Proteomic characterization of mouse cartilage degradation in vitro

Abstract

Objective.To develop proteomics to analyze mouse cartilage degradation and correlate transcriptional and translational responses to catabolic stimuli.Methods. Proteomic techniques were used to analyze catabolism in mouse femoral head cartilage. Using specific methods to prepare cartilage extracts and conditioned media for 2-dimensional polyacrylamide gel electrophoresis and subsequent tandem mass spectrometry, we identified novel proteins and fragments released into the media of control, interleukin-1␣ (IL-1␣)-treated, and all-trans-retinoic acid (RetA)-treated explants. Fluorescence 2-dimensional difference gel electrophoresis was used to quantify protein expression changes. We also measured changes in messenger RNA (mRNA) expression to distinguish transcriptional and posttranslational regulation of released proteins.Results. Differentially abundant proteins in the media of control and treated explants included fragments of thrombospondin 1 and connective tissue growth factor. IL-1␣ stimulated release of the cartilage degeneration marker matrix metalloproteinase 3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of the extracellular matrix proteins cartilage oligomeric matrix protein, link protein, and matrilin-3 into the media, which was accompanied by a dramatic reduction in the corresponding mRNA transcript levels. Gelsolin, which has been implicated in cytoskeletal reorganization in arthritis synovial fibroblasts but has not been previously associated with cartilage pathology, was regulated by IL-1␣ and RetA.Conclusion. In this first analysis of mouse cartilage degradation and protein release using proteomics, we identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and genetically modified mouse cartilage will provide insights into the mechanisms of cartilage degeneration.

Proteomics makes progress in cartilage and arthritis research

“…In contrast, extraction with GuHCl results in greater representation of matrix components ( Fig. 4; Belluoccio et al, 2006;Wilson et al, 2008b). Sequential NaCl and GuHCl extraction can enrich sub-proteomes of interest and facilitate deeper mining of the cartilage proteome (Wilson and Bateman, 2008).…”

“…3). Proteolysis of CTGF is increased in IL-1 treated femoral heads, together with candidate proteases MMP-3 and MMP-13 (Wilson et al, 2008b). The CTGF expressed in OA cartilage and synovium may therefore be subject to post-translational regulation by MMPs (Smith et al, 2008).…”

Effect of thiram on avian growth plate chondrocytes in culture

“…Although proteomic changes conceivably underlie various skeletal malformation yet studying skeletal cell proteomics with respect to toxic substances has been limited. The preponderance of extracellular matrix (ECM) molecules such as collagens and highly anionic and nonhomogeneous proteoglycans which interfere with analytical procedures involving isoelectric focusing and 2D gel electrophoresis (Belluoccio et al, 2006;Wilson et al, 2008;Ruiz-Romero and Blanco, 2009;Gharbi et al, 2011). The cell culture system circumvents some of these problems by taking advantage of the fact that the ECM proteins are secreted into the culture media and those retained in the cell layer are much lower in concentration compared with in vivo obtained tissue proteins to interfere with 2D gel electrophoresis.…”

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

“…41 THBS1 fragments were found in degenerated mouse cartilage and THBS2 functions as an endogenous regulator of inflammation in RA. 42,43 Taken together, expression of several genes coding for molecules involved in focal ahesions, ECM composition and ECM-receptor interaction was repressed in HSE stimulated alginate bead cultures.…”