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, βig-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 TGFβ-induced protein βig-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

Depletion of Collagen IX Alpha1 Impairs Myeloid Cell Function

The trabecular extracellular matrix (ECM) forms a three-dimensional scaffold to stabilize the bone marrow, provide substrates for cell-matrix interactions and retain, present or release signals to modulate hematopoietic stem and progenitor cell development. However, the impact of trabecular ECM components on hematopoiesis has been poorly studied. Using collagen IX alpha1 - deficient (Col9a1((-/-))) mice, we revealed that a lack of collagen IX alpha1 results in a disorganized trabecular network enriched in fibronectin, and in a reduction in myeloid cells, which was accompanied by a decrease in colony-stimulating factor 1 receptor expression on monocytes from the bone marrow. In contrast, B-cell numbers in the bone marrow and T-cell numbers in the thymus remained unchanged. Alterations in the bone marrow microenvironment may not only reduce myeloid cell numbers, but also have long-term implications for myeloid cell function. Mice were infected with Listeria moncytogenes to analyze the function of myeloid cells. In this case, an inadequate macrophage-dependent clearance of bacterial infections was observed in Col9a1((-/-)) mice in vivo. This was mainly caused by an impaired interferon-gamma/tumor necrosis factor-alpha-mediated activation of macrophages. The loss of collagen IX alpha1 therefore destabilizes the trabecular bone network, impairs myeloid cell differentiation, and affects the innate immune response against Listeria. Stem Cells 2018;36:1752-176