Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkappaB alpha phosphorylation and degradation - PubMed (original) (raw)

Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkappaB alpha phosphorylation and degradation

P Schwenger et al. Mol Cell Biol. 1998 Jan.

Abstract

Many actions of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) on gene expression are mediated by the transcription factor NF-kappaB. Activation of NF-kappaB by TNF and IL-1 is initiated by the phosphorylation of the inhibitory subunit, IkappaB, which targets IkappaB for degradation and leads to the release of active NF-kappaB. The nonsteroidal anti-inflammatory drug sodium salicylate (NaSal) interferes with TNF-induced NF-kappaB activation by inhibiting phosphorylation and subsequent degradation of the IkappaB alpha protein. Recent evidence indicated that NaSal activates the p38 mitogen-activated protein kinase (MAPK), raising the possibility that inhibition of NF-kappaB activation by NaSal is mediated by p38 MAPK. We now show that inhibition of TNF-induced IkappaB alpha phosphorylation and degradation by NaSal is prevented by treatment of cells with SB203580, a highly specific p38 MAPK inhibitor. Both p38 activation and inhibition of TNF-induced IkappaB alpha degradation were seen after only 30 s to 1 min of NaSal treatment. Induction of p38 MAPK activation and inhibition of TNF-induced IkappaB alpha degradation were demonstrated with pharmacologically achievable doses of NaSal. These findings provide evidence for a role of NaSal-induced p38 MAPK activation in the inhibition of TNF signaling and suggest a possible role for the p38 MAPK in the anti-inflammatory actions of salicylates. In addition, these results implicate the p38 MAPK as a possible negative regulator of TNF signaling that leads to NF-kappaB activation.

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Figures

FIG. 1

FIG. 1

NaSal inhibits TNF-induced IκBα phosphorylation and degradation. (A) COS-1 cells were either treated for 1 h with NaSal (20 mM) or left untreated. They were then either left unstimulated (Ctrl) or stimulated for the indicated times with TNF (20 ng/ml). Lysates were blotted with antibodies against IκBα (top panel) or with antibodies to phosphorylated IκBα (pIκB-α; bottom panel). (B) COS-1 cells were treated as described above, and lysates were blotted with an anti-IκBα antibody.

FIG. 2

FIG. 2

Selective inhibition of TNF-induced IκBα degradation by NaSal. COS-1 cells were preincubated for 1 h in the presence (+) or absence (−) of NaSal (20 mM). They were then left untreated (Ctrl) or treated with TNF (20 ng/ml) or IL-1 (4 ng/ml) for 15 min. In the last two lanes, an initial 15-min TNF treatment was immediately followed by a 15-min IL-1 treatment. Lysates were blotted with anti-IκBα antibody.

FIG. 3

FIG. 3

Inhibition of TNF-induced IκBα phosphorylation and degradation by NaSal is prevented by SB203580. (A) COS-1 and HT-29 cells were preincubated for 1.5 h in the presence (+) or absence (−) of SB203580 (10 μM). The cells were then incubated for 1 h in the presence or absence of NaSal (20 mM) and subsequently incubated for 5 min in the presence or absence of TNF (20 ng/ml). Lysates were blotted with antibodies against phosphorylated IκBα (pIκB-α; top panel) and with antibodies to IκBα (bottom panel). (B) COS-1 and HT-29 cells were treated as described above except that the duration of TNF treatment was 15 min instead of 5 min. Lysates were blotted with anti-IκBα antibody.

FIG. 4

FIG. 4

Kinetics of inhibition of IκBα degradation and of p38 MAPK activation by NaSal. (A) COS-1 cells were either left untreated (−) or treated for the indicated times with NaSal (20 mM) and then stimulated for 15 min with TNF (20 ng/ml). Lysates were blotted with anti-IκBα antibody. (B) COS-1 cells were treated for the indicated times with NaSal (20 mM) alone, lysed, and blotted with anti-phospho-p38 MAPK antibody (pp38; top panel) and with anti-p38 MAPK antibody (p38; bottom panel).

FIG. 5

FIG. 5

Inhibition of IκBα degradation and induction of p38 MAPK activation by different doses of NaSal. (A) COS-1 cells were treated for 15 min with the indicated doses of NaSal and then left unstimulated (−) or stimulated (+) for 15 min with TNF (20 ng/ml). Lysates were blotted with anti-IκBα antibody. (B) COS-1 cells were treated for 15 min with the indicated doses of NaSal alone, lysed, and blotted with anti-phospho-p38 MAPK antibody (pp38; top panel) and with anti-p38 MAPK antibody (p38; bottom panel).

FIG. 6

FIG. 6

Kinetics of p38 MAPK activation by NaSal and TNF. COS-1 cells were either left untreated (Ctrl) or treated for the indicated times with TNF (20 ng/ml) or NaSal (20 mM). Lysates were blotted with anti-phospho-p38 MAPK antibody (pp38; top panel) and with anti-p38 MAPK antibody (p38; bottom panel).

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