Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts

摘要

Introduction Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs. Methods Obs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D₃ (VitD₃). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors. Results The expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively ( P 3 stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia ( P 3 ( P 3-stimulated leptin mRNA and protein levels by 70% ( P =0.004) and 60% ( P 3 greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK ( P 3 stimulation, whereas DKK2 is regulated mainly by VitD₃ rather than hypoxia.