Peroxynitrite-modified collagen-II induces p38/ERK and NF-kappaB-dependent synthesis of prostaglandin E2 and nitric oxide in chondrogenically differentiated mesenchymal progenitor cells.
OBJECTIVE
Peroxynitrite (ONOO(-)) is formed in the inflamed and degenerating human joint. Peroxynitrite-modified collagen-II (PMC-II) was recently discovered in the serum of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore we investigated the cellular effects of PMC-II on human mesenchymal progenitor cells (MPCs) as a model of cartilage and cartilage repair cells in the inflamed and degenerating joint.


METHODS
MPCs were isolated from the trabecular bone of patients undergoing reconstructive surgery and were differentiated into a chondrogenic lineage. Cells were exposed to PMC-II and levels of the proinflammatory mediators nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) measured. Levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappaB) activation were measured by enzyme linked immunosorbent assay (ELISA) together with specific MAPK and NF-kappaB inhibitors.


RESULTS
PMC-II induced ()NO and PGE(2) synthesis through upregulation of iNOS and COX-2 proteins. PMC-II also lead to the phosphorylation of MAPKs, extracellularly regulated kinase 1/2 (ERK1/2) and p38 [but not c-Jun NH(2)-terminal kinase (JNK1/2)] and the activation of proinflammatory transcription factor NF-kappaB. Inhibitors of p38, ERK1/2 and NF-kappaB prevented PMC-II induced ()NO and PGE(2) synthesis, iNOS and COX-2 protein expression and NF-kappaB activation.


CONCLUSIONS
iNOS, COX-2, NF-kappaB and MAPK are known to be activated in the joints of patients with OA and RA. PMC-II induced iNOS and COX-2 synthesis through p38, ERK1/2 and NF-kappaB dependent pathways suggesting a previously unidentified pathway for the synthesis of the proinflammatory mediators, ()NO and PGE(2), further suggesting that inhibitors of these pathways may be therapeutic in the inflamed and degenerating human joint.
Whiteman
M
M
Department of Biochemistry, Yong Yoo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Republic of Singapore 117597. bchwml@nus.edu.sg
Spencer
J P E
JP
Zhu
Y Z
YZ
Armstrong
J S
JS
Schantz
J-T
JT
D001168:Arthritis / Q000503:physiopathology; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D024043:Collagen Type II; D008565:Membrane Proteins; D016328:NF-kappa B; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone