Dataset Information

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

ABSTRACT: BACKGROUND: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ?ig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGF?-induced protein ?ig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

SUBMITTER: Brachvogel B

PROVIDER: S-EPMC3650385 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

Brachvogel Bent B Zaucke Frank F Dave Keyur K Norris Emma L EL Stermann Jacek J Dayakli Münire M Koch Manuel M Gorman Jeffrey J JJ Bateman John F JF Wilson Richard R

The Journal of biological chemistry 20130324 19

<h4>Background</h4>Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function.<h4>Results</h4>Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage.<h4>Conclusion</h4>Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome.<h4>Significance</h4>We applied a proteomics approach to advance our understanding of collage ...[more]

PMID: 23530037

Similar Datasets

Project description:Primary chondrocytes dedifferentiate in serial monolayer with respect to their morphological and biosynthetic phenotype. They change from a round to a flattened fibroblast-like shape, and collagen I is secreted instead of the cartilage-specific collagen II. We analysed in detail the time course of dedifferentiation of mature bovine articular chondrocytes in monolayer for up to 32 weeks. Assessment of RNA expression by reverse transcription-PCR led to the identification of two novel phenotypical markers, the cartilage oligomeric matrix protein (COMP) and collagen IX, which are down-regulated faster than the widely accepted marker, collagen II. The different kinetics of COMP and collagen expression suggest differential regulation at the level of transcription. Immunostaining and metabolic labelling experiments confirmed the switch in the collagen expression pattern and the rapid down-regulation of de novo synthesis of COMP and collagen IX. Culture of chondrocytes in a three-dimensional matrix is known to stabilize the chondrocytic phenotype. We maintained cells for up to 28 weeks in an alginate bead system, which prevented dedifferentiation and led to a stabilization of collagen and COMP expression. Immunohistochemical analysis of the alginate beads revealed a similar distribution of matrix proteins to that found in vivo. Chondrocytes were transferred after a variable length of monolayer culture into the alginate matrix and the potential for redifferentiation was investigated. The re-expression of COMP and collagen IX was differentially regulated. The expression of COMP was re-induced within days after transfer into the three-dimensional matrix, while the expression of collagen IX was irreversibly down-regulated. In summary, these results demonstrate that the potential for redifferentiation decreases with increasing length of monolayer culture and show that the alginate bead system represents an attractive in vitro model to study the chondrocyte de- and re-differentiation processes, as well as extracellular matrix assembly.

Project description:BackgroundOsteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage.ResultsIn this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%), as well as participating in different biological functions including structural organization (19%), signal transduction and molecular signaling (11%), redox homeostasis (9%), transcription and protein synthesis (6%), and transport (6%). The identified proteins were assigned to one or more subcellular compartments.Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones.ConclusionsThis study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by immunolabeling in the chondrocytes from the middle, deep and calcified zones of normal articular cartilage, and superficial and deep zones of osteoarthritic cartilage.

Dataset Information

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

Publications

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets