Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

BACKGROUND: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ßig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. 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 cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFß-induced protein ßig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Growth-related structural, biochemical, and mechanical properties of the functional bone-cartilage unit.

Articular cartilage and subchondral bone act together, forming a unit as a weight-bearing loading-transmitting surface. A close interaction between both structures has been implicated during joint cartilage degeneration, but their coupling during normal growth and development is insufficiently understood. The purpose of the present study was to examine growth-related changes of cartilage mechanical properties and to relate these changes to alterations in cartilage biochemical composition and subchondral bone structure. Tibiae and femora of both hindlimbs from 7- and 13-week-old (each n = 12) female Sprague-Dawley rats were harvested. Samples were processed for structural, biochemical and mechanical analyses. Immunohistochemical staining and protein expression analyses of collagen II, collagen IX, COMP and matrilin-3, histomorphometry of cartilage thickness and COMP staining height were performed. Furthermore, mechanical testing of articular cartilage and micro-CT analysis of subchondral bone was conducted. Growth decreased cartilage thickness, paralleled by a functional condensation of the underlying subchondral bone due to enchondral ossification. Cartilage mechanical properties seem to be rather influenced by growth-related changes in the assembly of major ECM proteins such as collagen II, collagen IX and matrilin-3 than by growth-related alterations in its underlying subchondral bone structure. Importantly, the present study provides a first insight into the growth-related structural, biochemical and mechanical interaction of articular cartilage and subchondral bone. Finally, these data contribute to the general knowledge about the cooperation between the articular cartilage and subchondral bone.