ROS have been shown to have deleterious effects on cells and to contribute to chondrocyte death. Davies and colleagues [30
] demonstrated that OA cartilage has significantly more DNA damage than normal cartilage, and that this damage was mediated by IL-1 and, ultimately, by ROS. Porcine articular cartilage was harvested from normal tissue and compared with cartilage harvested from OA tissue and the number of single-stranded and double-stranded DNA breaks was analyzed. In cells from healthy cartilage, increasing concentrations of IL-1 correlated with increasing NO concentrations and increasing DNA damage. The increase in DNA damage was attenuated by incubation with the specific iNOS inhibitor 1400 W and the superoxide scavenger SOD, suggesting that superoxide may have a role in generating DNA breaks. It is not clear, however, what effect DNA damage has on OA cells and the disease process. The authors suggested that DNA damage could alter transcription by increasing errors, which could result in dysfunctional proteins, or alternatively by inhibiting the binding of transcription factors to promoter regions [30
There is some evidence that the degenerative activity attributed to an increase in NO concentration could be a result of an increase in the concentration of RNOS. Clancy and coworkers [31
] demonstrated that NO and peroxynitrite have opposing effects on nuclear factor-κB (NF-κB) activation in chondrocyte cultures. The transcription factor NF-κB is activated rapidly in response to inflammatory stimuli such as IL-1β and TNF-α and upregulates the transcription of a number of genes involved in cartilage degradation including iNOS, matrix metalloproteinases and COX-2, as well as IL-1β and TNF-α. Inactive NF-κB is sequestered in the cytoplasm by its inhibitor IκB. Upon activation, IκB is phosphorylated and degraded, which allows NF-κB to translocate to the nucleus and bind to its target DNA sequences. When bovine chondrocytes were incubated with IL-1β, 40% of the cells had active NF-κB, as visualized by positive immunostaining for a NF-κB subunit in the nucleus (Figure ). When cells were treated with IL-1β and the NO donor S
-nitrosocysteine ethyl ester, the number of cells with nuclear NF-κB decreased to 5%. However, incubation with IL-1β and peroxynitrite resulted in an increase in cells with activated NF-κB from 40% to 73%, illustrating opposing effects of NO and ROS (Figure ). These experiments suggest that NO is not required for immediate activation of NF-κB and suggest that its catabolic activity could be mediated in part through peroxynitrite [31
Figure 2 Effects of peroxynitrite and the NO donor SCNEE on IL-1β stimulated NF-κB (p65) nuclear translocation. NF-κB, nuclear factor-κB; NO, nitric oxide; PN, peroxynitrite; SCNEE, S-nitrosocysteine ethyl ester. Reproduced with (more ...)
Another group analyzed the differential roles of hydrogen peroxide and superoxide in IL-1-induced NF-κB activation. Mendes and colleagues [32
] found that IL-1 stimulation resulted in an increase in both hydrogen peroxide and superoxide in bovine articular chondrocytes, although only superoxide was required for NF-κB activation and iNOS expression. This conclusion is supported by the fact that SOD inhibited IL-1-induced IκB degradation. Like Clancy and coworkers [31
], this group also found that NO alone inhibits NF-κB activation and iNOS expression [33
], but they suggested that because the concentration of NO immediately after IL-1 stimulation appeared to be quite low, it was unlikely that significant quantities of peroxynitrite were generated. This led them to suggest that peroxynitrite is not likely to be required for NF-κB activation in chondrocytes. However, these results do not exclude the possibility that peroxynitrite is able to activate NF-κB, merely that it may not be required. These results clearly demonstrate the difficulty in teasing out the specific roles played by both NO and ROS in order to determine their involvement in IL-1-induced NF-κB activation.
Peroxynitrite also helps perpetuate the inflammatory process in mesenchymal progenitor cells (MPCs), which are used as a model of cartilage and cartilage repair cells. Whiteman and coworkers [34
] used MPCs to investigate the cellular role of peroxynitrite-modified collagen-II, a biomarker discovered in the serum of patients with both OA and rheumatoid arthritis. The authors showed that the addition of peroxynitrite-modified collagen-II to MPC cultures induced both iNOS expression and cyclo-oxygenase (COX)-2 synthesis and that specific iNOS and COX-2 inhibitors blocked this synthesis. Furthermore, the investigators demonstrated that this up-regulation was caused by the activation of NF-κB via mitogen-activated protein kinase signaling through p38 and ERK1/2. Whiteman and coworkers [34
] suggest that this newly identified proinflammatory pathway may be a target for the development of new therapies for the inflamed joint, reiterating the complexity of NO signaling and the need for continuing research to more fully elucidate the role of NO and its derivatives in cellular physiology and pathophysiology.