of aggrecan are associated with fibrocartilaginous regions, which develop at sites where tendons pass around bone and are subjected to compressive and shear forces (Gillard et al., 1979, Ralphs et al., 1991, Vogel et al., 1993). Similarly, aggrecan and other matrix molecules common to the cartilage phenotype are expressed at tendon insertion sites or entheses where tendon attaches to bone (Benjamin and Ralphs, 1995, Benjamin and Ralphs, 1997, Benjamin and McGonagle, 2001). In contrast, the proteoglycan concentration is significantly lower within proximal/tensional regions of tendon (Vogel et al., 1994, Vogel and Meyers, 1999, Rees et al., 2000).

Recent studies suggested that SZP (PRG4) and lubricin, an articular cartilage surface glycoprotein first identified by Swann et al., 1981a, Swann et al., 1985, may be the same molecule (Jay et al., 2001) which accumulates at the articular cartilage surface. Expression of SZP/lubricin (PRG4) is proposed to be important for preventing cell attachment to the articular surface of diarthrodal joints, in addition to maintaining lubrication properties at the articular cartilage–synovial interface as well as in teno-synovial tissues (Flannery et al., 1999, Marcelino et al., 1999). The significance of this function is highlighted by human camptodactyly–arthropathy–coxa vara-pericarditis (CACP) syndrome in which a number of abnormalities of tendons within the tenosynovial sheaths have been described, including significant adhesion formation and loss of tendon elasticity (Ochi et al., 1983). Mutations in the CACP gene, which encodes a secreted protein which is homologous to SZP/lubricin/MSF or PRG4 (Jay et al., 2000, Flannery et al., 1999), have been identified as the cause of CACP syndrome (Marcelino et al., 1999). In addition, an alternatively spliced form of SZP/PRG4 mRNA has been detected in human osteoarthritic cartilage and expression of this truncated variant has been proposed to contribute to joint pathology (Flannery et al., 1999).

PRG4 plays a significant role in the normal physiology of musculoskeletal tissues and the aberrant functioning of this molecule has been implicated in joint pathology. The present study was therefore conducted using young and mature tendons from compressed and tensional regions, as well as human pathological tendon to examine whether the expression and immunolocalisation of PRG4 occurs differentially in these distinct functional regions during development or the onset of disease. Our data demonstrate that PRG4 is present predominantly in fibrocartilaginous regions of tendon; in addition, its expression is modulated in tenocytes exposed to cytokines and growth factors. We also provide evidence that PRG4 may be expressed as an alternatively spliced variant in human pathological tendon.