Abstract
Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.
Highlights
The pathogenesis of osteoarthritis involves an imbalance between anabolic and catabolic pathways in chondrocytes
These data confirmed our previous observation that matrix metalloproteinases (MMPs)-1 synthesis in C-20/A4 chondrocytes upon IL-1b signalling is mediated by Nox4 oxidase activity [22]
We investigate the DNA fragmentation level and found a 80% increase of cell death (SubG1 population) of Nox4A transfected chondrocytes by flow cytometry after 5 days incubation with 10 ng/ml IL-1b compared to the control chondrocytes transfected with Nox4B gene (Figure 1D)
Summary
The pathogenesis of osteoarthritis involves an imbalance between anabolic and catabolic pathways in chondrocytes. The expression of matrix metalloproteinases (MMPs), chondrocyte hypertrophy and apoptosis are the main features of the pathology. Proinflammatory cytokines, such as interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) cause damages to cartilage via the synthesis and the secretion of MMPs, which in turn lead to matrix degradation [1,2,3,4,5]. ROS have been suggested to act as secondary messengers in bovine chondrocytes and are involved in AP-1 and NF-kappaB activation pathways leading to the transcription of cytokine-induced MMP-1 and MMP-13 metalloproteinases [7,8,9,10]. Hypertrophic chondrocytes undergo apoptosis leading to a dysregulation of matrix repair mechanism [11]
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