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: a–j: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced apoptosis and cellular degeneration in chondrocytes.

<p>Transmission electron microscopy was performed to study the effects of IGF-1 or/and PDGF-bb on IL-1β-stimulated primary chondrocytes. Untreated control cultures consist of vital, active cells containing a well-developed rough endoplasmic reticulum, mitochondria and a well-organized cytoplasm after 24 or 48 h in culture (a and b). In contrast, stimulation with IL-1β for 24 h led to degenerative changes including condensation of heterochromatin, swelling of the rough endoplasmic reticulum and mitochondria (c). Longer exposure (48 h) to IL-1β resulted in more severe degenerative features including formation of apoptotic bodies and cell lysis (d). Pre-treatment of IL-1β-stimulated chondrocytes with IGF-1 or/and PDGF-bb for 24 or 48 h inhibited the degenerative effects of IL-1β (e–j). After 48 h (f, h and j), chondrocytes exhibited as large, viable and flattened cells with numerous tiny cytoplasmic processes, mitochondria, rough endoplasmic reticulum and other cytoplasmic organelles compared to control chondrocytes. <b>B: a–e: Redifferentiation of IL-1β-treated chondrocytes in high-density culture by IGF-1 or/and PDGF-bb.</b> Primary chondrocytes were either left untreated (a) or were treated with 10 ng/ml IL-1β (b), pre-treated with 10 ng/ml IGF-1 (c), 10 ng/ml PDGF-bb (d) or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1 (e) for 12 h and then stimulated with IL-1β for another 24 h. The cells were transferred to high-density culture for seven days. Ultrastructural morphology was evaluated by electron microscopy. Control cultures showed characteristic features, including chondrocytes (c) embedded in a well-developed ECM (m) (<b>a</b>). Treatment with IL-1β for 24 h led to matrix breakdown and cell lysis (<b>b</b>). Pre-treatment with IGF-1 (<b>c</b>), PDGF-bb (<b>d</b>) or both growth factors in combination (<b>e</b>) resulted in a marked improvement of chondrocyte phenotype and the formation of cartilage nodules. The formation of a dense ECM (m) surrounding well-developed chondrocytes (c) was observed. ×5000; Bars: 1 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–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

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

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–D: Effects of PP1 and IGF-1 or/and PDGF-bb on IL-1β-induced c-Src phosphorylation 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, or with PP1 (10 µM) for 1 h and treated with 10 ng/ml IL-1β for the indicated times. The cell lysates were subjected to 10% SDS-PAGE (500 ng protein per lane), transferred to nitrocellulose membranes and then probed using anti-phospho-Src and anti-β-actin (as an indicator of protein loading in each lane) antibodies. Identical results were obtained in three independent experiments.</p

A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced Akt 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 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 immunoprecipitated with anti-IKK-α antibody followed by western blot analysis using anti-Akt, anti-phospho-specific Akt and anti-IKK-α antibodies. The results shown are representative of three independent experiments.</p

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

<p>Western blot analysis was performed to evaluate the effects of <i>IGF-1 or/and PDGF-bb</i> on IL-1β-induced inhibition of chondrogenic potential in chondrocytes. Whole cell lysates were probed with antibodies to collagen type II, cartilage specific proteoglycan (CSPG) and β1-integrin. Primary chondrocytes were treated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1, or were pre-treated with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1 for 12 h and then stimulated with IL-1β for 24. Untreated chondrocytes had strong production of collagen type II, CSPG and β1-integrin, and stimulation with IL-1β alone markedly reduced these proteins. Pre-treatment of the chondrocytes with IGF-1 or/and PDGF-bb, however inhibited adverse effects of IL-1β. This was confirmed by quantitative densitometry. Expression of the housekeeping gene β-actin remained un-affected.</p