Background/Purpose:

Stress granules (SGs) are ribonucleoprotein complexes assembled in the cytoplasm of eukaryotic cells to respond adequately to stress (e.g. heat shock, UV irradiation, oxidative stress etc.). SGs sequester mRNAs to enable the cell to reprogram its translational repertoire via mRNA triage. Translational stalling of selective mRNA populations by SGs has been shown to be critical in the pathogenesis of neurodegenerative diseases. Here we studied the induction and formation of SGs in response to inflammatory stimuli, translational stalling of COX-2 mRNAs by SGs, and the mechanism of SGs clearance in human osteoarthritis (OA) chondrocytes.

Methods:

Primary human OA chondrocytes were isolated from smooth cartilage obtained from OA patients who underwent total knee arthroplasty. Chondrocytes were treated with IL-1β (10ng/ml) for different periods of time. Chondrocyte lysate was prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Immunoblotting was done using specific antibodies. Activation of the integrated stress response (ISR) was determined by studying GRP78 expression levels, phosphorylation of eIF2α by Western immunoblotting, and formation and duration of SGs by immunofluorescence staining of G3BP1, TIA-1, TIAR, Staufen1 and HuR. Association of COX-2 mRNA with SGs was determined by FISH and RNA immunoprecipitation. mRNA decay was analyzed using Actinomycin-D chase assay. Role of specific proteins in IL-1b-induced SGs formation was assessed by RNAi mediated knockdown of target mRNAs. ROS generation was measured by flow cytometry using CellROX dye. Treatment with ammonium chloride and Bafilomycin was used to block SGs clearance via autophagy. Production of PGE₂ was determined by ELISA.

Results:

Human OA chondrocytes stimulated with IL-1β showed enhanced ROS production and the expression of the ER stress marker GRP78 and activation of the ISR through phosphorylation of eIF2α and the assembly of SGs. Immunofluorescence staining using antibodies specific for G3BP1, TIA-1, TIAR, Staufen1 and HuR as well as by transient transfection with the G3BP1-GFP plasmid showed robust assembly of SGs in IL-1b-stimulated OA chondrocytes. COX-2 mRNAs were mainly localized in SGs in complex with the RNA binding protein HuR. Sequestration in SGs stalled COX-2 mRNA translation and COX-2 protein levels increased only after the disassembly of SGs. Formation of SGs was TIA-1-dependant as its knockdown resulted in loss of SGs assembly and promoted early and enhanced translation of COX-2 mRNA and protein expression. Production of PGE₂ mirrored the COX-2 protein expression. Inhibition of autophagy blocked SGs clearance and inhibited COX-2 protein expression without affecting the COX-2 mRNA levels in OA chondrocytes.

Conclusion: To our knowledge, this is the first report showing IL-1β induced assembly of SGs in human OA chondrocytes and describes a novel function of SGs in controlling the pro-inflammatory stimulus in in OA chondrocytes through subcellular trafficking of COX-2 mRNAs to SGs. These data demonstrate the importance of RNA binding proteins and SGs assembly in OA pathogenesis and identify specific RBPs/SGs as potential therapeutic targets

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-1-induced-stress-granules-sequester-cox-2-mrna-and-regulates-its-expression-in-human-oa-chondrocytes/