Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis

Introduction: Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects. Methods: Human Col II was treated with HNE at different molar ratios (MR) (1: 20 to 1: 200; Col II: HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and alpha 1 beta 1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE(2)), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappa B) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-kappa B-p65 levels were analyzed by Western blotting. The formation of alpha 1 beta 1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation. Results: Col II modification by HNE at MR approximately 1: 20, strongly induced ICAM-1, alpha 1 beta 1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-kappa B-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1: 200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited alpha 1 beta 1 integrin and Col II expression as well as ERK1/2 and NF-kappa B-p65 phosphorylation, but, in contrast, markedly elicited PGE(2) release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of alpha 1 beta 1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1: 20 or approximately 1: 200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug. Conclusions: Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes.