Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Advanced Glycation End Products (AGEs)-Induced Expression of IL-6 and IL-8 in Human Osteoarthritis Chondrocytes Correlates with the Induction of Endoplasmic Reticulum Stress.
Rasheed, Zafar, Akhtar, Nahid, Haqqi, Tariq M.
During aging, non-enzymatic glycation of protein and other molecules results in the accumulation of advanced glycation end products (AGEs) in articular cartilage. Here, we investigated (a) whether AGEs induce endoplasmic reticulum stress (ERS) in chondrocytes; (b) whether the induction of proinflammatory cytokine IL-6 and chemokine IL-8 is related to ERS; and (c) determined the signal transduction pathways required for the AGEs-mediated induction of ERS and the expression of IL-6 and IL-8 in chondrocytes.
Human chondrocytes were derived from OA cartilage by enzymatic digestion (OA chondrocytes) and were stimulated with AGE-modified BSA (AGE-BSA). Expression of IL-6 and IL-8 was determined by qRT-PCR and the production of IL-6 or IL-8 in the culture medium was quantified by ELISA. Western immunoblotting was used to analyze the expression of GRP78 and phosphorylation of eukaryotic initiation factor-2a (eIF2a),both markers of ER stress, IkBa degradation, and the activation of mitogen-activated protein kinases (MAPKs). Activation of nuclear factor (NF)-kB was determined using a highly sensitive and specific ELISA. Studies to elucidate the involved pathways were executed using transfection of OA chondrocytes with siRNAs specific for RAGE and eIF2-a and specific inhibitors of MAPKs and NF-kB.
AGE-BSA induced the expression of the GRP78 with concomitant increase in the expression of IL-6 and IL-8. Transfection of OA chondrocytes with RAGE specific siRNAs inhibited the AGE-BSA-induced expression of GRP78, IL-6 and IL-8. In OA chondrocytes with SiRNA-mediated knockdown of RAGE, significant activation of MAPKs was not observed upon challenge with AGE-BSA. Inhibition of p38-MAPK (SB202190) blocked the AGE-BSA-induced expression of GRP78 in OA chondrocytes. Treatment of OA chondrocytes with SB202190 (p38 inhibitor) or PD98059 (ERK inhibitor) inhibited the AGE-BSA induced IL-6 or IL-8 mRNA and protein expression. Similar results were obtained when OA chondrocytes were treated with 2-aminopurine (an inhibitor of eIF2a). In contrast JNK inhibitor SP600125 had no effect on AGE-BSA-induced IL-6 expression but inhibited the expression of IL-8. NF-kB inhibitor Bay 117082 (IKKa/b inhibitors), Parthenolide (IKKb inhibitor), NEMO-BDBP (IKKg inhibitor) or MG-132 (proteasome inhibitor) potently suppressed the AGE-BSA induced IL-6 and IL-8 mRNA and protein expression in OA chondrocytes but had no effect on the expression of GRP78.
This is the first study to demonstrate that AGEs via RAGE mediated activation of p38-MAPK induce ER stress and stimulate the expression of IL-6 and IL-8 in OA chondrocytes. Our results also demonstrate that AGEs differentially induced the expression of IL-6 and IL-8 with expression of IL-6 was independent of the JNK activation but the expression of IL-8 required the JNK activation. Activation of NF-kB was an absolute requirement for ER stress-induced expression of both IL-6 and IL-8 but not of GRP78 via RAGE. Thus, our results provide important insights into the mechanisms of AGE-BSA-induced ER stress in human OA chondrocytes. Our data also suggests that AGEs-induced ER stress may contribute to the pathogenesis OA.
To cite this abstract, please use the following information:
Rasheed, Zafar, Akhtar, Nahid, Haqqi, Tariq M.; Advanced Glycation End Products (AGEs)-Induced Expression of IL-6 and IL-8 in Human Osteoarthritis Chondrocytes Correlates with the Induction of Endoplasmic Reticulum Stress. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :632