Abstract
Osteoarthritis is a chronic disease characterized by the loss of articular cartilage in synovial joints through a process of extracellular matrix destruction that is strongly associated with inflammatory stimuli. Chondrocytes undergo changes to their protein translational capacity during osteoarthritis, but a study of how disease-relevant signals affect chondrocyte protein translation at the transcriptomic level has not previously been performed. In this study, we describe how the inflammatory cytokine interleukin 1-β (IL-1β) rapidly affects protein translation in the chondrocytic cell line SW1353. Using ribosome profiling we demonstrate that IL-1β induced altered translation of inflammatory-associated transcripts such as NFKB1, TNFAIP2, MMP13, CCL2, and CCL7, as well as a number of ribosome-associated transcripts, through differential translation and the use of multiple open reading frames. Proteomic analysis of the cellular layer and the conditioned media of these cells identified changes in a number of the proteins that were differentially translated. Translationally regulated secreted proteins included a number of chemokines and cytokines, underlining the rapid, translationally mediated inflammatory cascade that is initiated by IL-1β. Although fewer cellular proteins were found to be regulated in both ribosome profiling and proteomic data sets, we did find increased levels of SOD2, indicative of redox changes within SW1353 cells being modulated at the translational level. In conclusion, we have produced combined ribosome profiling and proteomic data sets that provide a valuable resource in understanding the processes that occur during cytokine stimulation of chondrocytic cells.
Highlights
Osteoarthritis is a chronic disease characterized by the loss of articular cartilage in synovial joints through a process of extracellular matrix destruction that is strongly associated with inflammatory stimuli
Using ribosome profiling we demonstrate that IL-1 induced altered translation of inflammatory-associated transcripts such as NFKB1, TNFAIP2, MMP13, CCL2, and CCL7, as well as a number of ribosomeassociated transcripts, through differential translation and the use of multiple open reading frames
Results from the SW1353 cellular proteomic data showed that SOD2 was one of the highest ranked, differentially expressed proteins following exposure to IL-1 (Table S1)
Summary
Ribosome-protected mRNA suitable for RNA-Seq analysis can be isolated from SW1353 cells. We observed clustered ribosome-protected reads outside of the normal transcript protein coding sequence (CDS), within discrete regions of the 3Ј-UTR, for a limited set of genes Examples of these were reads observed in the 3Ј-UTR for TNFAIP2 (Fig. 4B) and for SOX9 (Fig. 4F) ( note that SOX9 was only in the top 200 differentially translated transcripts when all three reading frames were examined together and not when focusing on frame 1 from the triplet periodicity data). Concentrations of IL-1 as low as 0.1 ng/ml resulted in an increase in ROS activity (Fig. 5C, right panel) but still induced SOD2 at similar levels to the 10 ng/ml dose after 24 h These data along with the increase in NFKB1 mRNA expression and translation (Fig. 4A) suggest that IL-1 treatment activates NF-B and a cellular stress response within the SW1353 cells that is controlled through translational mechanisms
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