Download the small proteoglycan decorin binds to chondrocytes and prostatic

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