title: Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts.
abstract: BACKGROUND
  Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularisation 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 since they are up-regulated 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 either under 20% or 2% oxygen tension in the presence or not of 50 nM of 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factors (Hif)-1α and -2α was measured by real-time polymerase chain reaction and leptin production by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing (si) RNA technique. Signalling pathway of hypoxia-induced leptin was investigated by western blotting and mitogen-activated protein kinase (MAPK) inhibitors.
  
  
  RESULTS
  As expected, hypoxia stimulated the expression of Hif-1 and Hif-2. The expression of leptin and DKK2 in Obs was also stimulated 7-fold and 1.8-fold respectively (p<0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold under normoxia, it further stimulated it to 28- and 6.2-fold under hypoxia (p<0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in presence of VitD3 (p<0.02). Compared to Obs incubated in the presence of siScramble RNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (p=0.004) and 60% (p<0.02), respectively while it failed to significantly alter the expression of DKK2. SiHif-1α has no effect on these genes. Immunoblotting showed that VitD3 greatly stabilized Hif-2α under hypoxic condition. The increase in leptin expression under hypoxia was also regulated, in addition to the role of Hif-2α, by p38 MAPK (p<0.03) and PI 3-kinase (p<0.05). Finally, we demonstrated that the expression of leptin and DKK2 were not related to each other under hypoxia.
  
  
  CONCLUSIONS
  Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin particularly under VitD3 stimulation whereas DKK2 is mainly regulated by VitD3 rather than hypoxia.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Bouvard
authors.forename: Elie
authors.initials: E
authors.lastname: Abed
authors.forename: Mélissa
authors.initials: M
authors.lastname: Yéléhé-Okouma
authors.forename: Arnaud
authors.initials: A
authors.lastname: Bianchi
authors.forename: Didier
authors.initials: D
authors.lastname: Mainard
authors.forename: Patrick
authors.initials: P
authors.lastname: Netter
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Jouzeau
authors.forename: Daniel
authors.initials: D
authors.lastname: Lajeunesse
authors.forename: Pascal
authors.initials: P
authors.lastname: Reboul
mesh_terms: D000368:Aged; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001842:Bone and Bones / Q000473:pathology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D020738:Leptin / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000473:pathology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014807:Vitamin D / Q000494:pharmacology*; D014815:Vitamins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D036341:Intercellular Signaling Peptides and Proteins; D020738:Leptin; D015675:Osteocalcin; D019869:Phosphatidylinositol 3-Kinases; D014807:Vitamin D; D014815:Vitamins; D048051:p38 Mitogen-Activated Protein Kinases; D000099162:Endothelial PAS Domain-Containing Protein 1; C498622:DKK2 protein, human; C497442:HIF1A protein, human