Background/Purpose: Nrf2 is a redox sensitive transcription factor that regulates the expression of phase II antioxidant enzymes and cytoprotective genes and is crucial for maintaining the cartilage integrity. However, mechanisms underlying the cartilage/chondroprotective effects of Nrf2 remains largely unknown. Here, we examined the critical role of Nrf2 in protecting human OA chondrocytes against oxidative damage and induction of apoptosis under pathological conditions.

Methods:

Deidentified and discarded OA cartilage was obtained at the time of total joint arthroplasty and chondrocytes were prepared by enzymatic digestion. qRT-PCR and immunoblotting with validated antibodies were used to assess the gene and protein expression respectively. Nrf2 activation was determined by luciferase reporter assay. Chondrocytes were transfected with Nrf2 overexpression plasmid, ARE reporter vector or siRNAs using nucleofection. ROS levels were measured by H₂DCF-DA staining. Apoptosis was measured by TUNEL assay. Agarose gel electrophoresis was used to analyze DNA fragmentation.

Results:

Expression of Nrf2 and its downstream targets HO-1, NQO1, and SOD2 was significantly higher in damaged OA cartilage compared with the smooth cartilage of the same patient. A luciferase reporter assay demonstrated that IL-1β-stimulation was a potent inducer of Nrf2 activity in OA chondrocytes. Interestingly, pretreatment of OA chondrocytes with antioxidants significantly inhibited the IL-1β-mediated activation of Nrf2/ARE signaling indicating that the activity was due to oxidative stress. Over-expression of Nrf2 in OA chondrocytes significantly suppressed the IL-1β-mediated generation of ROS, production of H₂O₂ and expression of NOX4 whereas Nrf2 knockdown significantly enhanced the basal as well as induced levels of ROS and expression of NOX4. OA chondrocytes with overexpression of Nrf2 showed inhibition of both the extrinsic and intrinsic apoptotic pathways as IL-1β-induced DNA fragmentation, activation of Caspase-3, cleavage of PARP, cleavage of Caspase-8,-9, release of cytochrome-c, suppression of mitochondrial ROS production and mitochondrial dysfunction were not observed. Further, enhanced expression of Nrf2 stimulated the expression of anti-apoptotic proteins-Bcl2, Bcl-xl and Mcl-1-and significantly suppressed the expression of pro-apoptotic proteins-Bax, Bad and Bid-in IL-1β stimulated OA chondrocytes. Nrf2 overexpression enhanced the phosphorylation of ERK1/2 and its downstream target proteins-Elk-1, P70S6K and P90RSK in OA chondrocytes, whereas pharmacological inhibition of ERK1/2 activation enhanced the ROS generation and increased apoptosis in IL-1β-stimulated OA chondrocytes.

Conclusion: Taken together, these results show that Nrf2 is a stress response protein in OA chondrocytes with an anti-apoptotic function. IL-1β-induced stress causes activation of Nrf2 which activates the ERK1/2/ELK1-P70S6K-P90RSK signaling pathway to inhibit apoptosis of OA chondrocytes. These activities of Nrf2 make it a promising candidate for the development of novel therapies for the management of OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nrf2-inhibits-apoptosis-and-suppresses-oxidative-stress-by-activating-nox4erk12elk1-signaling-axis-in-human-chondrocytes/