Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis.
BACKGROUND
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 α1β1 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 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-κB-p65 levels were analyzed by Western blotting. The formation of α1β1 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, α1β1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-κB-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 α1β1 integrin and Col II expression as well as ERK1/2 and NF-κB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of α1β1 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.
Orthopaedic Research Laboratory, Hôpital du Sacré-Coeur de Montréal, Department of Surgery, University of Montreal, 5400 Gouin Blvd, West, Montreal, QC H4J 1C5, Canada. rana.el.bikai@umontreal.ca
Rana
R
El-Bikai
Mélanie
M
Welman
Yoran
Y
Margaron
Jean-François
JF
Côté
Luke
L
Macqueen
Michael D
MD
Buschmann
Hassan
H
Fahmi
Qin
Q
Shi
Karim
K
Maghni
Julio C
JC
Fernandes
Mohamed
M
Benderdour
D000368:Aged; D000447:Aldehydes / Q000378:metabolism*; D015153:Blotting, Western; D002336:Carnosine / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000187:drug effects / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D024043:Collagen Type II / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000447:Aldehydes; D015815:Cell Adhesion Molecules; D024043:Collagen Type II; D012333:RNA, Messenger; D002336:Carnosine; C027576:4-hydroxy-2-nonenal