We have previously shown that pyridinyl imidazole inhibitor of p38 MAPK SB203580 20 stimulates LPS-induced NO production 19. SB220025 is a recently developed potent and specific inhibitor of p38 MAPK with an IC₅₀ value of 60 nM in kinase activity assay 21. Figure 1A shows that SB220025 had a concentration dependent stimulatory effect on LPS-induced NO production and maximal effect (50%) was achieved at drug concentration of 0,5 μM. The effect of SB220025 was similar to the effect of SB203580 (1 μM) (Fig. 1B). A structurally related control compound SB202474, which does not inhibit p38 MAPK 22, had no effect on NO production. The stimulatory effect of SB220025 was maximal when the compound was added to cells 1 h after LPS (Fig 2A). This result is in line with our previous report in which we showed that the stimulatory effect of SB203580 was maximal when the compound was added 1 h after LPS 19. The levels of activated p38 peaked in 30 min after LPS, were still high at 1 h and declined gradually thereafter so that activated p38 could be detected even 4 h after LPS (Fig. 2B). Thus, the stimulation of LPS-induced iNOS production by SB220025 could result from inhibition of p38, even when the compound was added to cells 1–2 h after LPS. SB220025 had a clear stimulatory effect also on iNOS protein expression, whereas the negative control compound SB202747 had no effect (Fig. 3A). Interestingly, SB220025 did not increase LPS-induced iNOS mRNA levels when measured 4 h after addition of LPS, whereas a 100% increase in iNOS mRNA levels was observed when measured 10 h after addition of LPS (Fig. 3B).

Because SB220025 had no effect on iNOS mRNA levels when measured 4 h after LPS, but significantly increased the mRNA levels when measured 10 h after LPS, we hypothesized that SB220025 might stabilize iNOS mRNA.

To study the effect of SB220025 on the stability of iNOS mRNA, the cells were treated with LPS or LPS+SB220025 and cells were incubated for 6 h. Thereafter total RNA was isolated at 2 h intervals. The iNOS mRNA levels in cells were reducing rapidly between 6–12 h after LPS stimulation. The amount of iNOS mRNA in LPS treated cells halved in about 3 h (Fig. 4). The reduction in the amount of iNOS mRNA was slower in cells treated with LPS+SB220025 (iNOS mRNA halved in about 4,5 h).

Actinomycin D (an inhibitor of transcription) was added to cells 6 h after LPS in an attempt to test whether the slowed disappearance of iNOS mRNA in cells treated with LPS+SB220025 was due to increased rate of transcription of iNOS gene or reduced degradation of mRNA. Interestingly, the level of mRNA was reducing at the same or slower rate in cells treated with LPS+actinomycin D compared with cells treated with LPS only, suggesting that no significant transcription of iNOS gene occurs in cells 6 – 12 h after LPS stimulation and that actinomycin D itself inhibits the degradation of iNOS mRNA. Thus, the slowed disappearance of iNOS mRNA in cells treated with SB220025 was most likely due to reduced degradation of mRNA.

There are four known isoforms of p38 MAPK (α, β, γ and δ) 6, and SB203580 has been shown to inhibit p38α and p38β but not p38γ and p38δ isoforms 23. p38α and p38β have been recently reported to differently regulate iNOS expression 24. Therefore we wanted to investigate whether J774.2 macrophages express p38α and p38β isoenzymes.

We used real-time RT-PCR to study the p38α and p38β mRNA expression in J774.2 macrophages. Both unstimulated and LPS stimulated cells expressed p38α mRNA at relatively high level as compared to GAPDH mRNA (Fig. 5A). In contrast, only low level expression of p38β mRNA was detected. In line with the mRNA result, Western blot showed p38α protein expression (Fig. 5B), whereas no p38β protein could be detected by Western blotting.

Opposite roles for p38 MAPK and JNK have recently been reported on thrombin induced iNOS expression in RAW264.7 macrophages 25. JNK and p38 MAPK have common target proteins and there is crosstalk between these signaling cascades 26. Furthermore, we have previously reported that JNK inhibition destabilizes iNOS mRNA 10. Therefore we hypothesized that the roles of JNK and p38 MAPK pathways on LPS-induced iNOS expression may be coupled. We continued by investigating whether inhibition of p38 MAPK modulates the activity of JNK.

LPS induced a rapid phosphorylation of JNK. The phosphorylation peaked at 0.5 h and declined rapidly thereafter, remaining <33% of the maximum when measured 2–8 h after LPS (Fig. 6). SB220025, when given 1 h after LPS, further increased the LPS-induced JNK phosphorylation compared with cells treated with LPS only. In SB220025-treated cells the amount of phosphorylated JNK remained >55% of the maximum level up to 4 h and declined thereafter. SB220025 alone did not activate JNK (Fig. 7).

JNK phosphorylates c-Jun at residues Ser63 and Ser73 27. In parallel to increased phosphorylation of JNK by SB220025, increased phosphorylation of c-Jun at Ser63 was observed (Fig. 8A). Similar results were obtained when phosphorylation of Ser73 was measured (data not shown). 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 phosphorylation 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 phosphorylation compared with cells treated with LPS only (Fig. 8B). 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 dephosphorylation.

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 (Fig. 9A). However, when cells were treated with a combination of SB220025 and SP600125 the level of NO production 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 (Fig. 9B). 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 levels.

Cycloheximide is widely used as an inhibitor of protein synthesis. However, cycloheximide also activates JNK 28. Therefore we continued by investigating whether cycloheximide has similar effect on iNOS mRNA expression as SB220025. Cycloheximide at 0,05–0,1 μg/ml concentrations increased LPS-induced JNK activity (Fig. 10A). Interestingly, cycloheximide had no significant effect on iNOS mRNA expression when measured 4 h after LPS, but increased iNOS mRNA levels >4 fold when measured 10 h after LPS (Fig. 10B). Furthermore, the effect of cycloheximide on iNOS mRNA expression was partially inhibited by SP600125 (Fig. 10C). These results show that the effect of cycloheximide on JNK activity and iNOS expression were very similar to the effect of SB220025.

Reagents were obtained as follows: anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), 4-ethyl-2-(4-methoxyphenyl)-5-(4-hydroxyphenyl)-5-(4pyridyl)-imidazole (SB202474), 5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)-imidazole(SB220025) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole (SB203580) (Calbiochem), rabbit polyclonal mouse iNOS, c-Jun, JNK1 and actin antibodies, goat polyclonal p38β antibody, donkey anti-goat antibody and goat anti-rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.), rabbit polyclonal p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), p38α, phospho-SAPK/JNK (Thr183/Tyr185) and phospho-c-Jun (Ser63) II antibodies (Cell Signaling technology). All other reagents were from Sigma.

J774.2 macrophages (The European Collection of Cell Cultures) were cultured at 37°C, 5% CO₂ atmosphere, in Dulbecco's Modified Eagle's Medium with glutamax-I (Cambrex Bioproducts) containing 10% of heat inactivated foetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (all from Invitrogen). Cells were seeded on 24 well plates for nitrite measurements and in 6 well plates for Western blot and RT-PCR and grown for 48 h prior to experiments.

At indicated time points the culture medium was collected for nitrite measurement, which was used as a measure of NO production. Culture medium (100 μl) was incubated with 100 μl of Griess reagent (0.1% napthalethylenediamine dihydrochloride, 1% sulphanilamine, 2.5% H₃PO₄) and the absorbance was measured at 540 nm.

At indicated time points cells were rapidly washed with ice cold PBS and solubilised in cold lysis buffer containing 10 mM Tris-base, 5 mM EDTA, 50 mM NaCl, 1% Triton-X-100, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM sodiumorthovanadate, 10 μg/ml leupeptin, 25 μg/ml aprotinin, 1.25 mM NaF, 1 mM sodiumpyrophosphate and 10 mM n-octyl-β-D-glucopyranoside. After incubation for 20 min on ice, lysates were centrifuged (14500 g, 15 min) and supernatants were mixed 1:4 with SDS loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.025% bromophenol blue, 5% β-mercaptoethanol) and boiled for 5 min. Protein concentrations in the samples were measured by the Coomassie blue method 40.

Protein (30 μg) was loaded on 10% SDS-polyacrylamide electrophoresis gel and electrophoresed for 4 h at 100 V in buffer containing 95 mM Tris-HCl, 960 mM glycine and 0.5% SDS. After electrophoresis the proteins were transferred to Hybond ECL™ nitrocellulose membrane (Amersham) with semi-dry blotter at 2.5 mA/cm² for 60 min. After transfer the membrane was blocked in TBS/T (20 mM Tris-base pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibody in the blocking solution at 4°C overnight. Thereafter the membrane was washed 4× with TBS/T for 5 min, incubated with secondary antibody in the blocking solution for 0.5 h at room temperature and washed 4× with TBS/T for 5 min. Bound antibody was detected using SuperSignal^® West Pico chemiluminescent substrate (Pierce) and FluorChem™ 8800 imaging system (Alpha Innotech). The quantitation of chemiluminescent signal was carried out with FluorChem™ software v. 3.1.

At indicated time points cell monolayers were rapidly washed with ice cold PBS and cells were homogenised using QIAshredder™ (QIAGEN Inc.). RNA extraction was carried out with RNeasy^® kit for isolation of total RNA (QIAGEN inc.). Total RNA (25 ng) was reverse transcribed to cDNA using TaqMan Reverse Transcription reagents and random hexamers (Applied Biosystems). Reverse transcriptase (RT) reaction parameters were as follows: incubation at 25°C for 10 min, RT at 48°C for 30 min and RT inactivation at 95°C for 5 min. cDNA obtained from the RT reaction (amount corresponding approximately 1 ng of total RNA) was subjected to PCR using TaqMan^® Universal PCR Master Mix and ABI PRISM^® 7000 Sequence detection system (Applied Biosystems). GAPDH and iNOS primer and probe sequences and concentrations were optimised according to manufacturers guidelines in TaqMan^® Universal PCR Master Mix Protocol Part Number 4304449 Rev. C and were as follows: 5'-CCTGGTACGGGCATTGCT-3', 5'-GCTCATGCGGCCTCCTT-3' (forward and reverse mouse iNOS primer respectively, both 300 nM), 5'-CAGCAGCGGCTCCATGACTCCC-3'(mouse iNOS probe 150 nM, containing 6-FAM as 5'-reporter dye) and 5'-GCATGGCCTTCCGTGTTC-3', 5'-GATGTCATCATACTTGGCAGGTTT-3' (forward and reverse mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer respectively, both 300 nM), 5'-TCGTGGATCTGACGTGCCGCC-3'(mouse GAPDH probe 150 nM, containing 6-FAM as 5'-reporter dye) (Metabion). Primers and probes for p38α (product Mm00442497_m1) and p38β (product Mm00440955_m1) (Applied Biosystems) were used as recommended by the manufacturer. PCR reaction parameters were as follows: incubation at 50°C for 2 min, incubation at 95°C for 10 min and thereafter 40 cycles of denaturation at 95°C for 15 sec and annealing and extension at 60°C for 1 min. Each sample was determined in duplicate.

A standard curve method was used to determine the relative iNOS and GAPDH mRNA levels as described in Applied Biosystems User Bulletin #2. In short, a standard curve for each gene was created using mRNA isolated from LPS-stimulated J774.2 macrophages. Isolated RNA was reverse transcribed as described. Dilution series were made from obtained cDNA ranging from 10 ng to 1 pg and were subjected to real time PCR as described. Obtained threshold cycle values were plotted against dilution factor to create a standard curve. Relative mRNA levels in test samples were then calculated from the standard curve.

Results are expressed as mean ± standard error of mean (S.E.M.). When indicated, statistical significance was calculated by analysis of variances supported by Bonferroni multiple comparisons test. Differences were considered significant at P < 0.05.