Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress.
OBJECTIVE
It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs.


METHODS
Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL.


RESULTS
COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment.


CONCLUSIONS
Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.
Kishimoto
H
H
Department of Orthpaedic Surgery, Kinki University, Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan. kiss_kissy@hotmail.com
Akagi
M
M
Zushi
S
S
Teramura
T
T
Onodera
Y
Y
Sawamura
T
T
Hamanishi
C
C
D000111:Acetylcysteine / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002358:Cartilage, Articular / Q000187:drug effects / Q000473:pathology* / Q000503:physiopathology; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D024121:Collagen Type X / Q000096:biosynthesis / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006984:Hypertrophy / Q000139:chemically induced / Q000473:pathology / Q000503:physiopathology; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D051127:Scavenger Receptors, Class E / Q000172:deficiency / Q000235:genetics
D000975:Antioxidants; D024121:Collagen Type X; D050659:Core Binding Factor Alpha 1 Subunit; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein; D006861:Hydrogen Peroxide; D000469:Alkaline Phosphatase; D000111:Acetylcysteine