Novel elements of the chondrocyte stress response identified using an in vitro model of mouse cartilage degradation

Proteolytic destruction of the cartilage extracellular matrix is a key event in joint pathologies such as osteoarthritis. It is becoming increasingly evident that cartilage degradation is propagated by pro-inflammatory mediators such as interleukin-1 (IL-1). However, the contribution of inflammation to chondrocyte dysfunction and cartilage degeneration in OA is not fully understood. Here we used label-free quantitative proteomics to evaluate the chondrocyte response to IL-1 within a native cartilage ECM, using mouse femoral heads cultured with and without IL-1. We analyzed both cartilage tissue proteome and soluble factors released into the media (secretome). In addition, we investigated the IL-1 response in wild-type cartilage and in TS5deltacat cartilage lacking active ADAMTS-5, a key enzyme involved in destruction of the aggrecan network. Our proteomics analysis was further validated using whole-genome mRNA expression profiling. LTQ-Orbitrap analysis identified 368 proteins and 728 proteins in the cartilage secretome and proteome, respectively (protein FDR < 0.01%). Bioinformatic annotation of IL-1 modulated proteins identified significant protein groups associated with ECM-receptor interactions, the inflammatory response, peptidase activity, oxidoreductase activity, stress response and endoplasmic reticulum (adj. p < 0.05). These proteins included proteases/inhibitors (Mmp12, Lgmn, Cstl1 and Cst3), matrix components (Chad, Kera and Crispld1) and ER stress proteins (Manf, Creld2) previously not associated with the cartilage IL-1 response. Results in wild-type and TS5deltacat cartilage were highly consistent overall, although several proteins, including Timp1 and Arg1, were differentially modulated between the genotypes at both protein and mRNA levels. Finally, proteins of interest (Vnn1 and Mmp12) were further investigated using immunohistochemistry.