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.

Design: 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.

Conclusion: 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.