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.
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).
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.
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.