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Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression.

Mutabaruka MS, Aoulad Aissa M, Delalandre A, Lavigne M, Lajeunesse D - Arthritis Res. Ther. (2010)

Bottom Line: Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect.Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP.These results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-beta1 observed in these cells compared to normal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de recherche en Arthose, Centre de recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Hôpital Notre-Dame, 1560 rue Sherbrooke Est, Montréal, QC H2L 4 M1, Canada. ms.mutabaruka@umontreal.ca

ABSTRACT

Introduction: Leptin is a peptide hormone with a role in bone metabolism and rheumatic diseases. The subchondral bone tissue plays a prominent role in the pathophysiology of osteoarthritis (OA), related to abnormal osteoblast (Ob) differentiation. Although leptin promotes the differentiation of Ob under normal conditions, a role for leptin in OA Ob has not been demonstrated. Here we determined if endogenous leptin produced by OA Ob could be responsible for the expression of the abnormal phenotypic biomarkers observed in OA Ob.

Methods: We prepared primary normal and OA Ob from subchondral bone of tibial plateaus removed for knee surgery of OA patients or at autopsy. We determined the production of leptin and of the long, biologically active, leptin receptors (OB-Rb) using reverse transcriptase-polymerase chain reaction, ELISA and Western blot analysis. We determined the effect of leptin on cell proliferation by BrdU incorporation and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and we determined by Western blot analysis phospho 42/44 MAPK (p42/44 Erk1/2) and phospho p38 levels. We then determined the effect of the addition of exogenous leptin, leptin receptor antagonists, inhibitors of leptin signaling or siRNA techniques on the phenotypic features of OA Ob. Phenotypic features of Ob were determined by measuring alkaline phosphatase activity (ALP), osteocalcin release (OC), collagen type 1 production (CICP) and of Transforming Growth Factor-beta1 (TGF-beta1).

Results: Leptin expression was increased approximately five-fold and protein levels approximately two-fold in OA Ob compared to normal. Leptin stimulated its own expression and the expression of OB-Rb in OA Ob. Leptin dose-dependently stimulated cell proliferation of OA Ob and also increased phosphorylated p42/44 Erk1/2 and p38 levels. Inactivating antibodies against leptin reduced ALP, OC, CICP and TGF-beta1 levels in OA Ob. Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect. Inhibition of endogenous leptin levels using siRNA for leptin or inhibiting leptin signaling using siRNA for OB-Rb expression both reduced ALP and OC about 60%. Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP.

Conclusions: These results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-beta1 observed in these cells compared to normal. Leptin also stimulated cell proliferation, and Erk 1/2 and p38 signaling. Taken together, these data suggest leptin could contribute to abnormal osteoblast function in OA.

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Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling. OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C) Leptin expression in response to siRNA. D) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.
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Figure 5: Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling. OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C) Leptin expression in response to siRNA. D) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.

Mentions: Last, using siRNA techniques, we next evaluated if inhibiting leptin or OB-Rb would abrogate the response of OA Ob to endogenous leptin production. Indeed, as shown in Figure 5A, siRNA against leptin reduced alkaline phosphatase activity about 60% compared to a scrambled RNA. A similar observation could be made for osteocalcin (Figure 5B). Likewise, inhibiting OB-Rb expression using siRNA techniques also reduced ALP and OC about 60% in OA Ob (Figure 5A and 5B). Figures 5C and 5D show that specific siRNA inhibition reduced leptin and OB-Rb expression 60 and 55% respectively in these cells compared to a scrambled RNA.


Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression.

Mutabaruka MS, Aoulad Aissa M, Delalandre A, Lavigne M, Lajeunesse D - Arthritis Res. Ther. (2010)

Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling. OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C) Leptin expression in response to siRNA. D) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875652&req=5

Figure 5: Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling. OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C) Leptin expression in response to siRNA. D) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.
Mentions: Last, using siRNA techniques, we next evaluated if inhibiting leptin or OB-Rb would abrogate the response of OA Ob to endogenous leptin production. Indeed, as shown in Figure 5A, siRNA against leptin reduced alkaline phosphatase activity about 60% compared to a scrambled RNA. A similar observation could be made for osteocalcin (Figure 5B). Likewise, inhibiting OB-Rb expression using siRNA techniques also reduced ALP and OC about 60% in OA Ob (Figure 5A and 5B). Figures 5C and 5D show that specific siRNA inhibition reduced leptin and OB-Rb expression 60 and 55% respectively in these cells compared to a scrambled RNA.

Bottom Line: Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect.Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP.These results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-beta1 observed in these cells compared to normal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de recherche en Arthose, Centre de recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Hôpital Notre-Dame, 1560 rue Sherbrooke Est, Montréal, QC H2L 4 M1, Canada. ms.mutabaruka@umontreal.ca

ABSTRACT

Introduction: Leptin is a peptide hormone with a role in bone metabolism and rheumatic diseases. The subchondral bone tissue plays a prominent role in the pathophysiology of osteoarthritis (OA), related to abnormal osteoblast (Ob) differentiation. Although leptin promotes the differentiation of Ob under normal conditions, a role for leptin in OA Ob has not been demonstrated. Here we determined if endogenous leptin produced by OA Ob could be responsible for the expression of the abnormal phenotypic biomarkers observed in OA Ob.

Methods: We prepared primary normal and OA Ob from subchondral bone of tibial plateaus removed for knee surgery of OA patients or at autopsy. We determined the production of leptin and of the long, biologically active, leptin receptors (OB-Rb) using reverse transcriptase-polymerase chain reaction, ELISA and Western blot analysis. We determined the effect of leptin on cell proliferation by BrdU incorporation and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and we determined by Western blot analysis phospho 42/44 MAPK (p42/44 Erk1/2) and phospho p38 levels. We then determined the effect of the addition of exogenous leptin, leptin receptor antagonists, inhibitors of leptin signaling or siRNA techniques on the phenotypic features of OA Ob. Phenotypic features of Ob were determined by measuring alkaline phosphatase activity (ALP), osteocalcin release (OC), collagen type 1 production (CICP) and of Transforming Growth Factor-beta1 (TGF-beta1).

Results: Leptin expression was increased approximately five-fold and protein levels approximately two-fold in OA Ob compared to normal. Leptin stimulated its own expression and the expression of OB-Rb in OA Ob. Leptin dose-dependently stimulated cell proliferation of OA Ob and also increased phosphorylated p42/44 Erk1/2 and p38 levels. Inactivating antibodies against leptin reduced ALP, OC, CICP and TGF-beta1 levels in OA Ob. Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect. Inhibition of endogenous leptin levels using siRNA for leptin or inhibiting leptin signaling using siRNA for OB-Rb expression both reduced ALP and OC about 60%. Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP.

Conclusions: These results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-beta1 observed in these cells compared to normal. Leptin also stimulated cell proliferation, and Erk 1/2 and p38 signaling. Taken together, these data suggest leptin could contribute to abnormal osteoblast function in OA.

Show MeSH
Related in: MedlinePlus