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Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development

2011; Elsevier BV; Volume: 11; Issue: 1 Linguagem: Inglês

10.1074/mcp.m111.014159

1535-9484

Richard Wilson, Emma L. Norris, Bent Brachvogel, Constanza Angelucci, Snežana Živković, Lavinia Gordon, Bianca C. Bernardo, Jacek Stermann, Kiyotoshi Sekiguchi, Jeffrey J. Gorman, John F. Bateman,

Cell Adhesion Molecules Research

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. 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. Cartilage is a unique tissue characterized by an abundant extracellular matrix (ECM) 1The abbreviations used are:ECMextracellular matrixLTQlinear trap quadrupoleFDRfalse discovery rateGdnHClguanidinium hydrochlorideDAVIDDatabase for Annotation, Visualization and Integrated DiscoveryGOgene ontologicalPnpost-natal day nERendoplasmic reticulumCOMPcartilage oligomeric matrix proteinTGFtransforming growth factor. 1The abbreviations used are:ECMextracellular matrixLTQlinear trap quadrupoleFDRfalse discovery rateGdnHClguanidinium hydrochlorideDAVIDDatabase for Annotation, Visualization and Integrated DiscoveryGOgene ontologicalPnpost-natal day nERendoplasmic reticulumCOMPcartilage oligomeric matrix proteinTGFtransforming growth factor. and a single cell type, the chondrocyte. However, the permanent hyaline cartilage, which provides the articulating surfaces of long bones and vertebrae, and the transient growth plate cartilage responsible for endochondral bone growth are uniform in neither cellular phenotype nor protein composition. In articular cartilage, the chondrocytes form morphologically distinct regions comprising a superficial region of flattened cells, a sparsely populated middle layer, and a deep zone of hypertrophic chondrocytes embedded in calcified cartilage at the chondro-osseous junction. In mature articular cartilage, these chondrocytes divide infrequently. In contrast, the active division and expansion of chondrocytes in growth plate cartilage is the primary mechanism for growth of the axial and appendicular skeletal elements (see Fig. 1). Growth plate chondrocytes enter the maturation process from a pool of reserve zone cells in the epiphyseal cartilage most distal to the chondro-osseous junction. These small round cells differentiate into discoid proliferating chondrocytes that align into columns and dictate the axis of bone growth. The chondrocytes then enter a post-mitotic prehypertrophic phase and expand in volume to form fully differentiated hypertrophic chondrocytes that provide a niche for vascular invasion and remodeling of the cartilage into bone (1Kronenberg H.M. Developmental regulation of the growth plate.Nature. 2003; 423: 332-336Crossref PubMed Scopus (2134) Google Scholar). extracellular matrix linear trap quadrupole false discovery rate guanidinium hydrochloride Database for Annotation, Visualization and Integrated Discovery gene ontological post-natal day n endoplasmic reticulum cartilage oligomeric matrix protein transforming growth factor. extracellular matrix linear trap quadrupole false discovery rate guanidinium hydrochloride Database for Annotation, Visualization and Integrated Discovery gene ontological post-natal day n endoplasmic reticulum cartilage oligomeric matrix protein transforming growth factor. In humans, disruption of chondrocyte maturation in the growth plate results in inherited skeletal dysplasias ranging in severity from mild dwarfism (e.g. metaphyseal chondrodysplasia, Schmid type) and early onset osteoarthritis (e.g. multiple epiphyseal dysplasia) to perinatal lethality (e.g. achondrogenesis). Although individually rare, these skeletal dysplasias collectively affect 2–5 per 10,000 live born (reviewed in Ref. 2Newman B. Wallis G.A. Skeletal dysplasias caused by a disruption of skeletal patterning and endochondral ossification.Clin. Genet. 2003; 63: 241-251Crossref PubMed Scopus (34) Google Scholar). Mutations underlying skeletal dysplasias frequently compromise the precise assembly and interaction of cartilage ECM components, highlighting the critical role of ECM networks in chondrocyte differentiation, organization, and survival (3Behonick D.J. Werb Z. A bit of give and take: The relationship between the extracellular matrix and the developing chondrocyte.Mech. Dev. 2003; 120: 1327-1336Crossref PubMed Scopus (97) Google Scholar). In addition to a loss of ECM integrity, endoplasmic reticulum (ER) stress and activation of the unfolded protein response contribute to the pathology (4Bateman J.F. Boot-Handford R.P. Lamandé S.R. Genetic diseases of connective tissues: Cellular and extracellular effects of ECM mutations.Nat. Rev. Genet. 2009; 10: 173-183Crossref PubMed Scopus (237) Google Scholar). Microarray analysis of microdissected mouse cartilage zones has generated differential mRNA expression profiles of chondrocyte subpopulations (5Belluoccio D. Bernardo B.C. Rowley L. Bateman J.F. A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage.Biochim. Biophys. Acta. 2008; 1779: 330-340Crossref PubMed Scopus (27) Google Scholar, 6Wang Y. Middleton F. Horton J.A. Reichel L. Farnum C.E. Damron T.A. Microarray analysis of proliferative and hypertrophic growth plate zones identifies differentiation markers and signal pathways.Bone. 2004; 35: 1273-1293Crossref PubMed Scopus (86) Google Scholar, 7James C.G. Stanton L.A. Agoston H. Ulici V. Underhill T.M. Beier F. Genome-wide analyses of gene expression during mouse endochondral ossification.PLoS One. 2010; 5: e8693Crossref PubMed Scopus (38) Google Scholar). However, proteomics level analysis of cartilage development is challenging because of the limited available tissue and dominance of poorly soluble matrix components (8Wilson R. The extracellular matrix: An underexplored but important proteome.Expert Rev. Proteomics. 2010; 7: 803-806Crossref PubMed Scopus (14) Google Scholar). Novel methods developed for proteomic analysis of growth plate and articular cartilage (9Belluoccio D. Wilson R. Thornton D.J. Wallis T.P. Gorman J.J. Bateman J.F. Proteomic analysis of mouse growth plate cartilage.Proteomics. 2006; 6: 6549-6553Crossref PubMed Scopus (30) Google Scholar, 10Pecora F. Forlino A. Gualeni B. Lupi A. Giorgetti S. Marchese L. Stoppini M. Tenni R. Cetta G. Rossi A. A quantitative and qualitative method for direct 2-DE analysis of murine cartilage.Proteomics. 2007; 7: 4003-4007Crossref PubMed Scopus (20) Google Scholar, 11Vincourt J.B. Lionneton F. Kratassiouk G. Guillemin F. Netter P. Mainard D. Magdalou J. Establishment of a reliable method for direct proteome characterization of human articular cartilage.Mol. Cell Proteomics. 2006; 5: 1984-1995Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), in particular using solubility-based tissue fractionation, have improved coverage of both the intracellular and extracellular cartilage proteome using both two-dimensional electrophoresis and capillary HPLC-tandem MS (12Wilson R. Diseberg A.F. Gordon L. Zivkovic S. Tatarczuch L. Mackie E.J. Gorman J.J. Bateman J.F. Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.Mol. Cell. Proteomics. 2010; 9: 1296-1313Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 13Wilson R. Bateman J.F. A robust method for proteomic characterization of mouse cartilage using solubility-based sequential fractionation and two-dimensional gel electrophoresis.Matrix Biol. 2008; 27: 709-712Crossref PubMed Scopus (20) Google Scholar). In this study we used mouse femoral head cartilage to identify novel proteins associated with chondrocyte differentiation and cartilage development in vivo. Developmental stages with clear differences in chondrocyte differentiation states were selected based on histomorphology and FACS. At post-natal day 3 (P3), femoral head cartilage is populated predominantly by immature reserve chondrocytes, which at 3 weeks (P21) are largely replaced by maturing prehypertrophic and hypertrophic cells. Proteomic analysis of P3 and P21 cartilage extracts using label-free quantitative mass spectrometry identified 703 nonredundant proteins with high confidence. Using the beta-binomial distribution to model the spectral count data, we identified 146 significant differentially expressed proteins (q < 0.01). To extract functional information from the group of differentially expressed proteins, enriched gene ontological (GO) terms were identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Many of the differentially expressed proteins were novel in the context of cartilage development, including ECM components, cell adhesion proteins, and a group of calcium-binding ER lumenal proteins, the reticulocalbins. The results of our proteomic analysis were validated by immunohistochemistry analysis of selected ECM components, revealing distinct regional expression patterns associated with zones of chondrocyte proliferation, maturation, and hypertrophy. 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. The dissected cartilage was rinsed in PBS, frozen on dry ice, and stored at −80 °C in batches of eight P3 hips and six P21 hips, equivalent to ∼10 mg of wet weight of tissue. Dissected femoral heads from P3 and P21 mice were also used to characterize the distribution of novel cartilage proteins by immunohistochemistry. To analyze P3 and P21 chondrocytes by FACS, femoral heads were incubated for 2 h at 37 °C in DMEM containing 5% FCS and 2 mg/ml bacterial collagenase (Worthington Biochemicals). Chondrocytes were resuspended in PBS containing 5% FCS (6 × 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). The corresponding IgG isotypes (Becton Dickinson) were used as negative controls. The P3 and P21 femoral head cartilage (three independent batches of tissue per developmental stage) was pulverized using a liquid nitrogen-cooled tissue grinder and transferred to Eppendorf tubes. Sequential protein extracts were prepared using a nondenaturing buffer (1 m NaCl in 100 mm Tris acetate, pH 8.0) followed by a chaotropic buffer (4 m GdnHCl, 65 mm DTT, 10 mm EDTA in 50 mm sodium acetate, pH 5.8) as described (13Wilson R. Bateman J.F. A robust method for proteomic characterization of mouse cartilage using solubility-based sequential fractionation and two-dimensional gel electrophoresis.Matrix Biol. 2008; 27: 709-712Crossref PubMed Scopus (20) Google Scholar). The guanidine-extracted proteins were further partitioned by molecular mass cut-off filtration through a 100-kDa cut-off ultracentrifugal column (Amicon). Protein extracts were precipitated with 9 volumes of ethanol, and protein pellets washed twice in 70% (v/v) ethanol and resuspended in 150 μl of solubilization buffer containing 7 m urea, 2 m thiourea, 4% CHAPS, and 30 mm Tris, pH 8.0. Protein concentrations were estimated using the Bradford assay (Pierce). Cartilage extracts were analyzed by SDS-PAGE to assess reproducibility and relative levels of cartilage proteins in each of the femoral head cartilage fractions. Aliquots of each extract equivalent to 2% of the total protein yield were heated in Laemmli buffer containing 50 mm dithiothreitol for 15 min at 65 °C and resolved through 4–12% acrylamide Bis-Tris NuPAGE gels (Invitrogen), and proteins were visualized by silver staining as described (14Wilson R. Belluoccio D. Bateman J.F. Proteomic analysis of cartilage proteins.Methods. 2008; 45: 22-31Crossref PubMed Scopus (30) Google Scholar). Protein samples for LC-MS/MS analysis were sequentially reduced and alkylated under nitrogen by incubation in 10 mm dithiothreitol (overnight at 4 °C) and then 50 mm iodoacetamide (2 h at 25 °C in the dark). Proteins were co-precipitated with 1 μg of trypsin (Promega) overnight at −20 °C in 1 ml of methanol. The trypsin-protein precipitates were washed once with chilled methanol, dried, and reconstituted in 100 mm ammonium bicarbonate, followed by trypsinization at 37 °C for 5 h, with the addition of 1 μg of trypsin after 2 h. Digests were terminated by freezing on dry ice. Each of the three fractions per sample was analyzed in duplicate by LC-MS/MS using an UltiMate 3000 HPLC system (Dionex) or a Tempo NanoLC system (Eksigent) in line with an LTQ-Orbitrap XL (ThermoFisher Scientific). Aliquots of tryptic peptides equivalent to 25% of the in-solution digests were loaded onto a 0.3 × 5-mm C18 trap column (Dionex) at 20 μl/min in 98% solvent A (0.1% (v/v) formic acid) and 2% solvent B (80% (v/v) acetonitrile, 0.1% (v/v) formic acid) for 5 min and subsequently back-flushed onto a pre-equilibrated analytical column (Vydac Everest C18 300 Å, 150 μm × 150 mm; Alltech) using a flow rate of 1 μl/min. Peptides were separated at 40 °C using three linear gradient segments (2–12% solvent B over 6 min, 12–50% solvent B over 65 min, and then 50–100% solvent B over 15 min), holding at 100% solvent B for a further 15 min before returning to 2% solvent B over 5 min. The LTQ-Orbitrap was fitted with a dynamic nanoelectrospray ion source (Proxeon) containing a 30-μm inner diameter uncoated silica emitter (New Objective). The LTQ-Orbitrap XL was controlled using Xcalibur 2.0 software (Thermo Electron) and operated in data-dependent acquisition mode whereby the survey scan was acquired in the Orbitrap with a resolving power set to 60,000 (at 400 m/z). MS/MS spectra were concurrently acquired in the LTQ mass analyzer on the seven most intense ions from the FT survey scan. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion (repeat count, 1; repeat duration, 30 s; exclusion list size, 500) were used. Fragmentation conditions in the LTQ were: 35% normalized collision energy, activation q of 0.25, 50-ms activation time, and minimum ion selection intensity of 500 counts. The acquired MS/MS data, together with MS/MS data acquired in our previous LC-MS/MS cartilage proteomics study (12Wilson R. Diseberg A.F. Gordon L. Zivkovic S. Tatarczuch L. Mackie E.J. Gorman J.J. Bateman J.F. Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.Mol. Cell. Proteomics. 2010; 9: 1296-1313Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), were analyzed using Mascot version 2.2.06 (Matrix Science). Proteome Discoverer version 1.2 (Thermo Scientific) was used to extract tandem mass spectra from Xcalibur raw files and submit searches to an in-house Mascot server according to the following parameters: S-carboxamidomethylation of cysteine residues specified as a fixed modification and cyclization of N-terminal glutamine to pyroglutamic acid, deamidation of asparagine, hydroxylation of proline, oxidation of methionine specified as variable modifications. Parent ion tolerance of 20 ppm and fragment ion mass tolerances of 0.8 Da were used, and enzyme cleavage was set to trypsin, allowing for a maximum of two missed cleavages. The database searched consisted of 46,137 sequences, comprising the UniProt complete proteome set for Mus musculus (45,889 sequences downloaded on May 23, 2011) and sequences for common contaminants appended (downloaded from the Max Plank Institute http://maxquant.org). The automatic Mascot decoy database search was performed for all data sets. The Mascot search results were loaded into Scaffold version 3.0.08 to assign probabilities to peptide and protein matches (15Searle B.C. Scaffold: A bioinformatic tool for validating MS/MS-based proteomic studies.Proteomics. 2010; 10: 1265-1269Crossref PubMed Scopus (395) Google Scholar). Peptide-spectrum matches were accepted if the peptide was assigned a probability greater than 0.95 as specified by the Peptide Prophet algorithm (16Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3886) Google Scholar). The probability threshold of 0.95 showed good discrimination between the predicted correct and incorrect peptide-spectrum assignments, and only peptides with charge states of +1, +2, and +3 were retained as confident identifications because the Peptide Prophet models were not a good fit to the data for charge states ≥4. 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 >0.99 by the Protein Prophet algorithm (17Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3621) Google Scholar). This threshold will constrain the protein false discovery rate (FDR) to <1%. The minimal list of proteins that satisfy the principal of parsimony is reported. To obtain sample level spectral count data, MS/MS data for the E0, E1, and E2 fractions for each replicate sample were recombined in silico using the Scaffold MudPIT function. Global normalization was applied to the raw spectral counts, and a pseudo count of one was added to reduce the effect of undersampling. Fold changes were estimated using the mean of the normalized counts for P21 relative to P3. The beta-binomial test (18Pham T.V. Piersma S.R. Warmoes M. Jimenez C.R. On the beta-binomial model for analysis of spectral count data in label-free tandem mass spectrometry-based proteomics.Bioinformatics. 2010; 26: 363-369Crossref PubMed Scopus (134) Google Scholar) was used to assess the differences between the P3 and P21 samples. This test models the magnitude of the differences in spectral counts between the sample groups and the variation between replicate measurements. QVALITY was applied to estimate q values for each protein (19Käll L. Storey J.D. Noble W.S. QVALITY: Non-parametric estimation of q-values and posterior error probabilities.Bioinformatics. 2009; 25: 964-966Crossref PubMed Scopus (80) Google Scholar), where a q value is the minimum FDR at which the protein can be called significant. A q value of 0.01 was used as the significance threshold in this study. Functional annotation and enrichment analysis was performed using the DAVID version 6.7 (20Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25346) Google Scholar). Protein lists were uploaded as the official gene symbols to the DAVID website (http://david.abcc.ncifcrf.gov/) using the complete mouse genome as background. Significantly enriched functional groups were ranked using the functional annotation clustering tool set to the high classification stringency. Femoral heads isolated from P3 and P21 C57/Bl6 mice were fixed with 4% (v/v) paraformaldehyde in PBS overnight at 4 °C, followed by incubation in decalcification solution (Immunocal) for 18 h at 4 °C. Tissues were washed in PBS and processed (Leica TP 1010 automated tissue processor) prior to embedding in liquid paraffin wax. Saggittal sections of femoral heads (5 μm) were cut using a Leica 1512 microtome and then baked at 60 °C for 1 h. Three-week neocartilage culture (in vitro chondrocyte culture day 21) specimens were processed as described (12Wilson R. Diseberg A.F. Gordon L. Zivkovic S. Tatarczuch L. Mackie E.J. Gorman J.J. Bateman J.F. Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.Mol. Cell. Proteomics. 2010; 9: 1296-1313Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Paraffin sections were dewaxed in xylene and bought to water with graded ethanol, followed by two rinses in tap water and two rinses in PBS. Two methods were used for antigen retrieval. For Urb and vitrin analysis, slides were incubated in 2 mg/ml pepsin for 30 min at 37 °C followed by 10 mm citrate buffer containing 0.05% Tween 20 at 60 °C for 30 min. The cooled slides were washed in PBS and then treated with 0.2% hyaluronidase (Type IV-S) for 60 min at 37 °C, followed by three PBS washes. For the CILP-2 analysis, the slides were heat treated with 10 mm Tris, 1 mm EDTA, pH 8, at 60 °C for 30 min. The cooled slides were treated with 30 μg/ml proteinase K in 50 mm Tris, pH 6.0, 5 mm CaCl2 for 30 min at 37 °C and then 0.2% hyaluronidase (Type IV-S) for 60 min at 37 °C. Endogenous peroxidases were inactivated for 30 min using 3% H2O2 (v/v) in PBS. 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 μg/ml), Urb (0.2 μg/ml) (21Manabe R. Tsutsui K. Yamada T. Kimura M. Nakano I. Shimono C. Sanzen N. Furutani Y. Fukuda T. Oguri Y. Shimamoto K. Kiyozumi D. Sato Y. Sado Y. Senoo H. Yamashina S. Fukuda S. Kawai J. Sugiura N. Kimata K. Hayashizaki Y. Sekiguchi K. Transcriptome-based systematic identification of extracellular matrix proteins.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12849-12854Crossref PubMed Scopus (102) Google Scholar), or CILP-2 (22Bernardo B.C. Belluoccio D. Rowley L. Little C.B. Hansen U. Bateman J.F. Cartilage Intermediate Layer Protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis.J. Biol. Chem. 2011; 286: 37758-37767Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) (2 μg/ml). Control sections were probed with the preimmune serum (CILP-2) or nonimmune rabbit IgGs (Urb and vitrin) at the same concentration as the primary antibodies. Biotinylated secondary antibody (Vectastain Elite ABC rabbit IgG kit) was applied to sections for 1 h, and sections washed in three changes of PBS and then incubated with ABC reagent (Vectastain Elite ABC rabbit IgG kit) for 30 min at room temperature. The sections were washed again in PBS, with immunohistochemical staining detected using ImmPact DAB substrate (Vector Laboratories) until color was detected. The reactions were terminated by rinsing in tap water, followed by dehydration of tissue sections using a graded ethanol series (50, 70, 90, and 100%). The images were captured at 10× magnification with a Nikon Eclipse 80i microscope. Endochondral ossification, the process by which cartilaginous precursors are remodeled into bone, begins in different skeletal elements at different developmental stages. Therefore, our first aim was to establish a developmental model suitable for proteomic analysis. In the mouse femur, secondary ossification of the distal epiphyseal cartilage (corresponding to the lower boxed region I in Fig. 1) begins early in post-natal development (23Blumer M.J. Longato S. Schwarzer C. Fritsch H. Bone development in the femoral epiphysis of mice: The role of cartilage canals and the fate of resting chondrocytes.Dev. Dyn. 2007; 236: 2077-2088Crossref PubMed Scopus (33) Google Scholar). This limits the available cartilage to the growth plate, which can only be collected by tissue sectioning and microdissection (9Belluoccio D. Wilson R. Thornton D.J. Wallis T.P. Gorman J.J. Bateman J.F. Proteomic analysis of mouse growth plate cartilage.Proteomics. 2006; 6: 6549-6553Crossref PubMed Scopus (30) Google Scholar). In contrast, the proximal femoral head at the hip (corresponding to the upper boxed region II in Fig. 1) remains devoid of trabecular bone until the onset of skeletal maturity (24Hankenson K.D. Hormuzdi S.G. Meganck J.A. Bornstein P. Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head.Mol. Cell. Biol. 2005; 25: 5599-5606Crossref PubMed Scopus (52) Google Scholar) and can be dissected as an intact explant of articular cartilage and underlying primary growth plate (25Stanton H. Golub S.B. Rogerson F.M. Last K. Little C.B. Fosang A.J. Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage.Nat. Protoc. 2011; 6: 388-404Crossref PubMed Scopus (58) Google Scholar). Developmental changes in the femoral head were characterized by hematoxylin and eosin staining of saggittal sections cut through P3 and P21 cartilage explants (Fig. 2A). In P3 cartilage, with the exception of the proliferative chondrocytes in the growth plate, most of the cells were round and immature, with little morphological distinction between the articular cartilage surface and the presumptive ossification center. In contrast, the P21 ossification center, also described in the femoral head a