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

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

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

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: a–e: Redifferentiation of IL-1β-treated chondrocytes in high-density culture by IGF-1 or/and PDGF-bb. 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) (a). Treatment with IL-1β for 24 h led to matrix breakdown and cell lysis (b). Pre-treatment with IGF-1 (c), PDGF-bb (d) or both growth factors in combination (e) 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.

Figure 2. Effects of IGF-1 or/and PDGF-bb on IL-1β-induced inhibition of cartilage ECM expression in chondrocytes.

Western blot analysis was performed to evaluate the effects of IGF-1 or/and PDGF-bb 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.

Figure 3. Effects of IGF-1 or/and PDGF-bb on IL-1β-induced inhibition of signaling proteins expression in chondrocytes.

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.

Figure 4. 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.

To determine whether IGF-1 or/and PDGF-bbexert 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.

Figure 5. Inhibition of IL-1β-induced NF-κB activation and nuclear translocation by IGF-1 or/and PDGF-bb in primary chondrocytes.

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 a combination of both growth factors (5 ng/ml each) for the indicated times and then treated with 10 ng/ml IL-1β for 30 min. Nuclear extracts were prepared and assayed for NF-κB activation by western blot analysis. The results shown are representative of three independent experiments.

Figure 6. IKK inhibitor suppresses IL-1β-induced inhibition of Shc, Erk 1/2, SOX-9 and stimulation of caspase-3 cleavage in chondrocytes in monolayer culture.

Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, 5 µM BMS-345541 (BMS) or were pre-stimulated for 12 h with 5 µM BMS followed by 10 ng/ml IL-1β for 24. Whole cell lysates were probed with antibodies to Shc, Erk1/2, SOX-9 and active caspase-3. Untreated cultures showed high expression of Shc, Erk1/2 SOX-9 and no cleavage of caspase-3, while incubation with IL-1β alone resulted in suppression of Shc, Erk1/2 as well as SOX-9 production and stimulation of caspase-3 cleavage. However, pre-treatment of cultures with BMS inhibited the adverse effects of IL-1β and chondrocytes produced large amounts of Shc, Erk1/2 as well as SOX-9 proteins and the production of active caspase-3 was inhibited.

Figure 7. A–C: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced IκB-α phosphorylation and degradation and p65 phosphorylation in chondrocytes in monolayer cultures.

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 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. Cytoplasmic 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-IκB-α, anti-IκB-α, anti-phospho-specific p65, anti-p65 antibodies and anti-β-actin (control). The results shown are representative of three independent experiments.

Figure 8. A–C: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced p65 phosphorylation and nuclear translocation in chondrocytes in monolayer cultures.

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.

Figure 9. A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced p65 acetylation in chondrocytes in monolayer cultures.

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.

Figure 10. 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.

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 Materials and Methods. To examine the effect of IGF-1 or/and PDGF-bb or 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.

Figure 11. A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced Akt activation in chondrocytes in monolayer cultures.

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.

Figure 12. A–D: Effects of PP1 and IGF-1 or/and PDGF-bb on IL-1β-induced c-Src phosphorylation in chondrocytes in monolayer cultures.

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.

Figure 13. 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.

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.