Oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) in cultured bovine articular chondrocytes increases production of intracellular reactive oxygen species (ROS) resulting in the activation of NF-kappaB.
OBJECTIVE
To examine the effect of oxidized low-density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine articular chondrocytes (BACs) and to investigate whether this increase occurs through binding to the receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, to ascertain whether the binding of ox-LDL to LOX-1 results in NF-kappaB activation.


METHODS
BACs were preincubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye that allows the monitoring of intracellular ROS production for DCF by spectrofluorometry. BACs were incubated with native LDL and ox-LDL (10, 50, and 100 microg/ml) for 5 min at 37 degrees C and DCF formation was observed. BACs were also preincubated with anti-LOX-1 mAb (40 microg/ml) or ascorbic acid (10 microM). Nuclear extracts from BACs treated for the indicated periods with 50 microg/ml ox-LDL, and preincubated with anti-LOX-1 mAb or ascorbic acid, were prepared and analyzed by electrophoretic mobility shift assay (EMSA).


RESULTS
ox-LDL induced a significant dose-dependent increase in ROS production after 5-min incubation with BACs (P < 0.001). ROS formation was markedly reduced in BACs preincubated with anti-LOX-1 mAb and ascorbic acid (P < 0.001). Activation in BACs of the transcription factor NF-kappaB was evident after 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 mAb and ascorbic acid.


CONCLUSIONS
ox-LDL binding to LOX-1 in BACs increased the production of intracellular ROS and activated NF-kappaB. Reduction of NF-kappaB activation by ascorbic acid indicates that the activation, at least in part, is ROS-dependent. These observations support the hypothesis that hypercholesterolemia is one of several risk factors for arthritis, and that lipid peroxidation products such as ox-LDL are involved in cartilage matrix degradation.
Nishimura
Shunji
S
Department of Orthpaedic Surgery, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan.
Akagi
Masao
M
Yoshida
Koji
K
Hayakawa
Sumio
S
Sawamura
Tatsuya
T
Munakata
Hiroshi
H
Hamanishi
Chiaki
C
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000032:analysis; D008077:Lipoproteins, LDL / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011973:Receptors, LDL / Q000032:analysis / Q000378:metabolism*; D051131:Receptors, Oxidized LDL; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D005452:Fluoresceins; D008077:Lipoproteins, LDL; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D011973:Receptors, LDL; D051131:Receptors, Oxidized LDL; C099252:oxidized low density lipoprotein; C037631:2',7'-dichlorofluorescein; D001205:Ascorbic Acid