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Comparative Proteomic Analysis of Normal, Collagen VI null and Collagen IX Null Mouse Cartilage


Wilson, RR, Comparative Proteomic Analysis of Normal, Collagen VI null and Collagen IX Null Mouse Cartilage, April 7th-April 12th 2013, GRC, Les Diablerets, Switzerland (2013) [Conference Edited]


Cartilage is a unique tissue that requires specialized methods for proteomic analysis. We have focused on developing methods for mouse cartilage protein extraction and fractionation for 2-D gel and nano-LC/MS based proteomics. This has led to the discovery of novel cartilage ECM components and protein fragments, new protein markers associated with cytokine-driven cartilage degradation, and changes in chondrocyte proteins and tissue composition associated with cartilage development in vitro and in vivo. The essential benefit of mouse tissues is the availability of modified strains expressing disease-causing mutations or lacking cartilage constituents with important or unknown functions. Differential analysis of wild-type and mutant cartilage analysis enables the effects of these mutations to be studied in greater detail at the protein level. Here we report the proteomic comparison of wild-type femoral head cartilage with tissues lacking either collagen VI or collagen IX, two proteins required for normal skeletal development and cartilage integrity in adulthood, to further investigate their roles in vivo. Collagen VI contributes to the biomechanical properties of chondrocyte pericellular environment in both growth plate and articular cartilage. Abnormal skeletal development in the absence of collagen VI in cartilage is associated with delayed ossification, however the molecular consequences of collagen VI ablation had not been previously assessed. Proteomic analysis revealed major differences in ECM composition in collagen VI-deficient cartilage, including elevated levels of the prototypic cartilage markers aggrecan, link protein and collagen II, as well as more recently-identified cartilage matrix proteins such as CILP2 and lactadherin. Delayed cartilage mineralization in the COL6A1 knockout was evident from reduced levels of tartrate-resistant acid phosphatase and inorganic pyrophosphatase, as well as proteins with as-yet unknown roles in endochondral ossification such as dentin matrix protein-1 and alpha-2 HS-glycoprotein. A major finding was the >10-fold reduction in the level of MMP13, a protein essential for matrix remodeling in the growth plate (p < 0.00005), in collagen VI-deficient cartilage. Our proteomic analysis confirms the key role for collagen VI in endochondral ossification and provides the first evidence for the molecular changes resulting from collagen VI ablation in cartilage. Collagen IX, together with COMP and matrilin-3, is essential for cartilage matrix assembly and proper organization of the growth plate chondrocytes. Collagen IX ablation resulted in reduced levels of matrilin-1, matrilin-4, thrombospondin-4 and epiphycan, in addition to the known collagen IX binding partners COMP and matrilin-3; whereas collagen XII and the TGFbeta-induced proteins BGH3 and fibronectin were increased. In addition to altered abundance of BGH3, we detected differential processing of BGH3, which has recently been shown to be a substrate of MMP9. These data were validated using quantitative real-time PCR, immunostaining and western blotting and shed new light on the collagen IX physical and genetic "interactome".

Item Details

Item Type:Conference Edited
Keywords:proteomics cartilage arthritis
Research Division:Biomedical and Clinical Sciences
Research Group:Clinical sciences
Research Field:Rheumatology and arthritis
Objective Division:Expanding Knowledge
Objective Group:Expanding knowledge
Objective Field:Expanding knowledge in the biological sciences
UTAS Author:Wilson, RR (Dr Richard Wilson)
ID Code:84156
Year Published:2013
Deposited By:Central Science Laboratory
Deposited On:2013-04-23
Last Modified:2013-07-04

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