Abstract
Roflumilast, a potent and selective phosphodiesterase 4 (PDE4) inhibitor, has been demonstrated to be an effective anti-inflammatory agent in airway inflammatory diseases. In the present study, we investigated the mechanism of anti-inflammatory effects of roflumilast in murine macrophage cell line RAW264.7 cells. Roflumilast inhibited NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β production via suppression of their gene expressions in lipopolysaccharide (LPS)-stimulated macrophages. To elucidate the mechanism by which roflumilast inhibits the production of inflammatory mediators, we examined the effect of roflumilast on the activation of nuclear factor-κB (NF-κB) in these cells. Roflumilast inhibited the DNA binding activity of NF-κB by preventing inhibitor κBα phosphorylation and degradation. The phosphorylation of mitogen-activated protein (MAP) kinases, including stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, was also markedly inhibited by roflumilast. Similar to the effects of roflumilast, treatment of either SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] or SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone], specific inhibitors of p38 MAP kinase and JNK, respectively, suppressed NO, TNF-α, and IL-1β production. Consistent with in vitro results, administration of roflumilast recovered the survival rate of LPS-treated mice, with concurrent suppression of plasma levels of nitrite/nitrate, TNF-α, and IL-1β. These results suggest that the inhibitory activity of roflumilast on the production of inflammatory mediators seems to be mediated via inhibition of NF-κB, p38 MAP kinase, and JNK activation in macrophages.
Footnotes
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This research was supported by a grant from the Center for Biological Modulators of the 21st Century Frontier Research & Development Program, The Ministry of Science and Technology, Taejon, Korea.
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doi:10.1124/jpet.105.092056.
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ABBREVIATIONS: PDE4, phosphodiesterase 4; TNF, tumor necrosis factor; IL, interleukin; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; iNOS, inducible nitric-oxide synthase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; Bay11-7082, (E)-3[(4-methylphenyl)sulfonyl]-2-propenentrile; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone; NOx, nitrate and nitrite; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; TBS-T, Tris-buffered saline-Tween 20; GaIN, d(+)galactosamine; EMSA, electrophoretic mobility shift assay; PD98059, 2′-amino-3′-methoxyflavone.
- Received July 4, 2005.
- Accepted August 25, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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