The cell signaling pathway for osteogenic/adipogenic differentiation in MSCs by resveratrol.

<p>The cell signaling pathway for osteogenic/adipogenic differentiation in MSCs by resveratrol.</p

Effect of resveratrol and nicotinamide on association of Sirt-1 proteins with PPAR-γ and NCoR in MSC high-density cultures.

<p>Cultures were treated with 0, 1, 10, 100 mM nicotinamide or pre-treated with 1 µM resveratrol for 4 h followed by co-treatment with nicotinamide over 14 days with osteogenic induction medium. Cultures were lysed and immunoprecipitated with anti-PPAR-γ (a), or anti-Sirt-1 (b, c). The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting using anti-NCoR (a, b) and anti- PPAR-γ (c). The same blots were re-probed with an antibody to anti-PPAR-γ (a), anti-Sirt-1 (b, c). Results shown are representative of three independent experiments.</p

Light microscopic evaluation of the effects of resveratrol and/or nicotinamide on osteoblastic differentiation of MSCs in monolayer culture.

<p><i>a–d: Light microscopic demonstration of osteoid-tissue formation with von Kossa staining (A) or adipose-tissue formation with Oil Red O staining (B).</i> 21 days in monolayer culture. In cultures were stimulated with osteogenic induction medium (A,B: a) and with various concentrations of resveratrol (0.1 µM (A,B: b), 1 µM (A,B: c), 10 µM (A,B: d)calcium deposition was observed (A: a–d), but adipogenesis was negative (B: a–d). Light microscopy demonstrated that MSC cultures treated with osteogenic medium and with the sirtuin inhibitor nicotinamide (1 mM (A, B: e), 10 mM (A,B: f) and 100 mM (A,B: g)), did not differentiate to osteoblastic cells (A, e–g), but differentiated into adipocytes (B, e–g), exhibiting cytoplasmic lipid droplet accumulation in the presence of osteogenic induction medium. In another approach, MSCs were pre-treated with 1 µM resveratrol and then co-treated with various concentrations of nicotinamide (1 mM (A,B: h), 10 mM (A,B: i) and 100 mM (A,B: j)) in osteogenic medium. Pre-treatment of MSCs with 1 µM resveratrol and co-treatment with 1 and 10 mM nicotinamide inhibited adipogenic differentiation of MSCs (B: h–i), favoring osteoblastic differentiation (A: h–i). However, co-treatment with 100 mM nicotinamide resulted in adipogenesis (B: j), but not in osteogenesis (A: j). Magnification: ×200, <i>bar</i> 30 µm.</p

Effects of IGF-1 or/and PDGF-bb on IL-1β-induced inhibition of signaling proteins expression in chondrocytes.

<p>To evaluate the effects of IGF-1 and/or PDGF-bb on IL-1β-induced inhibition of MAPK signaling proteins in chondrocytes, whole cell lysates were probed with antibodies to Shc, Erk1/2 and SOX-9. Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factor (5 ng/ml each) or pre-treated for 12 h with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both (5 ng/ml each) followed by 10 ng/ml IL-1β for 24. Untreated cultures showed high expression of Shc, Erk1/2, and SOX-9, while IL-1β alone resulted in inhibition of Erk1/2, Shc as well as SOX-9 production. However, pre-treatment of cultures with IGF-1 or/and PDGF-bb inhibited the adverse effects of IL-1β and chondrocytes produced large amounts of Shc, Erk1/2 and SOX-9 at levels similar to control cultures. The results were confirmed by quantitative densitometry.</p

Effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB-dependent pro-inflammatory, pro-apoptotic and matrix degrading gene products in chondrocytes.

<p>To determine whether <i>IGF-1</i> or/and <i>PDGF-bb</i>exert effects on IL-1β-induced NF-κB-dependent expression of pro-inflammatory, pro-apoptotic and matrix degrading gene products, primary chondrocytes were either stimulated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) or pre-stimulated for 12 h with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) followed by 10 ng/ml IL-1β for 24. Equal amounts of total proteins were separated by SDS-PAGE and analyzed by immunoblotting using antibodies raised against COX-2, MMP-9 and MMP-13 and active caspase-3. Stimulation with IL-1β resulted in production of COX-2, MMP-9, MMP-13 and caspase-3 cleavage. Pre-treatment with a combination of both IGF-1 or/and PDGF-bb downregulated COX-2, MMP-9, MMP-13 and cleaved caspase-3.</p

A–D: Effects of IGF-1 or/and PDGF-bb or IKK-inhibitor (BMS) on the IL-1β-induced IKK activation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb, a combination of both growth factors (5 ng/ml each) or 5 µM BMS-345541 (BMS) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IκB kinase (IKK)and then analyzed by an immune complex kinase assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028663#s2" target="_blank">Materials and Methods</a>. To examine the effect of <i>IGF-1 or/and PDGF-bb or</i> BMS on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS–PAGE and examined by western blot analysis using anti-IKK-α and anti-IKK-β antibodies. The results shown are representative of three independent experiments.</p

A–B: Effects of Src-, PI-3K-, AKT-inhibitors and IGF-1 or/and PDGF-bb on IL-1β-stimulated phosphorylation of NF-κB in chondrocytes.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with PP1 (10 µM), wortmannin (20 nM) and SH-5 (10 µM) for 1 h (A), or with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h (B) and then incubated with IL-1β for 30 min. Nuclear extracts were subjected to 10% SDS-PAGE (500 ng protein per lane), transferred to nitrocellulose membranes and then probed using an antiserum reactive with an anti-phospho-p65 or anti-PARP polyclonal antibody (housekeeping control). Similar results were obtained in three independent experiments.</p

Specific antisense oligonucleotides against Sirt-1 in MSCs lead to decreased Sirt-1 expression in monolayer culture as revealed by immunofluorescence microscopy.

<p>A: Mesenchymal stem cells either served as controls (not treated, without primary antibody, a–b; not treated, with primary antibody, c–d) or were transfected with sense (e–f) or antisense (g–h) with 1 µM in the presence of lipofectin for 24 h and resistant colonies were collected. The collected MSCs were subjected to immunolabeling with Sirt-1 antibodies and rhodamine-coupled secondary antibodies. Counterstaining was performed with DAPI to visualize the cell nuclei. It shows that Sirt-1 antisense but not control sense knocks down Sirt-1 protein levels in the nuclei of cells as visualized using an epifluorescence microscope. Images shown are representative of three independent experiments. ×160. <i>B:</i> cell extracts generated from the cells in <i>A</i> were used for immunoblotting assay with Sirt-1 or β-actin (loading control) antibodies. The immunoblot shown is representative of three independent experiments.</p

A–C: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced p65 phosphorylation and nuclear translocation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Nuclear extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS–PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-p65, anti-p65 antibodies and anti-PARP (control). The results shown are representative of three independent experiments.</p

A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced p65 acetylation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were prepared and immunoprecipitated with an anti-p65 antibody. Western blot analysis was then performed with an anti-acetyl-lysine antibody or with an anti-p65 antibody. The results shown are representative of three independent experiments.</p