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-1alpha (IL-1alpha)-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-1alpha 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-1alpha 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.