Analysis of cartilage development in normal mice and in mice lacking collagen IX or COMP

The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3 and COMP is essential for cartilage matrix stability, as multiple epiphyseal dysplasia patients with Col9a1, Col9a2, Col9a3, Comp and Matn3 mutations develop early onset osteoarthritis. Ablation of COMP and matrilin-3 expression in mice results in no overt phenotype, while the collagen IX knockout has severely disturbed growth plate organization, hypocellular regions and abnormal chondrocyte shape. This abnormal chondrocyte differentiation is likely to involve altered ECM remodeling and cell-matrix interactions but the mechanisms are presently unknown. To investigate the role of collagen IX and molecular basis of the collagen IX-/- cartilage phenotype we quantified protein expression differences between wild-type and knockout femoral head cartilage by nanoLC tandem mass spectrometry (LTQ-Orbitrap) at two different developmental stages, using 2-D electrophoresis, Western blotting, ELISA, immunofluorescence and real-time quantitative PCR to validate our results. Additional analysis was performed on COMP -/- cartilage to ascertain differences related specifically to the collagen IX phenotype. We identified 403 and 472 proteins (≥2 unique peptides) by nanoLC-MS/MS analysis of 3-day and 21-day cartilage, respectively. Collagen IX ablation was associated with dramatically reduced levels of COMP and matrilin-3, consistent with known interactions. In addition, matrilin-1, matrilin-4, epiphycan and thrombospondin-4 levels were significantly reduced in collagen IX-/- cartilage. The direct association between matrilin-4, but not thrombospondin-4, was confirmed by ELISA, providing the first evidence that collagen IX is the major binding partner for matrilin-4 in cartilage. In contrast, we found that reduced thrombospondin-4 levels in collagen IX cartilage were caused by decreased expression at the mRNA level. Increased levels of collagen XII, TGFbeta-induced protein (beta-igH3) and fibronectin were specifically associated with collagen IX ablation and not detected in COMP -/- cartilage, suggesting a specific role in the collagen IX phenotype. In addition we observed altered processing of beta-igH3, a recently-identified target of MMP9, providing preliminary evidence for altered MMP9 activity in the collagen IX null mouse. This first differential analysis of wild-type and mutant mouse cartilage demonstrates the feasibility for this novel approach to discovery of protein interactions in vivo, leading the way for proteomic analysis of mouse models of human chondrodysplasias and mutant cartilage lacking matrix components of important or unknown function.