18 research outputs found

    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.

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    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

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

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    <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-induced inhibition of Sirt-1 expression.

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    <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.

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    <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

    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.

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    <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