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THE SMALL PROTEOGLYCAN DECORIN BINDS TO CHONDROCYTES AND PROSTATIC CANCER CELLS THROUGH THE EGF RECEPTOR AND COMPETES WITH BIGLYCAN FOR THE SAME RECEPTOR *David Gerard, *Wenhua Liu, +*Gabriella Cs-Szabo +* Rush University Medical Center, Chicago, IL [email protected] INTRODUCTION: Human articular chondrocytes produce a variety of Non-labeled decorin demonstrated a definite ability to compete with small proteoglycans (PGs), including the structurally similar decorin and labeled decorin in binding to both cell types by completely abolishing biglycan. These molecules play regulatory roles in the assembly of the the binding of the RR-decorin at the 20 µg/ml concentration. matrix through binding to different matrix molecules, including fibrillar Competition with EGF receptor blocking antibody was concentrationcollagens, fibronectin and growth factors. Decorin was shown to be dependent because the antibody at 10 µg/ml concentration abated the RR involved in collagen fibrillogenesis and biglycan in the assembly of the fluorescence to a greater extent (about 95% of signal was lost) than at pericellular matrix network. We previously reported that the expression the 5 µg/ml level (50% signal loss). Biglycan-decorin competition was of both decorin and biglycan is increased in degenerated [1] and also observed in both cell types at a 20 µg/ml concentration of the osteoarthritic [2] cartilage. We also found that the addition of decorin to respective competitor. chondrocytes cultured in the presence of transforming growth factor- ß (TGF-ß) counteracted the effect of the growth factor on matrix synthesis Decorin significantly decreased the expression levels of aggrecan and [3]. Therefore, it is important to determine whether decorin can directly type II collagen. Message levels decreased to 50% of the control level by bind to chondrocytes and then, to delineate the biological effect and 8 hours of treatment. On the other hand, the message level of the cell potential consequences of this binding on chondrocyte homeostasis. cycle inhibitor molecule p21 was strongly upregulated (Fig 1). For this purpose, human articular chondrocytes were cultured in confluent monolayer and binding characteristics of decorin were determined. Because decorin was shown to bind to cancer cells through the EGF receptor [4] and downregulated cell proliferation [5], human prostatic cancer cell line 3 (PC-3) that is known to express high amounts of the EGF receptor on the cell surface, was also used as a model for chondrocytes. Additionally, because biglycan competes with decorin for the same binding site on the surface of fibrillar collagens, competition assays for chondrocyte binding were performed. Lastly, the effect of decorin on chondrocyte gene expression was also assessed. MATERIALS AND METHODS: PC-3 cells (ATCC) were continuously kept in culture, passaged by trypsinization and then cultured and treated similarly to chondrocytes. Human ankle joints with no known joint disease (Collins Grade 0-1) were received from six donors (65-75 years old) and used with the permission of the Institutional Review Board. Chondrocytes were harvested and cells released by sequential enzymatic digestion. Binding assays: Rhdecorin (EMP Genetech, Germany) and biglycan (Sigma) were labeled with Rhodamine-red (RR) according to the manufacturer’s instructions (Molecular Probes). Cells were cultured overnight in chamber slides; and then were subjected to RR-labeled decorin in different concentrations (1-5 µg/ml) for 30 min, 90 min, 3 hr, 6 hr, 12 hr, or 24 hr at 37°C. Competition assays were performed using RR-labeled decorin (5 µg/ml) and non-labeled decorin (1-20 µg/ml); EGF receptor blocking antibody (Sigma; 5-10 µg/mL) or biglycan (5-20 µg/ml) for 3 hr at 37°C. RRlabeled biglycan (5 µg/ml) was also used with non-labeled decorin (5-20 µg/ml). The amount of fluorescent label was observed under a fluorescent microscope (Nikon Eclipse E600) and/or a confocal microscope (Nikon Eclipse TE200), both of which are equipped with Metamorf software. Internalization of the RR-labeled protein was determined by stripping the cell surface with trypsin and/or by confocal microscopy. The expression of the EGF receptor on the cell surface was demonstrated by antibody staining. EGF receptor expression was determined by RT/PCR in both cell types. For gene expression studies, chondrocytes were cultured in alginate beads [6] for 2-, 4-, 8- and 24-hr supplemented with ITS, in the presence or absence of 5 µg/ml rhdecorin. The cells were then released from the beads and total RNA was isolated. Expression levels of aggrecan, collagen type II and cyclin-dependent kinase inhibitor p21 were determined by RT/PCR using GAPDH as a control [2]. Statistical analyzes were performed by ANOVA. 0 2 4 8 24 hr p21 GAPDH Figure 1. Decorin upregulates p21 message levels in chondrocytes DISCUSSION: In this study, we demonstrate for the first time that decorin binds to the cell surface of chondrocytes. The receptor is most likely the EGF receptor, because EGF receptor blocking antibodies successfully competed with decorin for binding. This is further corroborated by the fact that PC-3 cells rich in EGF receptors demonstrate similar binding of fluorescently labeled decorin. This EGF receptor binding and upregulation of cell cycle inhibitor p21 could explain the influence that decorin has upon cancer cell proliferation. Because decorin has a similar effect on chondrocytes, this may be responsible for initiating chondrocyte quiescence. The downregulation of aggrecan and collagen expression by decorin may imply that this small PG, which is upregulated by TGF-ß, may serve as a regulator for the growth factor’s effect by imposing an opposite effect on the expression levels of the major extracellular molecules of cartilage. Finally, the ability of biglycan and decorin to compete for the same receptor on the cell surface as well as for binding sites on other extracellular molecules, suggests that these molecules may complement each other’s biological effects REFERENCES: [1] Cs-Szabo, G. et al: In: Many Faces of Osteoarthritis. (Eds. Hascall and Kuettner), Birkhauser Verlag AG, Basel, p. 369, 2002; [2] Cs-Szabo, G. et.al.: Arthritis Rheum 40, 1037, 1997; [3] Liu, W. et al.: Trans ORS 28, 100, 2003; [4] Iozzo, RV. et al.: J Biol Chem 274, 4489, 1999; [5] Santra, M. et al.: J Clin Invest 100, 9, 1997; [6] Hauselmann, H.J. et.al.: Matrix 12, 116, 1992; ACKNOWLEDGEMENTS: This work was supported by the NIH (3P50AR-39239). Collaboration with the Gift of Hope Organ and Tissue Donor Network and A. Margulis, M.D. is greatly appreciated. RESULTS: PC-3 cells, which express high levels of the EGF receptor, were able to bind decorin in a concentration- and time-dependent manner. Chondrocytes were also shown to have EGF receptors on their cell membrane and were able to bind decorin in similar fashion to PC-3 cells. RR-labeled decorin was found to initiate binding upon the cell surface of both cell types within 90 min, with complete binding observed by 3 hr. Internalization of labeled decorin was also detected in 3 hr. Binding intensity reached a plateau at a 5 µg/ml concentration. 51st Annual Meeting of the Orthopaedic Research Society Paper No: 0135