Proteomic characterization of mouse cartilage degradation in vitro

Purpose: 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 two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and subsequent tandem mass spectrometry (MS), we identified novel proteins and fragments released into the media of control, interleukin-1α (IL-1) and all-trans-retinoic acid (RetA)-treated explants. Fluorescent difference gel electrophoresis (2-D DIGE) was used to quantify protein expression changes. We also measured changes in mRNA expression to distinguish transcriptional and post-translational regulation of released proteins. Results: We identified 20 differentially-abundant proteins in media from control and treated explants, including fragments of thrombospondin-1 and connective tissue growth factor. IL-1 stimulated release of the cartilage degeneration marker MMP-3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinaseassociated lipocalin. RetA stimulated release of extracellular matrix proteins COMP, link protein and matrilin-3 into the media, accompanied by dramatic reduction in corresponding mRNA transcripts levels. Gelsolin, implicated in cytoskeletal reorganization in arthritic synovial fibroblasts but not previously associated with cartilage pathology, was regulated by IL-1 and RetA. Conclusions: This first analysis of mouse cartilage degradation and protein release using proteomics has 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 mechanisms of cartilage degeneration.