Osteopontin inhibits HIF-2α mRNA expression in osteoarthritic chondrocytes

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 purified 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 quantification 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.

Keywords: CD44; HIF-2α; chondrocyte; osteoarthritis; osteopontin.

Figure 1.

Inhibition of endogenous osteopontin (OPN) expression by OPN small interfering RNA (siRNA). *P<0.05 compared with the control group.

Figure 2.

Cell viability detected by an MTT assay following treatment with recombinant human osteopontin (rhOPN) and osteopontin (OPN) small interfering RNA (siRNA). There were no significant differences (P>0.05) among the groups.

Figure 3.

Relative hypoxia-inducible factor (HIF)-2α mRNA expression was altered following treatment with recombinant human OPN (rhOPN) and osteopontin (OPN) small interfering RNA (siRNA). *P<0.05 vs. control group.

Figure 4.

CD44-blocking monoclonal antibody (mAb) suppressed the inhibitory effect of osteopontin (OPN) on the expression of hypoxia-inducible factor (HIF)-2α mRNA when compared with that in the recombinant human OPN (rhOPN) group (*P<0.05). Pretreatment with isotype control IgG1 had no significant effect on (HIF)-2α mRNA expression (P>0.05) when compared with that in the rhOPN group. HIF-2α, hypoxia-inducible factor-2α.