4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Abstract

Introduction: 4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.

Methods: Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.

Results: Our data showed that HNE at concentrations of up to 10 microM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 microM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.

Conclusion: Our study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.

Figure 1

4-Hydroxynonenal (HNE)-induced cell death. (a) Chondrocytes were pre-incubated for 1 hour with or without 200 μM N-acetyl-cysteine (NAC) followed by another incubation for 16 hours with increasing concentrations of HNE (0 to 30 μM). Chondrocytes were pre-incubated for 1 hour with or without (b) 20 μg/mL anti-Fas/CD95 antibody or (c) 50 and 100 μM INH₂BP followed by another incubation for 16 hours with or without 30 μM HNE. Cell viability was evaluated by MTT assay. Data are mean ± standard error of the mean (n = 8). Statistics: Student unpaired t test; ***P < 0.001 (30 μM HNE versus untreated cells), ^#P < 0.05 (30 μM HNE+inhibitor versus 30 μM HNE).

Figure 2

Nuclear morphology study for apoptosis. Chondrocytes were incubated for 16 hours with or without 30 μM 4-hydroxynonenal (HNE), stained with Hoechst 33258, and then analyzed by fluorescence microscopy.

Figure 3

Caspase-8, -9, and -3 activation by 4-hydroxynonenal (HNE). Chondrocytes were treated with 30 μM HNE for the indicated times, and enzymatic activities of caspase-8 (a), caspase-9 (b), or caspase-3 (c) were determined with commercial kits. (d) Chondrocytes were pre-incubated for 1 hour with or without 200 μM N-acetyl-cysteine (NAC) followed by another incubation for 16 hours with increasing concentrations of HNE (0 to 30 μM). Pro-caspase-8, pro-caspase-9, and pro-caspase-3 were analyzed by Western blot. Data are mean ± standard error of the mean (n = 8) and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

4-Hydroxynonenal (HNE) modified Bcl-2/Bax protein expression and induced cytochrome c release from mitochondria. Chondrocytes were treated with 30 μM HNE for the indicated times and then Bcl-2 (a) and Bax (b) protein levels were determined by commercial kit and Western blot, respectively. (c) Chondrocytes were pre-incubated with or without 200 μM N-acetyl-cysteine (NAC) for 1 hour followed by another incubation for 16 hours in the presence of increasing concentrations of HNE (0 to 30 μM). Cytochrome c level was assessed in cytosolic fractions with a kit. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

4-Hydroxynonenal (HNE) induced DNA fragmentation, poly (ADP-ribose) polymerase (PARP) cleavage, and apoptosis-inducing factor (AIF) translocation to the nucleus. Chondrocytes were pre-incubated for 1 hour with or without 200 μM N-acetyl-cysteine (NAC) and then incubated for another 16 hours with 30 μM HNE or with increasing concentrations of HNE (0 to 30 μM). (a) The cytoplasmic histone-associated DNA fragments were quantified with a kit. (b) Chondrocytes were pre-incubated for 1 hour with or without 200 μM NAC followed by another incubation with 30 μM HNE at different incubation times. PARP cleavage and AIF translocation in nuclear fractions were analyzed by Western blot. Data are mean ± standard error of the mean and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *P < 0.05, **P < 0.01.

Figure 6

4-Hydroxynonenal (HNE) induced Fas/CD95 and p53 protein expression and reduced Akt phosphorylation. Chondrocytes were incubated with 30 μM HNE for increasing incubation times. Total cell lysates or nuclear extracts (~20 μg) were subjected to Western analysis (n = 8) using antibodies anti-Fas/CD95, anti-p53, anti-phospho Akt, and anti-total Akt.

Figure 7

4-Hydroxynonenal (HNE) altered redox status and energy metabolism in osteoarthritis chondrocytes. Chondrocytes were treated for 16 hours with 30 μM HNE, and cellular extracts were subjected to different analysis to determine (a) GSSG/(GSSG+GSH) and NADP/(NADPH+NADP) ratios, (b) mNADP⁺-ICDH activity, (c) glucose uptake, and (d) ATP level. Data are mean ± standard error of the mean and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; **P < 0.01, ***P < 0.001. GSH, glutathione; GSSG, oxidized glutathione; mNADP⁺-ICDH, mitochondrial NADP⁺-dependent isocitrate dehydrogenase.

Figure 8

Glutathione-S-transferase A4-4 (GSTA4-4) controlled the 4-hydroxynonenal (HNE) cytotoxicity. Chondrocytes were transfected with (a) small interfering RNA (siRNA) GSTA4-4 or with (c) wild-type or mutant GSTA4-4 expression plasmids, and then cell viability was determined by MTT assay. (b) and (d) GSTA4-4 protein expression was evaluated respectively in cellular extracts of transfected chondrocytes with siRNA or expression plasmids of GSTA4-4 by Western blotting. Data are mean ± standard error of the mean and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001. CTL, control; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mut, mutant; wt, wild-type.