Abstract
Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.
Highlights
From the ‡Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Victoria 3052, Australia, the §Central Science Laboratory, University of Tasmania, Hobart, Tasmania 7001, Australia, the Protein Discovery Center, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia, the **Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany, the ‡‡Medical Faculty, Center for Biochemistry, University of Cologne, 50931 Cologne, Germany, the ¶¶Institute for Protein Research, Osaka University, Suita, Osaka 5650871, Japan, the Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia, and the §§Department of Pediatrics, University of Melbourne, Parkville, Victoria 3052, Australia
Meta-analysis of Two Cartilage Developmental Models—We recently identified a cohort of extracellular matrix (ECM) components and markers for chondrocyte differentiation by comparison of mouse juvenile epiphyseal cartilage (P3 knee cartilage) and 3-week high density chondrocyte cultures [12]
Of the 146 proteins that were differentially expressed in post-natal day 3 (P3) and P21 cartilage, a subset of 34 significant (q Ͻ 0.01) ECM and related proteins were annotated by the functional terms extracellular matrix (ECM), thrombospondin, type III repeat (TSP), EGF-like region, conserved site (EGF), leucine-rich repeat (LRR), polysaccharide binding (PSB), and positive regulation of cell adhesion (Adhesion)
Summary
Cartilage Dissection and Chondrocyte Preparation for Fluorescence-activated Cell Sorting—Femoral head cartilage was obtained from 3- and 21-day post-natal C57/Bl6 mice by dislocation of the hip joint, fracture at the femoral neck, and removal of the ligamentum teres at the insertion site. Chondrocytes were resuspended in PBS containing 5% FCS (6 ϫ 103 cells/ml in 50 ml) and incubated with primary conjugated antibodies specific for CD24a and CD200 (Becton Dickinson) for 15 min on ice. The cells were subsequently washed and co-stained with 7-aminoactinomycin D for dead cell detection, followed by flow cytometry (FACSCantoII). Protein identifications were accepted if the protein contained at least two unique peptides (in terms of amino acid sequence), and the protein was assigned a probability Ͼ0.99 by the Protein Prophet algorithm [17] This threshold will constrain the protein false discovery rate (FDR) to Ͻ1%. Tissue sections were blocked with goat serum (Vectastain Elite ABC rabbit IgG kit) in 1% BSA in PBS for 60 min prior to overnight incubation with rabbit polyclonal antibodies to vitrin (2 g/ml), Urb (0.2 g/ml) [21], or CILP-2 [22] (2 g/ml). The images were captured at 10ϫ magnification with a Nikon Eclipse 80i microscope
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