JNK phosphorylates c Jun at residues Ser63 and Ser73. In the parallel to increased phosphorylation of JNK by SB220025, increased phosphorylation of c Jun at Ser63 was observed. Similar results were obtained when phosphorylation of Ser73 was measured. This suggests that the increased phosphorylation of JNK resulted in functionally significant increase in the activity of JNK. To rule out the possibility, that increased c Jun phospho rylation was a result of reduced dephosphorylation, we tested whether the effect of SB220025 could be reversed with JNK inhibitor SP600125. Treatment with LPS and SB220025 induced a 6 fold increase in c Jun Ser63 phos phorylation compared with cells treated with LPS only. In contrast, the negative control compound SB202474 had no effect on c Jun phosphorylation.

The SB220025 stimulated increase in c Jun phosphorylation was almost completely reversed by SP600125, suggesting that the increase in c Jun phosphorylation was due to increased JNK activity and not due to reduced dephospho rylation. The stimulatory effect of SB220025 on LPS induced NO production and iNOS mRNA expression can be reversed by SP600125 To continue, we hypothesized that the stimulatory effect of SB220025 on LPS induced NO production was due to increased JNK activity and therefore we tested the effect of JNK inhibitor SP600125 on SB220025 stimulated NO production. SB220025 induced a clear increase in LPS stimulated NO production, whereas SP600125 inhibited NO production. However, when cells were treated with a combi nation of SB220025 and SP600125 the level of NO pro duction was comparable to levels produced by cells treated with LPS SP600126.

Thus, the effect of SB220025 was reversed by SP600125. The same result was observed at the level of iNOS mRNA expression. SB220025 increased the amounts of iNOS mRNA to almost two fold compared with cells treated with LPS only, whereas the negative control compound SB202474 had no effect. SP600125 alone reduced the LPS stimulated iNOS mRNA levels slightly. In addition, in the presence of the JNK inhibitor SP600125, SB220025 had no stimulatory effect on iNOS mRNA lev els. Cycloheximide increases JNK activity and iNOS mRNA expression Cycloheximide is widely used as an inhibitor of protein synthesis. However, cycloheximide also activates JNK. Therefore we continued by investigating whether cycloheximide has similar effect on iNOS mRNA expres sion as SB220025.

Cycloheximide at 0,05 0,1 g ml con centrations increased LPS induced JNK activity. Interestingly, cycloheximide had no significant effect on iNOS mRNA expression when measured 4 h after LPS, but increased iNOS AV-951 mRNA levels 4 fold when measured 10 h after LPS. Furthermore, the effect of cyclohex imide on iNOS mRNA expression was partially inhibited by SP600125. These results show that the effect of cycloheximide on JNK activity and iNOS expression were very similar to the effect of SB220025.