Article Text

AB0116 HIF-2alpha dependent rankl induction and osteoclastogenesis is augmented by inflammatory cytokines
  1. S. Revu1,
  2. X. Zheng2,
  3. V. Sunkari2,
  4. A. Krishnamurthy1,
  5. I. Botusan2,
  6. S.-B. Catrina2,
  7. A. Catrina1
  1. 1Department of Medicine, Rheumatology Unit
  2. 2Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden


Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and consecutive local hypoxia, leading to cartilage and bone destruction. Hypoxia promotes osteoclasts formation in vitro but its role in mediating bone destruction in the presence of chronic inflammation has not previously been investigated

Objectives We aimed to investigate the effect of hypoxia on the RANKL/OPG system and bone destruction in the presence of pro inflammatory stimuli.

Methods We investigated the in vitro effect of hypoxia on RANKL/OPG expression in osteoblast-like (Saos2) cells. Cells were cultured in normoxic (21% pO2) or hypoxic (0.5% pO2) conditions with or without TNFα. Expression of RANKL and OPG mRNA was detected by rtPCR. Cellular and soluble forms of RANKL and OPG proteins were determined by Western blot and ELISA respectively. Functional contributions of the hypoxia inducible factor (HIF)-1 and 2 alpha were investigated by selective siRNA transfection. To confirm the direct interaction between HIF and RANKL promoter we performed Chromatin Immunoprecipitationc (ChIP) assays for both HIF1 and HIF-2. Hypoxia direct effect on osteoclastogenesis was also evaluated on a dentine pit formation assay using peripheral blood mononuclear cells from RA patients. Statistical analysis was performed using one-way ANOVA.

Results Hypoxia exposure changes the bone metabolism balance by induction of cellular RANKL mRNA and protein expression and reduction of the soluble OPG protein levels. Concomitant exposure to TNFα has an additive effect inducing a further increase of the RANKL/OPG ratio. Small interfering RNA against HIF 2alpha but not HIF 1alpha was able to abolish hypoxia effect on cellular RANKL expression. ChIP assay confirmed a direct interaction between HIF-2 alpha and at least one hypoxia responsive element (HRE) in the RANKL promoter. Furthermore hypoxia-mimicking using prolyl hydroxylases inhibitors and TNF have an additive effect in promoting RANKL-independent osteoclastogenesis

Conclusions TNF and hypoxia are additive in promoting HIF2alpha dependent RANKL induction but also RANKL-independent osteoclastogenesis. Our findings propose hypoxia as an important player in the bone metabolism imbalance observed in the setting of chronic joint inflammation.

Disclosure of Interest None Declared

Statistics from

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.