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Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains

MATTHEW WHITEMAN^*,1, JEFFREY S. ARMSTRONG^*, NAM SANG CHEUNG^*, JIA-LING SIAU^*, PETER ROSE, JAN-THORSTEN SCHANTZ, DEAN P. JONES^|| and BARRY HALLIWELL^*

Departments of
^* Biochemistry,
Community, Occupational and Family Medicine,
Plastic Surgery and
^|| Bioengineering, Faculty of Medicine, National University of Singapore, Republic of Singapore; and
||Clinical Biomarkers Laboratory, Center for Clinical and Molecular Nutrition, Emory University School of Medicine, Atlanta, Georgia, USA

¹ Correspondence: Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Dr., Republic of Singapore 117597. E-mail: bchwml{at}nus.edu.sg

SPECIFIC AIMS

Peroxynitrite is an endogenous cytotoxic agent formed at sites of chronic inflammation from the reaction of nitric oxide with superoxide. Although the chemistry of peroxynitrite is well documented, the precise molecular events by which it can induce cell death are unclear. The aim of this study was to determine the molecular mechanisms of peroxynitrite-induced cell death in human articular chondrocytes, cells known to be exposed to high levels of peroxynitrite in the inflamed human joint. Cells were treated with authentic peroxynitrite or the peroxynitrite generator SIN-1; cytosolic and mitochondrial calcium mobilization, mitochondrial membrane potential (_m), caspase, and calpain protease activities were determined using confocal microscopy, flow cytometry, Western blot, and microplate fluorescence assays. Cell death was determined using phosphatidylserine, sub-G1 cell population, chromatin condensation, and cell body shrinkage analysis in the presence or absence of established inhibitors of caspases, calpains, and mitochondrial permeability transition.

PRINCIPAL FINDINGS

1. Peroxynitrite caused extensive intracellular calcium mobilization leading to mitochondrial calcium uptake and loss of mitochondrial membrane potential
Addition of 50 µM ONOO^– or the ONOO^– generator SIN-1 (1 mM) to human articular chondrocytes (HAC) caused extensive and rapid mobilization of intracellular calcium resulting in mitochondrial calcium accumulation (Fig. 1 ) concomitant with a loss of mitochondrial membrane potential. This effect was inhibited by the mitochondrial permeability transition inhibitor cyclosporine A and intracellular calcium chelator BAPTA-AM. However, the adenine nucleotide translocase ligand bongkrekic acid did not inhibit loss of mitochondrial membrane potential. Analysis of mitochondrial swelling using isolated rat liver mitochondria also showed ONOO^–-induced mitochondrial swelling that was inhibited with cyclosporine A but not bongrekic acid, further suggesting ONOO^– exerted its effects on mitochondria at least in part on a cyclosporine A-sensitive site.

View larger version (39K): [in this window] [in a new window]   Figure 1. Peroxynitrite caused extensive intracellular calcium mobilization leading to mitochondrial calcium uptake. Human articular chondrocytes were loaded with the mitochondrial calcium dye dihydrorhod-2AM (100 nM) and the cytosolic calcium dye Fluo-3AM (1 µM) in serum-free chondrocyte growth media for 30 min. Cells were then gently washed with warm (37°C) Earle’s balanced salt solution and SIN-1 (1 mM) was added. Calcium mobilization was observed using confocal microscopy at the times stated. Photographs are representative of 6 determinations carried out on separate days using freshly prepared reagents.

2. Peroxynitrite-induced cell death to give biochemical markers of apoptosis without the activation of caspase proteases
Addition of ONOO^– (50 µM) or SIN-1 (1 mM) to HAC caused phosphatidylserine externalization, DNA condensation, cell body shrinkage, and extensive DNA fragmentation with the formation of sub-G1 cell populations, suggesting that the pathway of ONOO^–-induced cell death involved apoptosis. However, treatment of HAC with inhibitors of caspases-2, -3, -6, -7. -8, or -9 prior to ONOO^– or SIN-1 addition did not inhibit cell death when measured using MTT, LDH, or sub-G1 assays; analysis of caspase activation by Western blot showed pro-caspase cleavage only in the positive control, staurosporine (0.5 µM). Furthermore, caspase activity in cell lysates was detected only in staurosporine-treated cells and not in cells treated with ONOO^– or SIN-1. Addition of ONOO^– or SIN-1 to cells treated with staurosporine did not result in inhibition of caspase activity. These data strongly suggest that caspase proteases did not mediate ONOO^–-induced chondrocyte cell death and that ONOO^– did not inhibit caspase activity.

3. Calpain proteases mediate peroxynitrite-induced cell death
Calpain-protease activation was observed within 60 min of addition of ONOO^– (50 µM) and SIN-1 (1 mM) to HAC (Fig. 2 A), and ONOO^–- or SIN-1-induced calpain activity was significantly inhibited with calpain inhibitors (Fig. 2A ). Treatment of HAC with calpain inhibitors (ALLN, ALLM, EST, calpeptin, or calapstatin) prior to ONOO^– or SIN-1 addition markedly reduced cell death measured by MTT and LDH assays (Fig. 2B ) and sub-G1 cell populations (Fig. 2C ), suggesting that calpain-proteases mediated ONOO^–-induced chondrocyte cell death.

View larger version (37K): [in this window] [in a new window]   Figure 2. Calpain proteases mediated peroxynitrite-induced cell death. A) Calpain activity: human articular chondrocytes were treated with ONOO– (50 µM) or SIN-1 (1 mM) and calpain-protease activity was detected after 60 min using a commercial kit (BioVision) according to the manufacturer’s instructions. B, C) Inhibition of cell death by calpain inhibitors. Chondrocytes were treated with 50 µM calpain inhibitors (ALLN, ALLM, EST, calpeptin, or calapstatin) for 1 h prior to ONOO– (50 µM) or SIN-1 (1 mM); cell death was analyzed at 18 h by MTT and LDH assays (B) and sub-G1 assays (C). Data are expressed as mean ±SD of 6 or more separated experiments. Where significance testing was performed, an independent t test (Student’s; 2 populations) was used; *P <0.1, **P <0.05, ***P <0.01 compared with ONOO–/SIN-1-treated cells.

CONCLUSIONS AND SIGNIFICANCE

ONOO^– is known to induce mitochondrial permeability transition in isolated rodent mitochondria, an effect potentiated by calcium, but the effects on human mitochondria in whole cells in situ are not known. In addition, ONOO^– induces cell death in noncartilaginous cell lines by activation of caspase proteases, but the involvement of other cell death proteases such as calpains in ONOO^–-mediated cytotoxicity is unknown. To address these issues, we investigated the cell death mechanisms induced by ONOO^– in human articular chondrocytes, cartilage cells known to be exposed to ONOO^– in the human joint.

The novel findings of our study are 1) ONOO^– mediated a marked elevation of cytoplasmic calcium levels that resulted in the accumulation of intracellular calcium into the mitochondria, 2) ONOO^–-induced elevation of cytosolic calcium collapsed the mitochondrial membrane potential (_m), and 3) ONOO^–-induced cell death was mediated by calpain rather than caspase proteases but still exhibited several biochemical markers of apoptosis.

Recently, inhibition of calpain proteases was shown to decrease joint erosion, inflammation, and ONOO^– formation in rodent models of arthritis; chondrocyte function or the mechanism for protection was not examined. Our study suggests that inhibition of cell death induced by ONOO^– by calpain protease inhibitors may serve as novel therapeutic strategies for preventing chondrocyte cell death in inflammatory and degenerative joint diseases such as osteoarthritis and rheumatoid arthritis.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic representation of the proposed mechanism of ONOO–-induced human articular chondrocyte (HAC) cell death in the inflamed human joint. Nitric oxide (•NO) and superoxide (O2•–) formed from activated inflammatory cells and resident joint cells react to form peroxynitrite (ONOO–). ONOO– enters the cell and causes rapid mobilization of intracellular calcium leading to sequestration of calcium into the mitochondria, resulting in mitochondrial permeability transition (MPT). Mitochondrial calcium release elevates cytoplasmic calcium levels resulting in activation of calpain proteases, which execute the cell death pathway without the involvement of caspase proteases. Inhibition of calpain proteases with calpain inhibitors ALLN, ALLM, EST, calpeptin, or calpastatin inhibit cell death. Mt, mitochondria; HAC, human articular chondrocyte; m, mitochondrial membrane potential; CSA, cyclosporine A.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1096fje; doi: 10.1096/fj.03-1096fje

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