Curcumin modulates nuclear factor kappaB (NF-kappaB)-mediated inflammation in human tenocytes in vitro: role of the phosphatidylinositol 3-kinase/Akt pathway

Abstract

Inflammatory processes play essential roles in the pathogenesis of tendinitis and tendinopathy. These events are accompanied by catabolic processes initiated by pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Pharmacological treatments for tendinitis are restricted to the use of non-steroidal anti-inflammatory drugs. Recent studies in various cell models have demonstrated that curcumin targets the NF-κB signaling pathway. However, its potential for the treatment of tendinitis has not been explored. Herein, we used an in vitro model of human tenocytes to study the mechanism of curcumin action on IL-1β-mediated inflammatory signaling. Curcumin at concentrations of 5-20 μm inhibited IL-1β-induced inflammation and apoptosis in cultures of human tenocytes. The anti-inflammatory effects of curcumin included down-regulation of gene products that mediate matrix degradation (matrix metalloproteinase-1, -9, and -13), prostanoid production (cyclooxygenase-2), apoptosis (Bax and activated caspase-3), and stimulation of cell survival (Bcl-2), all known to be regulated by NF-κB. Furthermore, curcumin suppressed IL-1β-induced NF-κB activation via inhibition of phosphorylation and degradation of inhibitor of κBα, inhibition of inhibitor of κB-kinase activity, and inhibition of nuclear translocation of NF-κB. Furthermore, the effects of IL-1β were abrogated by wortmannin, suggesting a role for the phosphatidylinositol 3-kinase (PI-3K) pathway in IL-1β signaling. Curcumin suppressed IL-1β-induced PI-3K p85/Akt activation and its association with IKK. These results demonstrate, for the first time, a potential role for curcumin in treating tendon inflammation through modulation of NF-κB signaling, which involves PI-3K/Akt and the tendon-specific transcription factor scleraxis in tenocytes.

Figures

FIGURE 1.

Effect of curcumin on IL-1β-induced cellular degeneration and apoptosis in tenocytes. A, electron microscopy was performed to demonstrate the effects of curcumin on IL-1β-stimulated tenocytes in monolayer culture. Untreated control tenocytes containing mitochondria, rough endoplasmic reticulum, and many other cell organelles are in panel a. In contrast, stimulation of tenocytes with 10 ng/ml of IL-1β for 12, 24, and 48 h resulted in degenerative changes of the cells. After 12 h, tenocytes became rounded and the nucleus contained more condensed chromatin (b). After 24 h multiple vacuoles, swelling of rough endoplasmic reticulum, and clustering of swollen mitochondria was visible (c). Longer incubations of 48 h led to the formation of apoptotic bodies and cell lysis (d). However, pre-treatment of IL-1β-stimulated tenocytes with curcumin inhibited the adverse effects of IL-1β (e–g) and after 48 h treatment (g) tenocytes demonstrated large, flattened cells with numerous microvilli-like processes and mitochondria were comparable with control cultures. Bar, 1 μm. B, to quantify cellular degradation and apoptosis in these cultures, 100 cells from 20 microscopic fields were counted. The number of degraded and apoptotic cells was highest in cultures stimulated with IL-1β alone. In contrast to this, pre-treatment of IL-1β-stimulated cultures with curcumin inhibited the cellular degradation and apoptotic effects of IL-1β and the number of degraded and apoptotic cells remained significantly lower over the entire culture period (*).

FIGURE 2.

Effects of curcumin on IL-1β-induced inhibition of extracellular matrix compounds and signaling proteins expression in tenocytes. To evaluate the effects of curcumin on IL-1β-stimulated tenogenic inhibition in tenocytes, whole cell lysates (500 ng of protein/lane) were probed with antibodies to collagen type I (CI), collagen type III (CIII), decorin, tenomodulin (Tnmd), and the tenogenic specific transcription factor SCXA. Serum-starved human tenocytes (1 × 106 cells/ml) were exposed to 10 ng/ml of IL-1β alone, 5 μm curcumin alone, or were pre-treated with 5 μm curcumin for 4 h and followed either by incubation with IL-1β alone or incubation with IL-1β and curcumin, or left untreated for 24 h. Each experiment was performed in triplicate. Expression of the β-actin housekeeping gene was not affected by IL-1β and/or curcumin.

FIGURE 3.

Effects of curcumin on IL-1β-induced NF-κB-dependent pro-inflammatory, anti-apoptotic, and pro-apoptotic gene products in tenocytes. A, to evaluate whether curcumin exerts effects on IL-1β-induced NF-κB-dependent expression of pro-inflammatory (COX-2) and matrix-degrading (MMP-1, -9, and -13) gene products, serum-starved tenocytes (1 × 106 cells/ml) were exposed to 10 ng/ml of IL-1β alone, 5 μm curcumin alone, or were pre-treated with 5 μm curcumin for 4 h and followed either by incubation with IL-1β alone or incubation with IL-1β and curcumin, or left untreated for 24 h. Each experiment was performed in triplicate. Expression of the β-actin housekeeping gene was not affected by IL-1β and/or curcumin. B, to determine whether curcumin treatment actively stimulates the production of anti-apoptotic gene products (Bcl-2, TRAF1, and Bcl-xL), serum-starved human tenocytes (1 × 106 cells/ml) were exposed to 10 ng/ml of IL-1β alone, 5 μm curcumin alone, or were pre-treated with 5 μm curcumin for 4 h and followed either by incubation with IL-1β alone or incubation with IL-1β and curcumin, or left untreated for 24 h. Synthesis of the β-actin housekeeping gene remained unaffected. C, whole cell lysates of serum-starved human tenocytes (1 × 106 cells/ml) were exposed to 10 ng/ml of IL-1β alone, 5 μm curcumin alone, or were pre-treated with 5 μm curcumin for 4 h and followed either by incubation with IL-1β alone or incubation with IL-1β and curcumin, or left untreated and evaluated with Western blot analysis to examine the effect on the pro-apoptotic proteins Bax and caspase-3. Expression of the housekeeping protein β-actin remained unaffected.

FIGURE 4.

Curcumin suppression of IL-1β-induced NF-κB activation. A, serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 10 ng/ml of IL-1β for the indicated times or preincubated with 5 μm curcumin for 0, 5, 10, 20, 40, and 60 min, co-treated with 10 ng/ml of IL-1β for 30 min, and then probed for phospho-p65 by Western blot analysis using antibodies to phospho-specific p65 and poly(ADP-ribose) polymerase (PARP) (control). B, serum-starved human tenocytes were either incubated with curcumin at various concentrations (0, 1, 2, 5, 10, and 15 μm) for 4 h or preincubated with curcumin at various concentrations for 4 h followed by 10 ng/ml of IL-1β stimulation for 30 min. The nuclear extracts (500 ng of protein/lane) were probed for phospho-p65 by Western blot analysis using antibodies to phospho-specific p65 and PARP (control). Expression of PARP remained unaffected in the nuclear extracts. The results shown are representative of three independent experiments.

FIGURE 5.

Effect of wortmannin on IL-1β-induced NF-κB activation. A, serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 20 nm wortmannin for 0, 5, 10, 20, 40, and 60 min or preincubated with 20 nm wortmannin for 0, 5, 10, 20, 40, and 60 min, co-treated with 10 ng/ml of IL-1β for 30 min, and then probed for phospho-p65 by Western blotting using antibodies to phospho-specific p65 and poly(ADP-ribose) polymerase (PARP) (control). B, serum-starved tenocytes were preincubated with wortmannin at various concentrations (0, 1, 10, 20, 30, and 40 nm) for 1 h followed by 10 ng/ml of IL-1β stimulation for 30 min. Nuclear extracts (500 ng of protein/lane) were probed for phospho-p65 by Western blotting using antibodies to phospho-specific p65 and PARP (control). Expression of PARP remained unaffected in nuclear extracts. The results shown are representative of three independent experiments.

FIGURE 6.

Effect of curcumin on the IL-1β-induced phosphorylation and degradation of IκBα and the phosphorylation and translocation of p65. Serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 10 ng/ml of IL-1β for the indicated times or pre-treated with 5 μm curcumin for 4 h, followed by 10 ng/ml of IL-1β stimulation for the indicated times. Cytoplasmic (A) and nuclear extracts (B) were prepared, fractionated with SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκBα, anti-phosphospecific IκBα, anti-p65, and anti-phosphospecific p65 antibodies. The results shown are representative of three independent experiments. PARP, poly(ADP-ribose) polymerase.

FIGURE 7.

Immunocytochemical analysis of p65 localization after treatment with IL-1β as revealed by immunofluorescence microscopy. Tenocyte cultures either served as controls (not treated) or were treated with IL-1β alone for 10 min or co-treated with 5 μm curcumin and 10 ng/ml of IL-1β for 40 min before immunolabeling with phospho-p65 antibodies and rhodamine-coupled secondary antibodies. Data shown are representative of three independent experiments. Original magnification, ×160.

FIGURE 8.

Effect of curcumin on IL-1β-induced acetylation of p65. Serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 10 ng/ml of IL-1β for the indicated times or pre-treated with 5 μm curcumin for 4 h and then exposed to 10 ng/ml of IL-1β. Whole cell extracts were prepared, immunoprecipitated (IP) with anti-p65 antibody, and subjected to Western blot analysis using anti-acetyl-lysine antibody. The same blots were re-probed with anti-p65 antibody. IB, immunoblot.

FIGURE 9.

A, effects of curcumin treatment on IL-1β-induced IκB kinase activation. Serum-starved primary human tenocytes were either stimulated with 10 ng/ml of IL-1β for the indicated times or pre-treated with 5 μm curcumin for 4 h and then co-treated with IL-1β (10 ng/ml) for the indicated times. Whole cell extracts were immunoprecipitated with an antibody against IκB kinase (IKK)-α and analyzed by an immune complex kinase assay. To examine the effect of curcumin on the expression level of IKK proteins, whole cell extracts (500 ng of protein/lane) were fractionated by SDS-PAGE and examined using Western blot analysis with anti-IKK-α and anti-IKK-β antibodies. Data shown are representative of three independent experiments. B, direct effect of curcumin treatment on IL-1β-induced IκB kinase activation. Serum-starved human tenocytes (1 × 106 cells/ml) were treated with 10 ng/ml of IL-1β. Whole cell extracts were prepared and immunoprecipitated with anti-IKK-α antibody. The immunocomplex kinase assay was performed in the absence or presence of curcumin at the indicated concentrations. Data shown are representative of three independent experiments.

FIGURE 10.

Effect of curcumin on IL-1β-induced Akt phosphorylation. Serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 10 ng/ml of IL-1β for the indicated times or pre-treated with 5 μm curcumin for 4 h and exposed to 10 ng/ml of IL-1β for the indicated times. Whole cell extracts were analyzed by Western blot analysis using anti-phospho-specific Akt (row I). Cell extracts were immunoprecipitated (IP) with anti-IKK-α antibody and the immunoprecipitates were separated (500 ng of protein/lane) by SDS-PAGE and analyzed by immunoblotting (IB) using anti-Akt antibody (row II) or with anti-IKK-α antibody (row III, as a loading control). Results shown are representative of three independent experiments.

FIGURE 11.

Effects of curcumin treatment on IL-1β-induced PI-3K/p85. Serum-starved human tenocytes (1 × 106 cells/ml) were either stimulated with 10 ng/ml of IL-1β for the indicated times or pre-treated with 5 μm curcumin for 4 h and exposed to 10 ng/ml of IL-1β for the indicated times. Whole cell extracts (500 ng of protein/lane) were analyzed by Western blot analysis using anti-PI-3K/p85. Expression of the housekeeping protein β-actin remained unaffected. Results shown are representative of three independent experiments.

FIGURE 12.

Effects of wortmannin on curcumin-mediated inhibition of NF-κB activated by IL-1β. Serum-starved human tenocytes (1 × 106 cells/ml) were pre-treated with different concentrations of wortmannin for 1 h, treated with 5 μm curcumin for 4 h, and then exposed to 10 ng/ml of IL-1β for 30 min. After these treatments, nuclear extracts were prepared and analyzed for NF-κB and poly(ADP-ribose) polymerase (PARP) (control) as described under “Experimental Procedures.”