Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics*

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.

[1]  R. Tuan,et al.  The intermediate filament vimentin regulates chondrogenesis of adult human bone marrow‐derived multipotent progenitor cells , 2009, Journal of cellular biochemistry.

[2]  Susumu Goto,et al.  KEGG for representation and analysis of molecular networks involving diseases and drugs , 2009, Nucleic Acids Res..

[3]  C. Flannery,et al.  002 IDENTIFICATION OF A NOVEL HTRA1-SUSCEPTIBLE CLEAVAGE SITE IN HUMAN AGGRECAN: EVIDENCE FOR THE INVOLVEMENT OF HTRA1 IN AGGRECAN PROTEOLYSIS IN VIVO , 2009 .

[4]  S. Grässel,et al.  Genetic mouse models for the functional analysis of the perifibrillar components collagen IX, COMP and matrilin-3: Implications for growth cartilage differentiation and endochondral ossification. , 2009, Histology and histopathology.

[5]  F. Berenbaum,et al.  Proteomics: addressing the challenges of osteoarthritis. , 2009, Drug discovery today.

[6]  M. Paulsson,et al.  Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms* , 2009, The Journal of Biological Chemistry.

[7]  F. Luyten,et al.  Cartilage repair: past and future – lessons for regenerative medicine , 2009, Journal of cellular and molecular medicine.

[8]  J. Bateman,et al.  Proteomics makes progress in cartilage and arthritis research. , 2009, Matrix biology : journal of the International Society for Matrix Biology.

[9]  L. Lohmander,et al.  Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases. , 2009, Osteoarthritis and cartilage.

[10]  Forest M White,et al.  Mechanical Injury and Cytokines Cause Loss of Cartilage Integrity and Upregulate Proteins Associated with Catabolism, Immunity, Inflammation, and Repair* , 2009, Molecular & Cellular Proteomics.

[11]  E. Blain.  Involvement of the cytoskeletal elements in articular cartilage homeostasis and pathology , 2009, International journal of experimental pathology.

[12]  R. Savarirayan,et al.  Employing molecular genetics of chondrodysplasias to inform the study of osteoarthritis. , 2009, Arthritis and rheumatism.

[13]  J. Mateos,et al.  Mitochondrial Dysregulation of Osteoarthritic Human Articular Chondrocytes Analyzed by Proteomics , 2009, Molecular & Cellular Proteomics.

[14]  A. Rowan,et al.  Emerging roles of serine proteinases in tissue turnover in arthritis. , 2008, Arthritis and rheumatism.

[15]  P. Dundr,et al.  Distribution of chondrocytes containing alpha-smooth muscle actin in human normal, osteoarthrotic, and transplanted articular cartilage. , 2008, Pathology, research and practice.

[16]  M. Mann,et al.  Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast , 2008, Nature.

[17]  D. Tabb,et al.  Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry. , 2008, Journal of proteome research.

[18]  J. Bateman,et al.  A robust method for proteomic characterization of mouse cartilage using solubility-based sequential fractionation and two-dimensional gel electrophoresis. , 2008, Matrix biology : journal of the International Society for Matrix Biology.

[19]  A. Vetere,et al.  Galectin-1 in cartilage: expression, influence on chondrocyte growth and interaction with ECM components. , 2008, Matrix biology : journal of the International Society for Matrix Biology.

[20]  J. Lawler,et al.  Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen. , 2008, The American journal of pathology.

[21]  N. Miosge,et al.  Nidogen-1 and nidogen-2 in healthy human cartilage and in late-stage osteoarthritis cartilage. , 2008, Arthritis and rheumatism.

[22]  D. Belluoccio,et al.  Proteomic analysis of cartilage proteins. , 2008, Methods.

[23]  Johanne Martel-Pelletier,et al.  Cartilage in normal and osteoarthritis conditions. , 2008, Best practice & research. Clinical rheumatology.

[24]  D. Deforce,et al.  Differential proteome analysis of normal and osteoarthritic chondrocytes reveals distortion of vimentin network in osteoarthritis. , 2008, Osteoarthritis and cartilage.

[25]  Jiang Wu,et al.  Comparative proteomic characterization of articular cartilage tissue from normal donors and patients with osteoarthritis. , 2007, Arthritis and rheumatism.

[26]  A. Forlino,et al.  A quantitative and qualitative method for direct 2‐DE analysis of murine cartilage , 2007, Proteomics.

[27]  D. Eyre,et al.  Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse. , 2007, Matrix biology : journal of the International Society for Matrix Biology.

[28]  B. Caterson,et al.  Macromolecular Organization and In Vitro Growth Characteristics of Scaffold-free Neocartilage Grafts , 2007, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  Kozo Nakamura,et al.  Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes* , 2007, Journal of Biological Chemistry.

[30]  C. Little,et al.  Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair. , 2007, The Journal of clinical investigation.

[31]  R. Mazzarella,et al.  Identification of an ADAMTS-4 Cleavage Motif Using Phage Display Leads to the Development of Fluorogenic Peptide Substrates and Reveals Matrilin-3 as a Novel Substrate* , 2007, Journal of Biological Chemistry.

[32]  S. Glasson.  In vivo osteoarthritis target validation utilizing genetically-modified mice. , 2007, Current drug targets.

[33]  E. Roos,et al.  Serum levels of Cartilage Oligomeric Matrix Protein (COMP) increase temporarily after physical exercise in patients with knee osteoarthritis , 2006, BMC musculoskeletal disorders.

[34]  Didier Mainard,et al.  Establishment of a Reliable Method for Direct Proteome Characterization of Human Articular Cartilage* , 2006, Molecular & Cellular Proteomics.

[35]  M. Goldring.  Update on the biology of the chondrocyte and new approaches to treating cartilage diseases. , 2006, Best practice & research. Clinical rheumatology.

[36]  H. Knull,et al.  Glycolytic enzyme interactions with yeast and skeletal muscle F-actin. , 2006, Biophysical journal.

[37]  K. Resing,et al.  Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics*S , 2005, Molecular & Cellular Proteomics.

[38]  F. Blanco,et al.  Proteomic characterization of human normal articular chondrocytes: A novel tool for the study of osteoarthritis and other rheumatic diseases , 2005, Proteomics.

[39]  M. Paulsson,et al.  The matrilins – adaptor proteins in the extracellular matrix , 2005, FEBS letters.

[40]  Guy Perrière,et al.  MADE4: an R package for multivariate analysis of gene expression data , 2005, Bioinform..

[41]  A. Wallace,et al.  Proteomic Analysis of Articular Cartilage Shows Increased Type II Collagen Synthesis in Osteoarthritis and Expression of Inhibin βA (Activin A), a Regulatory Molecule for Chondrocytes* , 2004, Journal of Biological Chemistry.

[42]  R. Zimmer,et al.  Functional Genomics of Osteoarthritis: On the Way to Evaluate Disease Hypotheses , 2004, Clinical orthopaedics and related research.

[43]  M. Mastrogiacomo,et al.  URB expression in human bone marrow stromal cells and during mouse development. , 2004, Biochemical and biophysical research communications.

[44]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[45]  R. Timpl,et al.  Alternative Splicing in the Aggrecan G3 Domain Influences Binding Interactions with Tenascin-C and Other Extracellular Matrix Proteins* , 2004, Journal of Biological Chemistry.

[46]  D. Eyre,et al.  Covalent Cross-linking of the NC1 Domain of Collagen Type IX to Collagen Type II in Cartilage* , 2004, Journal of Biological Chemistry.

[47]  R. Aebersold,et al.  A statistical model for identifying proteins by tandem mass spectrometry. , 2003, Analytical chemistry.

[48]  G. Wallis,et al.  Skeletal dysplasias caused by a disruption of skeletal patterning and endochondral ossification , 2003, Clinical genetics.

[49]  A. Freemont,et al.  Annexin VIII Is Differentially Expressed by Chondrocytes in the Mammalian Growth Plate During Endochondral Ossification and in Osteoarthritic Cartilage , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[50]  Alexey I Nesvizhskii,et al.  Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. , 2002, Analytical chemistry.

[51]  T. Linsenmayer,et al.  Immunohistological analysis of transglutaminase factor XIIIA expression in mouse embryonic growth plate , 2002, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[52]  N. Morris,et al.  Developmental Distribution of Collagen Type XII in Cartilage: Association with Articular Cartilage and the Growth Plate , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[53]  K. Iyama,et al.  Molecular Cloning and Biological Activity of a Novel Lysyl Oxidase-related Gene Expressed in Cartilage* , 2001, The Journal of Biological Chemistry.

[54]  M. Paulsson,et al.  Molecular Structure, Processing, and Tissue Distribution of Matrilin-4* , 2001, The Journal of Biological Chemistry.

[55]  M. Paulsson,et al.  Molecular Structure and Tissue Distribution of Matrilin-3, a Filament-forming Extracellular Matrix Protein Expressed during Skeletal Development* , 2000, The Journal of Biological Chemistry.

[56]  C. Colnot,et al.  Cellular and subcellular distribution of galectin-3 in the epiphyseal cartilage and bone of fetal and neonatal mice. , 1999, Cellular and molecular biology.

[57]  N. Yanagihara,et al.  Progressive association of a "soluble" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells. , 1999, Cell motility and the cytoskeleton.

[58]  B. Månsson,et al.  Chondroadherin expression changes in skeletal development. , 1998, The Biochemical journal.

[59]  M. Paulsson,et al.  Primary structure of matrilin‐3, a new member of a family of extracellular matrix proteins related to cartilage matrix protein (matrilin‐1) and von Willebrand factor 1 , 1997, FEBS letters.

[60]  C. A. Poole.  Review. Articular cartilage chondrons: form, function and failure , 1997 .

[61]  R. Burgeson,et al.  Type XII and XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability. , 1994, The Journal of biological chemistry.

[62]  F. Reinholt,et al.  Ultrastructure of hypertrophic cartilage: Histochemical procedures compared with high pressure freezing and freeze substitution , 1994, Calcified Tissue International.

[63]  J. Ralphs,et al.  Cytoskeleton of cartilage cells , 1994, Microscopy research and technique.

[64]  P. Schnegelsberg,et al.  Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[65]  P. Schmid,et al.  Variable and multiple expression of Protease Nexin-1 during mouse organogenesis and nervous system development. , 1993, Development.

[66]  J. Bereiter-Hahn,et al.  Microcompartmentation of glycolytic enzymes in cultured cells. , 1992, European journal of cell biology.

[67]  Y. Benjamini,et al.  More powerful procedures for multiple significance testing. , 1990, Statistics in medicine.

[68]  L. Vaughan,et al.  Cartilage contains mixed fibrils of collagen types II, IX, and XI , 1989, The Journal of cell biology.

[69]  D. Farrell,et al.  Localization of protease nexin‐1 on the fibroblast extracellular matrix , 1988, Journal of cellular physiology.

[70]  D. Farrell,et al.  Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1. , 1987, The Biochemical journal.

[71]  E. Hunziker,et al.  Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state , 1984, The Journal of cell biology.

[72]  H. Moor,et al.  Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion , 1984, The Journal of cell biology.

[73]  D. Low,et al.  Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[74]  J. Thyberg,et al.  Cartilage proteoglycan aggregates. Electron-microscopic studies of native and fragmented molecules. , 1978, The Biochemical journal.

[75]  P. Benya,et al.  Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture , 1978, Cell.

[76]  M. A. Bowie,et al.  Observations on Respiration in Articular Cartilage * , 1941, Annals of the rheumatic diseases.

[77]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[78]  P. Dundr,et al.  Expression of actin isoforms in human auricular cartilage. , 2006, Folia biologica.

[79]  C. A. Poole.  Articular cartilage chondrons: form, function and failure. , 1997, Journal of anatomy.

[80]  H. Knull,et al.  Association of glycolytic enzymes with the cytoskeleton. , 1992, Current topics in cellular regulation.

[81]  C. A. Poole,et al.  Morphological and functional interrelationships of articular cartilage matrices. , 1984, Journal of anatomy.