The homologous DDRs are composed of
The homologous DDRs are composed of an N-terminal discoidin homology domain, followed by a sequence of ∼220 amino acids unique to the DDRs, a transmembrane domain, a large juxtamembrane domain, and a conserved cytoplasmic tyrosine kinase domain. In a previous study, we demonstrated that DDR activation by collagen I is a consequence of direct DDR-collagen interaction via the discoidin domain, and we mapped the collagen I binding site on the DDR2 discoidin domain (Leitinger, 2003). In another study, we mapped the DDR2 binding site on collagen II to the collagen II D2 period (Leitinger et al., 2004).
In this study, we sought to define whether collagen X is a ligand for the DDRs. We were particularly interested in the interaction between DDR2 and collagen X, because DDR2 is known to function in cartilage: DDR2 −/− mice have shorter long bones due to reduced chondrocyte proliferation (Labrador et al., 2001); DDR2 is associated with osteoarthritis in a mouse model of the disease (Xu et al., 2005); and in our previous study, we found that the cartilage-specific collagen II was a much better ligand for DDR2 than for DDR1 (Leitinger et al., 2004). Whether DDR1 has any function in cartilage is currently not known. We found that collagen X is primarily a ligand for DDR2. The characterisation of collagen X receptors and their respective binding site/s is essential for our understanding of how collagen X regulates chondrocyte metabolism and will provide important clues to the mechanism and functional significance of cell adhesion to collagen X.
Discussion Despite nearly two decades of investigations, the functional role of collagen X deposition in the growth plate ECM remains ill-defined. Collagen X null-mutation studies have provided support to the notion that the collagen X network is important in maintaining the compositional and organisational integrity of the ECM, but also pointed to the involvement of the hypertrophic ECM in hematopoiesis (Kwan et al., 1997, Gress and Jacenko, 2000). With its unique temporal and spatial MK-3207 HCl sale pattern and its localisations in both the pericellular and interterritorial matrices, we hypothesised that the pericellular network of collagen X provides an important structural link between hypertrophic chondrocytes and the ECM. Cell signalling events following the binding of collagen X to the respective receptors are important mediators of processes in EO such as cell maturation and matrix turnover. This notion is supported by our recent report of the interaction between hypertrophic chondrocytes with purified collagen X via the collagen binding integrin α2β1 (Luckman et al., 2003). In order to further understand the biological significance of cell-collagen X interactions, we have examined the possibilities of involvement of other collagen receptors in cell binding to collagen X. The present study demonstrates that collagen X is a ligand for DDR2. DDR2 has been observed to bind to a number of fibrillar collagens (types I, II, III and V), but does not recognise the basement membrane collagen IV (Shrivastava et al., 1997, Vogel et al., 1997). Collagen X thus represents the first non-fibrillar ligand for DDR2. The solid phase binding assays (Fig. 2) and receptor autophosphorylation data (Fig. 3) show that collagen X is primarily a ligand for DDR2, not DDR1. However, as DDR1 showed weak binding to collagen X, DDR1 may act as a low affinity collagen X receptor. These results are similar to our earlier data on the interaction of the DDRs with the cartilage-specific collagen II (Leitinger et al., 2004). For a physiologically relevant interaction, ligand and receptor have to be expressed in the same place. Collagen X expression is restricted to the growth plate of long bones, while DDR2 is expressed widely in a number of tissues throughout the body. Our data presented in Fig. 4, Fig. 5 show that hypertrophic chondrocytes, the source of collagen X deposition, express mRNA for DDR2 and that the DDR2 protein can also be detected in hypertrophic chondrocytes. These findings are consistent with an interaction of DDR2 with collagen X in the growth plate and strongly support the idea that DDR2 is a physiological receptor for collagen X. In an earlier study, DDR2 mRNA was detected in proliferating murine chondrocytes in vivo, and the elimination of DDR2 in the mouse led to a bone growth defect due to reduced chondrocyte proliferation (Labrador et al., 2001). Our present RT-PCR data are consistent with the report by Labrador et al. (2001). However, we have not been able to localise DDR2 protein in the proliferation zone of the murine growth plate by immunohistochemical methods. Fig. 5F indicates that the initial DDR2 protein expression is at the junction of the proliferative and hypertrophic zones. Our observations in the growth plate therefore suggest a possible role of DDR2 in cell maturation rather than cell proliferation. In the articular cartilage, DDR2 protein is detected on all articular chondrocytes whereas collagen X expression is restricted to the pericellular area of the deep zone (hypertrophic) chondrocytes (Fig. 5A and B). The reason why DDR2 is not found on growth plate proliferative chondrocytes would require further investigation. Thus, DDR2 fulfils important functions in bone growth, and our present study forms the basis for future studies into the effect of DDR2 signalling on hypertrophic chondrocytes and EO. In particular, our hypothesis that DDR2 regulates chondrocyte maturation will need to be tested.