The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

University of Reading, Reading, England, United Kingdom
Cellular Signalling (Impact Factor: 4.32). 05/2007; 19(4):705-14. DOI: 10.1016/j.cellsig.2006.08.019
Source: PubMed


At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint.

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    • "Alterations of the mitochondrial membrane potential (« m ) were determined by fluorescence microscopy using 1 ␮g/mL rhodamine 123 (Rhod123; Sigma–Aldrich), a fluorescent molecule that is commonly used for detecting mitochondrial membrane potential (Whiteman et al., 2007). Spores of P. expansum (1 × 10 6 spores/mL) were cultured at 23 • C in PDB medium. "
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    • "During apoptotic stimulation, alteration of the Bcl-2/Bax protein ratio may affect mitochondrial cytochrome c release (Hildeman et al., 2003). The ratio of these two protein levels can be predictive of whether or not a cell undergoes apoptosis (Gross et al., 1999); a decrease in Bcl-2/Bax ratio is a decisive correlated in apoptosis or cell death (Whiteman et al., 2007; Kim et al., 2010; Wiebe et al., 2010). Once released into the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1 (Apaf1), thereby promoting the activation of caspase (Zimmermann et al., 2001). "
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