title: Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development.
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.
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia; Central Science Laboratory, University of Tasmania, Hobart, Tasmania 7001, Australia. Electronic address: richard.wilson@utas.edu.au.
authors: Richard
authors: R
authors: Wilson
authors: Protein Discovery Center, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia.
authors: Emma L
authors: EL
authors: Norris
authors: Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; Medical Faculty, Center for Biochemistry, University of Cologne, 50931 Cologne, Germany.
authors: Bent
authors: B
authors: Brachvogel
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia.
authors: Constanza
authors: C
authors: Angelucci
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia.
authors: Snezana
authors: S
authors: Zivkovic
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia.
authors: Lavinia
authors: L
authors: Gordon
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia; Department of Pediatrics, University of Melbourne, Parkville, Victoria 3052, Australia.
authors: Bianca C
authors: BC
authors: Bernardo
authors: Medical Faculty, Center for Biochemistry, University of Cologne, 50931 Cologne, Germany.
authors: Jacek
authors: J
authors: Stermann
authors: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors: Kiyotoshi
authors: K
authors: Sekiguchi
authors: Protein Discovery Center, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia.
authors: Jeffrey J
authors: JJ
authors: Gorman
authors: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia. Electronic address: john.bateman@mcri.edu.au.
authors: John F
authors: JF
authors: Bateman
mesh_terms: D000818:Animals; D002356:Cartilage / Q000254:growth & development / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D020543:Proteome
chemical_list: D020543:Proteome