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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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Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin. OA osteoblasts were plated at 10,000 cells/cm2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A) Incorporation of BrdU by OA Ob in response to leptin; B) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. Values are the mean ± SEM of at least four separate experiments; *P < 0.05, **P < 0.01.
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Figure 3: Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin. OA osteoblasts were plated at 10,000 cells/cm2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A) Incorporation of BrdU by OA Ob in response to leptin; B) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. Values are the mean ± SEM of at least four separate experiments; *P < 0.05, **P < 0.01.

Mentions: Since OA Ob expressed both leptin and leptin receptors, we tested if these cells could respond to exogenous leptin and we first determined the effect of leptin on cell proliferation. Figure 3A and 3B show that leptin dose-dependently (1 ng/ml to 10 μg/ml) stimulated cell proliferation and this effect plateaued at 100 ng/ml leptin when assessing proliferation using BrdU incorporation or MTT assay respectively. We next evaluated if the effect of leptin on cell proliferation was via the Erk 1/2 MAPK pathway as we previously showed with insulin-like growth factor 1 [45]. Indeed, in response to exogenous leptin, phospho p42/44 MAPK levels rose (Figure 3C). This effect was again dose-dependent and also plateaued around 100 ng/ml (Figure 3D). In addition, we evaluated the role of leptin on the p38 pathway. Again, leptin dose-dependently stimulated phospho p38 levels (Figure 3E) and this effect was significant at doses as low as 1 μg/ml (Figure 3F).


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)

Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin. OA osteoblasts were plated at 10,000 cells/cm2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A) Incorporation of BrdU by OA Ob in response to leptin; B) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. Values are the mean ± SEM of at least four separate experiments; *P < 0.05, **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin. OA osteoblasts were plated at 10,000 cells/cm2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A) Incorporation of BrdU by OA Ob in response to leptin; B) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program [46]. Values are the mean ± SEM of at least four separate experiments; *P < 0.05, **P < 0.01.
Mentions: Since OA Ob expressed both leptin and leptin receptors, we tested if these cells could respond to exogenous leptin and we first determined the effect of leptin on cell proliferation. Figure 3A and 3B show that leptin dose-dependently (1 ng/ml to 10 μg/ml) stimulated cell proliferation and this effect plateaued at 100 ng/ml leptin when assessing proliferation using BrdU incorporation or MTT assay respectively. We next evaluated if the effect of leptin on cell proliferation was via the Erk 1/2 MAPK pathway as we previously showed with insulin-like growth factor 1 [45]. Indeed, in response to exogenous leptin, phospho p42/44 MAPK levels rose (Figure 3C). This effect was again dose-dependent and also plateaued around 100 ng/ml (Figure 3D). In addition, we evaluated the role of leptin on the p38 pathway. Again, leptin dose-dependently stimulated phospho p38 levels (Figure 3E) and this effect was significant at doses as low as 1 μg/ml (Figure 3F).

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