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

Bouvard B, Abed E, Yéléhé-Okouma M, Bianchi A, Mainard D, Netter P, Jouzeau JY, Lajeunesse D, Reboul P - Arthritis Res. Ther. (2014)

Bottom Line: The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing (si) RNA technique.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.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: 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.

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Related in: MedlinePlus

Alteration of the expression of selective gene targets under hypoxia. Human osteoarthritis (OA) osteoblasts were incubated for 24 hours either under normoxia (Nox) or hypoxia (Hox; 2% O2). Vascular endothelial growth factor (VEGF) (A), Sirt1(B) and transforming growth factor β1 (TGFbeta) (C). *P <0.05, statistically significant compared to Nox (n =4 to 6 experiments).
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Fig1: Alteration of the expression of selective gene targets under hypoxia. Human osteoarthritis (OA) osteoblasts were incubated for 24 hours either under normoxia (Nox) or hypoxia (Hox; 2% O2). Vascular endothelial growth factor (VEGF) (A), Sirt1(B) and transforming growth factor β1 (TGFbeta) (C). *P <0.05, statistically significant compared to Nox (n =4 to 6 experiments).

Mentions: We first sought to determine whether hypoxia would affect OA Obs by evaluating the expression of selective gene targets. Figures 1A to 1C show that our experimental conditions stimulated expression of genes known to be induced by hypoxia (namely, VEGF (P <0.05) (Figure 1A), Sirt1 (P <0.05) (Figure 1B) and TGF-β1 P <0.05) (Figure 1C)), thus validating their relevance to hypoxia.Figure 1


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

Bouvard B, Abed E, Yéléhé-Okouma M, Bianchi A, Mainard D, Netter P, Jouzeau JY, Lajeunesse D, Reboul P - Arthritis Res. Ther. (2014)

Alteration of the expression of selective gene targets under hypoxia. Human osteoarthritis (OA) osteoblasts were incubated for 24 hours either under normoxia (Nox) or hypoxia (Hox; 2% O2). Vascular endothelial growth factor (VEGF) (A), Sirt1(B) and transforming growth factor β1 (TGFbeta) (C). *P <0.05, statistically significant compared to Nox (n =4 to 6 experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302570&req=5

Fig1: Alteration of the expression of selective gene targets under hypoxia. Human osteoarthritis (OA) osteoblasts were incubated for 24 hours either under normoxia (Nox) or hypoxia (Hox; 2% O2). Vascular endothelial growth factor (VEGF) (A), Sirt1(B) and transforming growth factor β1 (TGFbeta) (C). *P <0.05, statistically significant compared to Nox (n =4 to 6 experiments).
Mentions: We first sought to determine whether hypoxia would affect OA Obs by evaluating the expression of selective gene targets. Figures 1A to 1C show that our experimental conditions stimulated expression of genes known to be induced by hypoxia (namely, VEGF (P <0.05) (Figure 1A), Sirt1 (P <0.05) (Figure 1B) and TGF-β1 P <0.05) (Figure 1C)), thus validating their relevance to hypoxia.Figure 1

Bottom Line: The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing (si) RNA technique.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.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus