Effect of ligand-receptor interaction of osteopontin-CD44 on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro.

Abstract

To investigate the effect of ligand-receptor interaction of osteopontin (OPN)-CD44 on the expression of hyaluronic acid (HA) in the cultured human knee osteoarthritis (OA) chondrocytes via interfering the reaction between OPN and CD44 ligand-receptor. The OA chondrocytes and normal chondrocytes were obtained from knee joint cartilage tissues in the patients with knee OA and malignant tumor respectively. The normal chondrocytes and OA chondrocytes were detected and analyzed, and then the intervention analysis of OA chondrocytes was carried out. The OA chondrocytes were divided into 4 groups: a negative control group, which was cultured with complete medium without any molecular intervention reagent; an OPN intervention group, which was cultured with recombinant human OPN (rhOPN) for 24 hours; a CD44 blocking group, which were pretreated with CD44 receptor specific antagonist for 1 hour to block the binding of OPN-CD44, and then treated with rhOPN for 23 hours; a CD44 homotype group, which was pretreated with CD44 for 1 hour and then treated with rhOPN for 23 hours. In addition, the study for OPN-CD44 axis was also divided into 4 groups: an OA-negative control group (OA-NC group), a si-OPN intervention group, a rhOPN intervention group, and a rhOPN + CD44 antibody (Ab) group. Western blotting, real-time PCR, and enzyme linked immunosorbent assay (ELISA) were used to detect the protein and mRNA expression levels of OPN, CD44, hyaluronate synthase (HAS), and HA, respectively. The protein expression levels of OPN, CD44, and HAS1 and the secretion levels of HA in the OA chondrocytes were higher than those in the normal chondrocytes. Compared with the OPN intervention group, the expression levels of HAS1, HAS2, HAS3 and HA in the CD44 blocking group were lower than those in OPN intervention group (all P<0.05); but there was no significant difference in the expression levels of HAS1, HAS2, HAS3 and HA between the CD44 homotype group and the OPN intervention group (all P>0.05). The results of OPN-CD44 axis study showed that: compared with the OA-NC group, the expression of CD44 in the rhOPN intervention group was slightly lower, but the protein and mRNA levels of HAS1 were significantly increased (all P<0.05); compared with the OA-NC group, the expression of CD44 was up-regulated, but the protein and mRNA level of HAS1 were significantly inhibited in the si-OPN intervention group (all P<0.05); compared with the OA-NC group, the protein and mRNA levels of HAS1 in the rhOPN+CD44 Ab group were also significantly inhibited (all P<0.05). The OPN in OA chondrocytes can promote the expression of HAS1, and the OPN can stimulate the secretion of HAS and induce HA expression by reacting with CD44 ligand receptor. They constitute the axis of OPN/CD44/HAS1, which plays an important role in regulating the expression of HA in chondrocytes.