Abstract
Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1β-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1β-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1β activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1β-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1β-mediated catabolic response that could be therapeutically targeted in OA.
Highlights
Osteoarthritis (OA) is a chronic degenerative joint disease in which the articular cartilage is progressively degraded leading to the loss of joint function
Treatment of OA chondrocytes with the pro-inflammatory cytokine IL-1β, a major player in OA pathogenesis, initiates a cascade of signaling events that leads to the activation of nuclear factor kappa B (NFκB) and other transcription factors[27,28] culminating in the induction and expression of the catabolic factors and downregulation of anabolic factors[29]
We investigated the mechanism of delayed COX-2 protein expression in IL-1βstimulated OA chondrocytes
Summary
Osteoarthritis (OA) is a chronic degenerative joint disease in which the articular cartilage is progressively degraded leading to the loss of joint function. Increased IL-1βlevels in synovial fluid of OA patients have been detected and the cytokine is known to activate signaling pathways in chondrocytes leading to the production of catabolic mediators such as prostaglandin E2 (PGE2) that play a significant role in cartilage matrix degradation and joint dysfunction[4]. We demonstrate that IL-1βstimulated OA chondrocytes exhibited high levels of GRP78, a marker for ER stress, coupled with the expression and phosphorylation of eIF2αthat is known to trigger the assembly of SGs. Assembly of SGs in IL-1β-stimulated OA chondrocytes served to capture and stalling of COX-2 mRNAs translation and extended the half-life of COX-2 mRNAs. Our results show that translocation and localization of COX-2 mRNAs to SGs was principally mediated by the RNA binding protein HuR. Sequestered COX-2 mRNAs were principally associated with HuR in OA chondrocytes, knockdown of TIA-1 abolished SGs assembly and prevented the localization of COX-2 mRNAs to SGs suggesting that the SGs function as a common factor and provide the scaffold to coordinate the function of the two proteins
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