COX-2 and iNOS are critical in advanced glycation end product-activated chondrocytes in vitro.
BACKGROUND
The advanced glycation end products (AGEs) accumulate in joints of osteoarthritis patients. This study aimed to investigate the roles of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) pathways in AGE-mediated cartilage damage.


METHODS
Methylglyoxal-modified albumin was used as the source of AGE. Porcine and human chondrocytes were prepared from the joint cartilage of pigs and osteoarthritis patients. The activation of COX-2, iNOS, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and protein kinases was determined by Western blotting, kinase assay, electrophoretic mobility shift assay (EMSA) or transfection assay. Prostaglandin E(2) (PGE(2)) and NO concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and Griess reaction respectively. The enzymatic activity of COX was determined by measuring the conversion of arachidonic acid to PGE(2). The release of sulphated glycosaminoglycan and the intensity of Safranin O staining were used to measure cartilage degradation.


RESULTS
AGE potently induced COX-2-PGE(2) and iNOS-NO activation in porcine and human chondrocytes. Meanwhile, the upstream molecules regulating COX-2/iNOS activation, such as AP-1, NF-kappaB, extracellular signal regulated protein kinase (ERK) and c-jun N-terminal kinase (JNK), were activated by AGE. Although AGE could not activate p38 directly, by measuring COX enzyme activity, the inhibition of p38 resulted in suppressing AGE-induced conversion of arachidonic acid to PGE(2). Furthermore, successful blockage of either COX-2 or NOS activity significantly reduced AGE-mediated proteoglycan release and cartilage degradation.


CONCLUSIONS
This study highlights the significance of COX-2 and iNOS pathways in AGE-mediated OA pathogenesis and their potential as therapeutic targets that are beyond pain killing for OA treatment.
Huang
C-Y
CY
National Health Research Institute, Zhunan, Miaoli, Taiwan, ROC.
Hung
L-F
LF
Liang
C-C T
CC
Ho
L-J
LJ
D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism*; D013552:Swine; D055815:Young Adult
D017127:Glycation End Products, Advanced; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2