IGF-1 and PDGF-bb suppress IL-1β-induced cartilage degradation through down-regulation of NF-κB signaling: involvement of Src/PI-3K/AKT pathway.

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling. The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects

Resveratrol mediated modulation of Sirt-1/Runx2 promotes osteogenic differentiation of mesenchymal stem cells: potential role of Runx2 deacetylation.

Osteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD(+)-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation. Monolayer and high-density cultures of MSCs and pre-osteoblastic cells were treated with an osteogenic induction medium with/without the Sirt-1 inhibitor nicotinamide or/and resveratrol in a concentration dependent manner. MSCs and pre-osteoblastic cells differentiated to osteoblasts when exposed to osteogenic-induction medium. The osteogenic response was blocked by nicotinamide, resulting in adipogenic differentiation and expression of the adipose transcription regulator PPAR-γ (peroxisome proliferator-activated receptor). However, in nicotinamide-treated cultures, pre-treatment with resveratrol significantly enhanced osteogenesis by increasing expression of Runx2 (bone specific transcription factor) and decreasing expression of PPAR-γ. Activation of Sirt-1 by resveratrol in MSCs increased its binding to PPAR-γ and repressed PPAR-γ activity by involving its cofactor NCoR (nuclear receptor co-repressor). The modulatory effects of resveratrol on nicotinamide-induced expression of PPAR-γ and its cofactor NCoR were found to be mediated, at least in part, by Sirt-1/Runx2 association and deacetylation of Runx2. Finally, knockdown of Sirt-1 by using antisense oligonucleotides downregulated the expression of Sirt-1 protein and abolished the inhibitory effects of resveratrol, namely nicotinamide-induced Sirt-1 suppression and Runx2 acetylation, suggesting that the acetylated content of Runx2 is related to downregulated Sirt-1 expression. These data support a critical role for Runx2 acetylation/deacetylation during osteogenic differentiation in MSCs in vitro. (242 words in abstract)

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

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

Effect of resveratrol on nicotinamide and/or antisense oligonucleotide-induced specific inhibition of Sirt-1 expression, Runx2 acetylation, Runx2 target genes (i.e.osteocalcin) or activation of PPAR-γ during osteoblastic differentiation of MSCs in monolayer culture.

<p>Cells were either untreated or treated with resveratrol (1 µM), nicotinamide (10 mM) or with Sirt-1 <i>antisense</i> (1 µM) or sense oligonucleotides (1 µM) in the presence of lipofectin alone or cells were pre-treated with resveratrol for 4 h followed by co-treatment with Sirt-1 antisense or sense oligonucleotides in the presence of lipofectin for 24 h or/and with nicotinamide over 21 days with osteogenic induction medium in monolayer cultures. (A) Whole cell lysates (500 ng/lane) were fractionated and subjected to western blotting with antibodies against Sirt-1 and β-actin. Synthesis of the housekeeping protein β-actin was unaffected. (B) Whole-cell extracts were prepared, immunoprecipitated with an anti-Runx2 antibody, and subjected to western blot analysis using an anti–acetyl-lysine antibody. The same blots were re-probed with an antibody to anti-Runx2. Whole cell lysates (500 ng/lane) were fractionated and analyzed by immunoblotting using anti-osteocalcin (C) or anti-PPAR-γ (D) antibodies and β-actin. Synthesis of the housekeeping protein β-actin was unaffected.</p

Effect of resveratrol on nicotinamide-induced inhibition of Sirt-1 expression.

<p><i>A: Sirt-1 protein expression during osteogenesis in monolayer cultures.</i> 21 days monolayer cultures of osteogenic induced fat tissue derived MSCs. Whole cell lysates (500 ng/lane) were probed for Sirt-1. MSCs express high levels of Sirt-1 before and after induction of osteogenic differentiation. Synthesis of the housekeeping protein β-actin was unaffected. Sirt-1 control peptide was used as a control (co pep.). M = Marker for molecular weights. <i>B–C: Effect of resveratrol on NA-induced inhibition of Sirt-1 expression during osteogenesis in monolayer culture.</i> 14 days osteogenic induction culture of control MSCs, cells treated with 0.1, 1, 10 µM resveratrol or with 1, 10, 100 mM nicotinamide or pre-treated with 1 µM resveratrol for 4 h followed by co-treatment with nicotinamide. Whole cell lysates (500 ng/lane) were fractionated and subjected to western blotting with antibodies against Sirt-1. D: <i>Densitometric evaluation was performed for Sirt-1 expression from Fig. B–C.</i> Each experiment was performed in triplicate and mean values and standard deviation are indicated. Values were compared to the control and statistically significant values with <i>p</i><0.05 were designated by an asterisk (*).</p

Transmission electron microscopic (TEM) studies of the effects of resveratrol on osteoblastic differentiation of MSCs in high-density culture.

<p><i>A: a–d: 14 days in high-density culture.</i> Cultures were stimulated with osteogenic induction medium (a) and with various concentrations of resveratrol (0.1 µM (b), 1 µM (c), 10 µM (d)). TEM revealed details of ultrastructural changes that the MSCs underwent while differentiating into osteoblasts (Os). Cells contained high levels of nuclear euchromatin and a large number of sub-cellular organelles (mitochondria, rough ER, Golgi apparatus). Large quantities of thick extracellular matrix fibrils (arrows) were observed in the extracellular space (arrows). However, no significant differences in osteogenesis were observed at the ultrastructural level between resveratrol-treated and control MSC cultures. Magnification: 5000×, bar = 1 µm. <i>A: e–g: 14 days in high-density culture.</i> MSC cultures were treated with osteogenic medium and with the sirtuin inhibitor nicotinamide (1 mM (e), 10 mM (f) and 100 mM (g)). TEM clearly demonstrated that MSCs differentiated into adipocytes (F), exhibiting cytoplasmic lipid droplet accumulation (*) in the presence of osteogenic induction medium. The adipocytes produced high quantities of ECM (arrows) and were embedded in this well organized matrix. Magnification: 5000×, bar = 1 µm. <i>A: h–j: 14 days high-density culture.</i> MSCs were pre-treated with 1 µM resveratrol for 4 h and then co-treated with various concentrations of nicotinamide (1 mM (h), 10 mM (i) and 100 mM (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, favoring osteoblastic differentiation. However, co-treatment with 100 mM nicotinamide resulted in adipogenesis. Magnification: 5000×, bar = 1 µm. B: Adipocyte differentiation in the cultures was estimated by counting 100 cells from 20 different microscopic fields. The number of adipocytes was highest in cultures stimulated with 100 mM nicotinamide alone. However, cells pre-treated with resveratrol and co-treated with nicotinamide at 1 or 10 mM but not at 100 mM nicotinamide significantly decreased the number of adipocytes compared to the chemical by itself (*).</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