Lorraine Hart

Superinduction of COX‐2 mRNA by cycloheximide and interleukin‐1β involves increased transcription and correlates with increased NF‐κB and JNK activation

Many primary response genes, including cyclooxygenase‐2 (COX‐2), exhibit mRNA superinduction following agonist stimulation in the presence of translational blockers such as cycloheximide. This is widely assumed to result from mRNA stabilisation. However, superinduction of IL‐1β‐induced COX‐2 mRNA levels by cycloheximide in pulmonary type II A549 cells occurred by increased transcription and not by mRNA stabilisation. Furthermore, equivalent effects were observed on NF‐κB binding to COX‐2 promoter κB sites and activation of the Jun N‐terminal kinases (JNK), p54 and p46. These signalling pathways play important roles in COX‐2 induction and may therefore account for the observed increases in COX‐2 transcription. These data are consistent with negative feed‐back involving down‐regulation of NF‐κB by de novo IκBα synthesis and suggest that JNK activation may also be down‐regulated by a cycloheximide sensitive process.

The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1β-dependent PGE2 release via mechanistically distinct processes

In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E2 in response to pro‐inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL‐1β‐dependent release of PGE2. Following IL‐1β treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. PD09059, UO126 and SB203580 prevented IL‐1β‐induced PGE2 release at doses that correlated closely with published IC50 values. Small or partial effects at the relevant doses were observed on induction of cyclo‐oxygenase (COX) activity or COX‐2 protein suggesting that the primary effects were at the level of arachidonate availability. Neither PD098059 nor SB203580 showed any effect on IL‐1β‐induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX‐2. In contrast, UO126 was highly effective at inhibiting IL‐1β‐dependent arachidonate release, implicating MEK2 in the activation of the PLA2 that is involved in IL‐1β‐dependent PGE2 release. We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti‐inflammatory benefits.