EXPERIMENTAL AND THERAPEUTIC MEDICINE 9: 2415-2419, 2015 Abstract. The aim of the present study was to investigate the in vitro effect of osteopontin (OPN) on the expression of hypoxia-inducible factor-2α (HIF-2α) in chondrocytes and the role of OPN in osteoarthritis (OA). Cartilage was puriied from the tibial surfaces of patients with OA of the knee and cultured in vitro to obtain chondrocytes. Recombinant human OPN (rhOPN) and OPN small interfering RNA (siRNA) were used to treat the chondrocytes, and the changes in the expression levels of the HIF-2α gene were measured. An anti-CD44 blocking monoclonal antibody (mAb) was used to determine the probable ligand-receptor interactions. Reverse transcription-quantitative polymerase chain reaction assays were designed and validated with SYBR ® Green dyes for the simultaneous quantiication of the mRNA expression levels of OPN and HIF-2α. The mRNA expression level of HIF-2α was markedly decreased in the rhOPN-treated group compared with that in the control group; by contrast, OPN siRNA increased HIF-2α gene expression. CD44 blocking mAb suppressed the inhibitory effect of OPN on HIF-2α mRNA expression. The results of the present study suggest that OPN may play a protective role in OA by inhibiting HIF-2α gene expression in osteoarthritic chondrocytes through CD44 interaction. Introduction Osteoarthritis (OA) is a metabolically active, dynamic process that affects all joint tissues. The major clinical signs of the disease include destruction of the articular cartilage and changes to the underlying subchondral bone. However, the etiology of the disease remains poorly understood. Several biochemical and biomechanical factors are considered to play a role in the pathogenesis. Osteopontin (OPN), also known as early T cell activation gene-1 (Eta-1) is abundant in bone tissue and may be secreted by a number of different cell types including chondrocytes and synoviocytes (1-4). OPN is upregulated in human chondrocytes (5). A previous study found that the level of OPN mRNA isolated from human OA cartilage was increased as compared with that in normal cartilage (5). Furthermore, the expression level of OPN in the plasma, synovial fluid and articular cartilage is associated with progressive joint damage and may be a useful biomarker for determining the severity and progression of disease in knee OA (6,7). OPN interacts with a variety of cell surface recep- tors, including integrin and CD44 (8). The receptor CD44 has been implicated in the development and progression of OA, and CD44 present in articular cartilage has been associated with progressive knee OA joint damage (9,10). However, the role of OPN in the pathological changes in knee OA remains unknown. Articular cartilage is an avascular tissue that derives its nutritional and oxygen supply by a diffusion process from the synovial fluid and subchondral bone. Thus, articular cartilage is maintained in a low oxygen environment in the body (11). Chondrocytes are therefore adapted to these hypoxic conditions. A number of previous studies have shown that hypoxia triggers essential positive signals for the chondrocyte phenotype (12-14). Adaptation to this avascular environment is mediated by hypoxia-inducible factor (HIF)-1 and HIF-2 (12). The HIF protein family consists of α and β subunit members that func- tion by forming heterodimers (12). Two HIF isoforms (HIF-1 α and HIF-2α) mediate the response of cells to hypoxia (13,14). In a previous study, HIF-2α was demonstrated to be essential for the endochondral ossiication of cultured chondrocytes and embryonic skeletal growth in mice (15). Furthermore, HIF-2α expression has been found to be higher in osteoarthritic carti- lage than in non-diseased cartilage in mice and humans (15). Another study observed that HIF-2α increased the expression levels of genes encoding catabolic factors, including matrix metalloproteinases (MMPs) -1, 3, 9, 12 and 13, aggrecanase-1, nitric oxide synthase-2 and prostaglandin-endoperoxide synthase-2 in chondrocytes (16). Thus, HIF-2α is an important catabolic transcription factor in the process of OA development and may be considered as a therapeutic target for OA. Osteopontin inhibits HIF-2α mRNA expression in osteoarthritic chondrocytes CHAO CHENG 1 , FANG-JIE ZHANG 1 , JIAN TIAN 1 , MIN TU 1 , YI-LIN XIONG 1 , WEI LUO 1 , YU-SHENG LI 1 , BING-BING SONG 2 , SHU-GUANG GAO 1,3 and GUANG-HUA LEI 1,3 1 Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha, Hunan, 410008; 2 Hunan Province Environmental Monitoring Center, Changsha, Hunan 410019; 3 Orthopaedics Institute of Central South University, Changsha, Hunan 410008, P.R. China Received July 21, 2014; Accepted March 26, 2015 DOI: 10.3892/etm.2015.2434 Correspondence to: Professor Guang-Hua Lei, Department of Orthopaedics, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, P.R. China E-mail: guanghualei@yahoo.com Key words: chondrocyte, CD44, HIF-2α, osteopontin, osteoarthritis