Resveratrol modulates interleukin-1β-induced phosphatidylinositol 3-kinase and nuclear factor κB signaling pathways in human tenocytes

Abstract

Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1β-mediated inflammatory signaling. Resveratrol suppressed IL-1β-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1β-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1β-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1β-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB.

FIGURE 1.

Effect of resveratrol on IL-1β-induced mitochondrial changes and apoptosis in human tenocytes in monolayer cultures. A, shown is a morphological evaluation of human tenocytes in monolayer culture by transmission electron microscopy. Tenocytes were either left untreated (a–c) or were treated with IL-1β (10 ng/ml) for 24, 48, and 72 h (d–f) or pretreated with resveratrol (5 μm) for 4 h and then stimulated with IL-1β (10 ng/ml) for 24, 48, and 72 h (g–i). B, to quantify apoptosis and MC in these cell cultures, 100 cells from 20 microscopic fields were counted. The examination was performed in triplicate, and the results are provided as the mean values with S.D. from three independent experiments. ×5000; bar = 1μm. Values were compared with the control, and statistically significant values with p < 0.05 were designated by an asterisk (*).

FIGURE 2.

Effects of resveratrol and IL-1β on human tenocytes in high density cultures. Human tenocytes either served as a control (A) or were stimulated with IL-1β (10 ng/ml) alone (B) or pretreated with resveratrol (5 μm) for 4 h and then exposed to IL-1β (C) for 10 days in high density cultures. Morphological evaluation was performed by transmission electron microscopy. ×4000; bars, 1μm.

FIGURE 3.

Effects of resveratrol on IL-1β-induced NF-κB and PI3K activation in human tenocytes. Human tenocytes in monolayer cultures were pretreated with different concentrations of resveratrol (0, 0.1, 0.5, 1, 5, 10, and 20 μm) for 4 h and then treated with 10 ng/ml IL-1β for 30 min (A) or treated with 5 μm resveratrol for different times (0, 1, 12, 24, 48, and 72 h) and then stimulated with 10 ng/ml IL-1β for 30 min (B). In a different approach, untreated cells and cells pretreated with resveratrol (5 μm for 4 h) were exposed to various concentrations of IL-1β (0, 0.1, 1, 5, and 10 ng/ml) for 30 min (C) or were incubated with 10 ng/ml IL-1β for different times (0, 5, 10, 20, 40, and 60 min) (D). After completion of the experiments, whole cell lysates were prepared and analyzed by Western blotting with antibodies against phosphospecific-p65 and PI3K. Housekeeping protein β-actin served as a loading control in all experiments. Densitometric analyses were performed by setting control cultures as 1-fold.

FIGURE 4.

Effects of resveratrol on IL-1β-induced matrix degradation, proinflammatory and apoptotic signaling, and down-regulation of tenocyte-specific transcription factor SCXA in human tenocytes. A, monolayer cultures of human tenocytes were either left untreated or were treated with 10 ng/ ml IL-1β or 5 μm resveratrol for 24, 48, and 72 h or were pretreated with 5 μm resveratrol for 4 h and then co-treated with 10 ng/ ml IL-1β and resveratrol for 24, 48, and 72 h. Whole cell lysates were subjected to Western blotting with antibodies against collagen (Coll) types I and III, tenomodulin (Tnmd), and the tendon-specific transcription factor SCXA. B, the same tenocyte cultures as in A were subsequently also probed for expression of Cox-2, MMP-1, MMP-9, MMP-13, cleavage of PARP, and caspase-3. Housekeeping protein β-actin served as a loading control.

FIGURE 5.

Effects of resveratrol on IL-1β-induced Src and PI3K/Akt activation in human tenocytes. Serum-starved human tenocytes in monolayer cultures were either treated with 10 ng/ ml IL-1β or 5 μm resveratrol for 24, 48, and 72 h or pretreated with 5 μm resveratrol for 4 h and then co-treated with 10 ng/ml IL-1β and resveratrol for 24, 48, and 72 h or were left untreated. Whole cell lysates were prepared, and samples were examined by Western blot analysis with antibodies against PI3K, phosphorylated c-Src, and phosphorylated Akt. Housekeeping protein β-actin served as a loading control.

FIGURE 6.

Effects of resveratrol and c-Src, PI3K, and Akt inhibitors on IL-1β-induced phosphorylation of NF-κB in human tenocytes. Monolayer cultured serum-starved human tenocytes were either left untreated or treated with 10 ng/ml IL-1β alone for 1 h or pretreated with different concentrations of resveratrol (0.1, 1, 5, 10 μm) for 4 h or inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin (W), 10 nm), or Akt (SH-5, 1 μm) for 1 h and then co-treated with 10 ng/ml IL-1β for 1 h. Protein extracts from whole cell lysis were then probed by Western blotting with antibodies against NF-κB subunit p65 and phosphospecific-p65 as well as housekeeping protein β-actin.

FIGURE 7.

Effects of resveratrol and c-Src, PI3K, and Akt inhibitors on IL-1β-induced NF-κB-dependent proinflammatory, matrix-degrading, and apoptotic gene products in human tenocytes. Human tenocytes in monolayer cultures were incubated with serum-starved medium and were then treated with 10 ng/ml IL-1β alone for 1 h or pretreated with different concentrations of resveratrol (0.1, 1, 5, 10 μm) for 4 h or inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin (W), 10 nm) or Akt (SH-5, 1 μm) for 1 h and then co-treated with 10 ng/ml IL-1β for 1 h or were left untreated. Cells were lysed and subjected to Western blot analysis with antibodies specific for MMP-1, MMP-9, MMP-13, Cox-2, and cleaved caspase-3. Expression of β-actin was examined as a loading control.

FIGURE 8.

Effects of resveratrol and c-Src, PI3K, and Akt inhibitors on IL-1β-induced c-Src, PI3K, and Akt activation in human tenocytes. Monolayer cultured serum-starved human tenocytes were either left untreated or treated with 10 ng/ml IL-1β alone for 1 h or pretreated with different concentrations of resveratrol (0.1, 1, 5, 10 μm) for 4 h or inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin (W), 10 nm) or Akt (SH-5, 1 μm) for 1 h and then co-treated with 10 ng/ml IL-1β for 1 h. Cell lysates were then analyzed by immunoblotting using antibodies raised against c-Src, PI3K, phosphospecific-Akt, and β-actin.

FIGURE 9.

Effects of resveratrol and c-Src, PI3K, and Akt inhibitors on IL-1β-induced Sirt-1 inhibition in human tenocytes. Human tenocytes cultured in monolayer were either left untreated, treated with 5 μm resveratrol or 10 ng/ml IL-1β alone for 1 h, or pretreated with different concentrations of resveratrol (0.1, 1, 5, 10 μm) for 4 h or inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin (W), 10 nm), or Akt (SH-5, 1 μm) for 1 h and then co-treated with 10 ng/ml IL-1β for 1 h. Whole cell lysates were prepared and analyzed by Western blotting using antibodies against Sirt-1 and β-actin. Sirt-1 control peptide (Co Pep.) was used as a control for antibody specificity.

FIGURE 10.

Effects of resveratrol and c-Src, PI3K, Akt inhibitors on IL-1β-induced NF-κB p65 activation and nuclear translocation in human tenocytes. Serum-starved human tenocytes cultured in monolayer were either left untreated or treated with 10 ng/ml IL-1β alone for 1 h or prestimulated with 5 μm resveratrol or with inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin (W), 10 nm), and Akt (SH-5, 1 μm) for 5, 10, 20, 40, or 60 min and then co-treated with 10 ng/ml IL-1β for 1 h. Nuclear and cytoplasmic cell fractions were prepared and submitted to Western blot analysis using antibodies against p65, phosphospecific-p65, and β-actin or nuclear protein poly(ADP-ribose) polymerase (PARP) as a loading control.

FIGURE 11.

Effects of resveratrol and c-Src, PI3K, and Akt inhibitors on IL-1β-induced activation of NF-κB-regulated proinflammatory and apoptotic gene products in human tenocytes. Serum-starved human tenocytes in monolayer culture were either left untreated or treated with 10 ng/ml IL-1β alone for 1 h or prestimulated with 5 μm resveratrol or with inhibitors of c-Src (PP1, 1 μm), PI3K (wortmannin, 10 nm), and Akt (SH-5, 1 μm) for 5, 10, 20, 40, or 60 min and then co-treated with 10 ng/ml IL-1β for 1 h. Cytoplasmic cell fractions were prepared, and expression of MMP-9 and Cox-2 as well as cleavage of caspase-3 was determined by Western blot analysis. Housekeeping protein β-actin was used as a loading control.

FIGURE 12.

Effects of resveratrol and PI3K inhibitor wortmannin on IL-1β-induced activation of IKK and Akt in human tenocytes. Serum-starved human tenocytes in monolayer culture were either treated with IL-1β alone for 0, 5, 10, 20, 40, or 60 min or were pretreated with resveratrol (5 μm) or wortmannin (10 nm) for 1 h and then co-treated with IL-1β for 0, 5, 10, 20, 40, or 60 min. A, to determine the activation level of IKK, whole cell lysates were immunoprecipitated with an antibody against IKK and underwent immune complex kinase assay as described under “Experimental Procedures.” Extracts were then fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α, anti-IKK-β, and anti-phosphospecific-IκBα antibodies. B, cell lysates from the same cells were also examined for phosphorylation of Akt by immunoblotting with antibodies against Akt and phosphospecific-Akt. C, shown is the direct effect of resveratrol treatment on IL-1β-induced IκB kinase activation. Serum-starved human tenocytes were treated with 10 ng/ml IL-1β. The cell extracts were prepared and immunoprecipitated with anti-IKK-α antibodies. The immunocomplex kinase assay was performed in the absence or presence of resveratrol at the indicated concentrations. Data shown are representative of three independent experiments.

FIGURE 13.

Effects of resveratrol on IL-1β-induced acetylation of NF-κB and PI3K in human tenocytes. Human tenocytes in monolayer culture were either treated with IL-1β alone for 0, 5, 10, 20, 40, or 60 min or were pretreated with resveratrol (5 μm) for 1 h and then co-treated with IL-1β for 0, 5, 10, 20, 40, or 60 min. After completion of the experiments, cell extracts were immunoprecipitated (IP) with anti-p65 antibody (A) or anti-PI3K antibody (B) and then analyzed by Western blotting (IB) using antibodies against acetyl-lysine, p65, and PI3K.

FIGURE 14.

Effects of resveratrol and PI3K-inhibitor wortmannin on IL-1β-induced suppression of Sirt-1/SCXA interaction in human tenocytes. Serum-starved human tenocytes in monolayer culture were either treated with IL-1β alone for 0, 5, 10, 20, 40, or 60 min or pretreated with resveratrol (5 μm) or wortmannin (10 nm) for 1 h and then co-treated with IL-1β for 0, 5, 10, 20, 40, or 60 min. Whole cell lysates were immunoprecipitated with an antibody against Sirt-1 and then immunoblotted with an antibody against SCXA or vice versa. The same immunoprecipitates were then reblotted with SCXA or Sirt-1 antibody. Sirt-1 control peptide (Co Pep) was used as a control for antibody specificity.

FIGURE 15.

Schematic diagram showing IL-1β-induced proinflammatory and apoptotic signaling pathways modulated by resveratrol.