RAW 264.7 cells (ECACC, Salisbury, UK) were maintained in 25 cm² flasks in DMEM medium supplemented with 2 mM L-glutamine and 10% v/v FCS, without antibiotics, at 37°C in a humidified atmosphere of 95% air and 5% CO₂. For Western blotting, cells were grown in 25 cm² flasks, whilst for the measurement of NO the cells were grown to 95% confluence in 96-well plates and stimulation carried out within these plates. Cells were stimulated by replacing the culture medium with medium containing LPS, LPS with phorbol-12-myristate-13-acetate (PMA) or PMA alone in the presence or absence of various inhibitors.

Inhibitors used were: the PKC inhibitors Gö 6983 (Go) and Bisindolymalemide I (Bis); the JAK2 inhibitor AG-490; the p38 MAP kinase inhibitor SB 203580 (Calbiochem, Nottingham, UK). Bis shows high selectivity for PKC α, β_I, β_II, γ, δ and ε isoforms at 20 μm 19 whilst Go inhibits PKC α, β, γ, δ and ζ isoforms at 10 μm 20. AG-490 was used at 10 mM, a concentration previously shown to inhibit JAK2 21, and SB 204580 at 10 mM, a concentration previously shown to inhibit the p68 MAP kinase family 22. For the purpose of specific inhibition of PKC translocation, the following MALY-TAT linked peptides (kindly supplied by Dr M. Lindsay, AstraZeneca, Charnwood, UK) were used: MALYO1 (TAT- RFARKGALRQKNHEVK), MALY1O (TAT-EAVSLKPT), MALY II (TAT-LSETKPAV0) at concentrations previously shown to inhibit translocation of PKC isoforms 23. For Western blotting cells were incubated for 0, 1, 2, 3 or 5 hours, whilst for the NO assay, cells were incubated for 24 hours.

RAW 264.7 cells were harvested in ice cold PBS after stimulation with LPS from 0 to 5 h. Cells were then lysed in 70 μl of buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.25% v/v noident P-40, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) in de-ionised water (dH₂O) for 20 min on ice, to yield the cytoplasmic cellular fraction, as described previously 24. The samples were microfuged at 12,000 g for 15 sec to pellet the unlysed nuclei and the supernatant (cytoplasmic fraction) was collected. The nuclei were lysed in 15 μl of buffer B (20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 0.42 M NaCl, 0.5 mM DTT, 25% v/v Glycerol, 0.5 mM PMSF in diH₂O) for 20 min on ice, and microfuged or centrifuged at 12,000 g for 60 sec to pellet the cellular debris. The supernatant (nuclear fraction) was collected and 60 μl of buffer C (20 mM HEPES pH 7.9, 50 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF) was added to it. At this stage the protein concentration of the samples was assessed by BioRad protein assay ™ (Biorad, UK).

Samples (10 μg) were separated by 10% SDS-PAGE and proteins transferred to a nitrocellulose membrane (Amersham-Pharmacia, Amersham, UK) by electroblotting. Equal protein loading was confirmed by Ponceau S staining of the membrane. Non-specific protein binding was blocked by incubation of the membrane in PBS-T + 1% w/v milk overnight at 4°C. Membranes were then washed twice in PBS-T for 5 min before incubation for 1 hour at room temperature (RT) with rabbit anti-p65 antibody (1:4000, Santa Cruz, Wembley, UK). Membranes were then washed twice in phosphate buffered saline (pH 7.4) containing 0.05% v/v Tween 20 (PBS-T) and 1% w/v milk for 5 min followed by an hour incubation at room temperature (RT) with goat anti-rabbit HRP conjugate (1:4000, Dako, UK). All antibodies incubations were carried out in PBS-T containing 1% w/v milk. Membranes were washed three times for 5 min in PBS-T before incubation with ECL substrate (Amersham-Pharmacia, UK), followed by exposure to an autoradiographic film and subsequent semi-quantification of band intensity by densitometry (UVP Ltd, Cambridge, UK).

NO levels were assessed by nitrite quantification as described previously 25. Briefly, 90 μl of sample (cell culture medium) was incubated for 5 min in dark at RT with 90 μl of suphanilamide (1% w/v in 4 M HCl). 90 μl of napthylethylenediamine (1% w/v in dH₂O) was then added and a further 5 min incubation was carried out in dark at RT. Absorbance was read at 540 nm.

All reagents were purchased from Sigma (Poole, UK) unless otherwise stated above.

Data are reported as mean ± SEM. Statistical analysis was performed in Prism 5 (Graph Pad Software, Inc. San Diego, USA) using one way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test (TMCT) when ANOVA indicated a statistical significance existed.

Constitutive NO production by RAW 264.7 macrophages (Fig. 1) was at the lower limit of the sensitivity of the assay (3 μM,). LPS induced a concentration-dependent increase in NO production with a maximum response at 50 μg/ml at 24 h (Figure 1b.). For subsequent experiments, LPS (1 μg/ml) was chosen for its ability to stimulate high levels of NO production whilst not possessing the significant (p < 0.001) toxicity seen with 10 and 50 μg/ml (38% and 51% reduction in viability respectively).

LPS stimulation induced NO production with nitrite levels peaking at 33 μM (p < 0.001, Fig. 1), in agreement with the results of others 2627. In contrast, PMA alone (0.5–500 ng/ml) had no effect on NO production but significantly attenuated LPS-induced NO production by ~50% (Fig. 1) even at concentrations (50 ng/ml) previously shown to activate NF-κB.

LPS (1 μg/ml) induced a significant 4-fold induction of p65 nuclear translocation which was maintained for up to 5 hours (p < 0.05, Fig. 2). PMA alone also significantly induced NF-κB activation (data not shown), however, combined effect of PMA and LPS-stimulated NF-κB showed an 8-fold increase within 30 minutes PMA reduced the duration of LPS-stimulated p65 nuclear translocation from > 5 to less than 2 hours (Fig. 3).

Effects on PKC isoforms degradation have been reported following PMA treatment at the concentration of PMA used in this study (50 ng/ml) 28. The induction of NOS2 activity upon activation of PKC by LPS stimulation has also been demonstrated previously using a non-selective inhibitor of all PKC isoforms 16. However, the role of specific PKC isoforms has remained unclear. The MALY peptides have been reported to mimic the PKC variable regions 1 and 2 (V1-2). These regions are necessary for binding PKC to the receptors for activated C kinase (RACK) and thereby prevent nuclear-cytoplasmic translocation of specific PKC isoforms 29. The addition of a TAT sequence (GGGGYGRKKRRQRRR-GGGG) to the MALY peptides ensures that they are transported into the nucleus, an important facet for some PKC enzymes. Pre-treatment with these translocation inhibitor-peptides had no effect on NO production in these cells (Table 1). In addition, the PKC inhibitor Gö 6983 (Go, 10 μM) had no effect on LPS-stimulated NO production (Fig. 4).

In contrast, bisindolymalemide I (Bis, 20 μM) completely inhibited LPS (1 μg/ml) -stimulated NO production (Fig. 4). This was not due to an inhibitory effect on peak NF-κB p65 nuclear translocation as Bis (20 μM) had no effect on LPS-stimulated nuclear translocation at 3 hr (Fig. 5). This time point was chosen as it represented the time at which NF-κB p65 nuclear translocation was at its peak (Fig. 2). The results also contrast with the effect of PMA which returned p65 nuclear translocation to baseline within 2 hr.

AG-490, a potent inhibitor of JAK2, caused a concentration-dependent inhibition of LPS-stimulated NO production and this was significant at 10 μM (Fig. 6). JAK2 has been shown to be activated in response to a wide variety of stimuli 30 and AG-490 to block induced NOS2 expression in IL-1β/TNFα/IFNγ-stimulated human epithelial-like colon carcinoma DLD-1 cells 31 and in IFNγ/LPS stimulated RAW cells 32. This present finding extends the previous studies and further indicates the involvement of JAK2 in NF-κB-induced LPS-stimulated NO production and NF-κB activation in RAW cells.

p38 MAP kinase has previously been implicated in NF-κB activation 33 and in IFNγ/LPS stimulated NOS2 expression in RAW 264.7 cell, although this has not been reported for LPS-stimulated cells alone 34. SB203580 inhibited LPS-stimulated NO production in a concentration-dependent manner with an IC₅₀ of ~3 μM indicating selectivity of this effect. Maximal inhibition (~35%) was seen at 10 μM (Fig. 7).