Abstract
In contrast to aspirin, salicylate, its active metabolite, possesses profound anti-inflammatory properties without blocking cyclooxygenase. Inhibition of the transcription factor nuclear factor-κB (NF-κB) has been discussed to play a role in the anti-inflammatory profile of salicylate. However, NF-κB-independent effects of salicylate have been assumed but have up to now been poorly investigated. Therefore, the aim of the present study was to investigate NF-κB-independent anti-inflammatory mechanisms of salicylate in human umbilical vein endothelial cells using interleukin-4 (IL-4) as NF-κB-independent proinflammatory stimulus and P-selectin as inflammatory read-out parameter. Using quantitative real-time reverse transcription-polymerase chain reaction, we found that salicylate decreases IL-4-induced P-selectin expression. As judged by Western blot analysis, salicylate increased endothelial heme oxygenase-1 (HO-1) protein levels. Using both the HO-1 inhibitor tin(II) protoporphyrin IX and HO-1 antisense oligonucleotides, we causally linked the induction of HO-1 to the decrease of P-selectin. Moreover, we were interested in the signaling mechanisms leading to the up-regulation of HO-1 by salicylate. c-Jun NH2-terminal kinase (JNK) was found to be activated by salicylate, and we could causally link this activation to the induction of HO-1 by using the JNK inhibitor 1,9-pyrazoloanthrone. By applying activator protein-1 (AP-1) decoys, it was shown that the transcription factor AP-1 is crucially involved in the up-regulation of HO-1 downstream of JNK. In summary, our study introduces HO-1 as novel NF-κB-independent anti-inflammatory target of salicylate in human endothelial cells. Moreover, we elucidated the JNK/AP-1 pathway as crucial for the induction of HO-1 by salicylate.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.102251.
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ABBREVIATIONS: COX, cyclooxygenase; NF-κB, nuclear factor-κB; IL-4, interleukin-4; HO-1, heme oxygenase-1; AP-1, activator protein-1; HUVEC, human umbilical vein endothelial cell(s); TNF-α, tumor necrosis factor-α; SnPP, tin(II) protoporphyrin IX; NaSal, sodium salicylate; SP600125, 1,9-pyrazoloanthrone; PCR, polymerase chain reaction; JNK, c-Jun N-terminal kinase; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; RT, reverse transcription.
- Received February 3, 2006.
- Accepted April 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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