title: 4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Orthopaedic Research Laboratory, Hôpital du Sacré-Caeur de Montréal, Department of Surgery, University of Montreal, 5400 Gouin Blvd, West, Montreal, QC, H4J 1C5, Canada. france.vaillancourt@umontreal.ca
authors.forename: France
authors.initials: F
authors.lastname: Vaillancourt
authors.forename: Hassan
authors.initials: H
authors.lastname: Fahmi
authors.forename: Qin
authors.initials: Q
authors.lastname: Shi
authors.forename: Patrick
authors.initials: P
authors.lastname: Lavigne
authors.forename: Pierre
authors.initials: P
authors.lastname: Ranger
authors.forename: Julio C
authors.initials: JC
authors.lastname: Fernandes
authors.forename: Mohamed
authors.initials: M
authors.lastname: Benderdour
mesh_terms: D000368:Aged; D000447:Aldehydes / Q000633:toxicity*; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D019902:Chondrocytes / Q000378:metabolism / Q000473:pathology*; D015853:Cysteine Proteinase Inhibitors / Q000633:toxicity*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D000447:Aldehydes; D015853:Cysteine Proteinase Inhibitors; D005982:Glutathione Transferase; D020169:Caspases; C027576:4-hydroxy-2-nonenal
references.citation: Science. 1997 Feb 21;275(5303):1132-6
references.pmid: 9027315
references.citation: Arthritis Rheum. 1998 Sep;41(9):1632-8
references.pmid: 9751096
references.citation: Trends Biochem Sci. 2001 Nov;26(11):657-64
references.pmid: 11701324
references.citation: FASEB J. 1999 Nov;13(14):1985-90
references.pmid: 10544181
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):55-63
references.pmid: 6606682
references.citation: Arthritis Rheum. 2002 Feb;46(2):394-403
references.pmid: 11840442
references.citation: Free Radic Biol Med. 2001 Apr 15;30(8):884-94
references.pmid: 11295531
references.citation: Eur J Biochem. 1999 Sep;264(3):687-701
references.pmid: 10491114
references.citation: Med Res Rev. 2008 Jul;28(4):569-631
references.pmid: 18058921
references.citation: Arthritis Rheum. 2006 Jan;54(1):271-81
references.pmid: 16385544
references.citation: Nat Rev Cancer. 2002 Sep;2(9):647-56
references.pmid: 12209154
references.citation: Free Radic Biol Med. 1991;11(1):81-128
references.pmid: 1937131
references.citation: Free Radic Biol Med. 2008 Jul 15;45(2):111-8
references.pmid: 18456001
references.citation: Arch Biochem Biophys. 1999 Dec 1;372(1):29-36
references.pmid: 10562413
references.citation: J Biol Chem. 2001 May 11;276(19):16168-76
references.pmid: 11278619
references.citation: Arthritis Rheum. 1998 Feb;41(2):284-9
references.pmid: 9485086
references.citation: Arch Biochem Biophys. 2001 Aug 15;392(2):197-207
references.pmid: 11488593
references.citation: J Cell Biochem. 2007 Apr 1;100(5):1217-31
references.pmid: 17031850
references.citation: J Rheumatol. 2000 Feb;27(2):455-62
references.pmid: 10685814
references.citation: Histol Histopathol. 2002 Oct;17(4):1239-67
references.pmid: 12371151
references.citation: Am J Pathol. 1995 Jan;146(1):75-85
references.pmid: 7856740
references.citation: Free Radic Biol Med. 2007 Jun 1;42(11):1661-70
references.pmid: 17462534
references.citation: Arthritis Res. 2001;3(2):107-13
references.pmid: 11178118
references.citation: Arthritis Rheum. 1986 Aug;29(8):1039-49
references.pmid: 3741515
references.citation: Biofactors. 2005;24(1-4):151-7
references.pmid: 16403975
references.citation: Nature. 1998 Jan 29;391(6666):496-9
references.pmid: 9461218
references.citation: Biochemistry. 2006 Oct 10;45(40):12253-64
references.pmid: 17014078
references.citation: Biochem Biophys Res Commun. 1995 Jan 5;206(1):15-21
references.pmid: 7529496
references.citation: J Cell Sci. 2000 Feb;113 ( Pt 4):635-41
references.pmid: 10652256
references.citation: Biochem Biophys Res Commun. 1994 Apr 15;200(1):142-8
references.pmid: 7513156
references.citation: Cell Death Differ. 2003 Jul;10(7):772-81
references.pmid: 12815460
references.citation: Alcohol Clin Exp Res. 2001 Jun;25(6):862-71
references.pmid: 11410723
references.citation: J Biol Chem. 2003 Nov 14;278(46):45154-9
references.pmid: 12960146
references.citation: Osteoarthritis Cartilage. 2004 Feb;12(2):131-41
references.pmid: 14723872
references.citation: Front Biosci. 1999 Oct 15;4:D662-70
references.pmid: 10525481
references.citation: Brain Res Brain Res Rev. 2005 Apr;48(2):157-65
references.pmid: 15850654
references.citation: Neuropharmacology. 2007 Oct;53(5):687-98
references.pmid: 17889908
references.citation: Arthritis Rheum. 1998 Jul;41(7):1266-74
references.pmid: 9663485
references.citation: Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8621-5
references.pmid: 1528871