p38 Mitogen-activated Protein Kinase Regulates Cyclooxygenase-2 mRNA Stability and Transcription in Lipopolysaccharide-treated Human Monocytes*

p38 mitogen-activated protein kinase (MAPK) is activated by inflammatory stimuli such as bacterial lipopolysaccharide (LPS), interleukin-1, and tumor necrosis factor. We have previously shown that the pyridinyl imidazole SB 203580, which inhibits it, blocks the interleukin-1 induction of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 1 and 3 mRNAs in fibroblasts. Here we explore the role of p38 MAPK in the response of human monocytes to LPS. 0.1 μm SB 203580 significantly inhibited the LPS induction of COX-2 and tumor necrosis factor protein and mRNAs. The activity of MAPK-activated protein kinase-2 (a substrate of p38 MAPK) in the cells was commensurately reduced. Some isoforms of c-junN-terminal kinase (which is also activated by LPS) are sensitive to SB 203580; the inhibitor had little effect on monocyte c-junN-terminal kinases up to 2 μm. We investigated the mechanism of inhibition of COX-2 induction. Transcription (measured by a nuclear run-on assay) was 60% inhibited by SB 203580 (2 μm). Importantly, we found that p38 MAPK was essential for stabilizing COX-2 mRNA: when cells stimulated for 4 h with LPS were treated with actinomycin D, COX-2 mRNA decayed slowly. Treatment of stimulated cells with 2 μm SB 203580 caused a rapid disappearance of COX-2 mRNA, even with actinomycin D present. We conclude p38 MAPK plays a role in the transcription and stabilization of COX-2 mRNA.

p38 mitogen-activated protein kinase (MAPK) 1 is a member of the MAPK family and is activated by the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), by bacterial lipopolysaccharide (LPS), and by a range of cellular stresses (1)(2)(3)(4)(5). Although originally characterized as a stress or inflammatory kinase, it is likely to have diverse functions because it is also activated in platelets by thrombin and collagen (6) and in T cells upon activation by various stimuli (7) and is constitutively active in liver (8,9). Little is known about the physiological functions it controls. One substrate is MAPKactivated protein kinase-2 (MAPKAPK-2) (10,11), which in turn phosphorylates the small heat shock protein hsp27 (12) and the cAMP-response element binding protein (13). Other putative targets are the MAPK integrating kinase (14,15) and the transcription factors CHOP (16), myocyte enhancer factor 2C (17), and activating transcription factor 2 (4).
Besides the original p38 MAPK (called ␣), a closely similar ␤ form has been described (18) as well as two more distantly related enzymes that also contain the TGY motif: stress-activated protein kinase 3 (or p38␥) (19 -21) and stress-activated protein kinase 4 (or p38␦) (22)(23)(24). The p38␣ and p38␤ MAPKs are inhibited by a class of pyridinyl imidazole compounds of which the best characterized is SB 203580 (11). These were first identified as inhibitors of TNF (and IL-1) production by LPS-activated monocytes (25) and were later shown to inhibit p38 MAPK (5,11). The pyridinyl imidazoles inhibited TNF (and IL-1) protein production with relatively little effect on the levels of mRNA induced, and it was suggested that they worked mainly by inhibiting translation (25,26). Our laboratory used SB 203580 to investigate the role of p38 MAPK in cellular responses to IL-1 (27). We found that the drug strongly inhibited the induction of cyclooxygenase-2 (COX-2), collagenase (matrix metalloproteinase 1), and stromelysin (matrix metalloproteinase 3) by IL-1 at the protein and mRNA levels within the range of its IC 50 for p38 MAPK (0.1-0.5 M) (27). There was comparatively little inhibition of secondary cytokine production; IL-6 was only inhibited 30 -50% (at 1 M), and IL-8 was unaffected. The inhibition of induction by IL-1 of COX-2 and matrix metalloproteinase in fibroblasts at the mRNA level was in contrast to the inhibition of TNF and IL-1 protein at the translational level in LPS-activated monocytes. However, a recent report showed that LPS induction of COX-2 mRNA was inhibited by SB 203580 in monocytes (28). It was possible that p38 MAPK regulated the expression of different genes at different levels, depending upon the cell type and the nature of the stimulus.
We have therefore investigated the effects of SB 203580 on the induction of both COX-2 and TNF in monocytes stimulated by LPS and found each to be similarly inhibited at both the protein and mRNA levels. We then went on to show that in the case of COX-2, the p38 MAPK inhibitor impaired transcription and destabilized the mRNA.
Doubts have recently been raised concerning the specificity of SB 203580 (29,30) because it can inhibit certain isoforms of c-jun N-terminal kinase (JNK), another MAPK family member activated by the same stimuli as p38 MAPK. We have therefore checked the sensitivity of the LPS-activated JNKs in human monocytes to the inhibitor. LPS from Salmonella typhimurium was from Sigma-Aldrich Company, Ltd. (Poole, United Kingdom) and was used at a concentration of 10 ng ml Ϫ1 . Sheep anti-rabbit MAPKAPK-2 antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The rabbit antiserum to the Cterminal peptide of p38␣ MAPK (ISFVPPLDQEEMES) has been described previously (6 Isolation and Culture of Human Monocytes-For each experiment, human peripheral blood monocytes were freshly prepared from the buffy coat fraction of a unit of blood from a single donor. Mononuclear cells were prepared by Ficoll-Hypaque centrifugation on a Lymphoprep gradient, and monocytes were isolated by centrifugal elutriation on a Beckman JE6 elutriator (High Wycombe, United Kingdom) using RPMI 1640 medium with L-glutamine and 1% heat-inactivated fetal calf serum. Monocyte purity was routinely assessed by fluorescence-activated cell-sorting forward/side scatter measurements in a Becton Dickinson FACScan, and cells of Ͼ80% purity with Յ10% T cells were collected. Monocytes collected in this way were typically found to be 75% pure as judged by flow cytometry using fluorochrome-conjugated anti-CD-14 monoclonal antibodies. Monocytes were cultured in the same medium used for elutriation at 37°C in a humidified atmosphere containing 5% CO 2 .
p38 MAPK and MAPKAPK-2 Kinase Assays-Cells (5 ϫ 10 6 ) were stimulated for the time period indicated with 10 ng ml Ϫ1 LPS and lysed for 10 min on ice in 1 ml of lysis buffer (20 mM HEPES, pH 7.4, 50 mM sodium ␤-glycerophosphate, 2 mM EGTA, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 3 g ml Ϫ1 aprotinin, 10 M E64, and 2 g ml Ϫ1 pepstatin A). Samples were clarified by centrifugation at 4°C for 10 min at 16,000 ϫ g. p38 MAPK was immunoprecipitated by the addition of 5 l of rabbit antiserum to the C terminus of p38 MAPK. Samples were incubated at 4°C for 1 h with mixing before the addition of 30 l of a 50% slurry of protein A beads in lysis buffer, and samples were incubated for an additional 2 h. The beads were washed four times in 1 ml of wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 25 mM sodium ␤-glycerophosphate, 10 mM sodium tetrapyrophosphate, 1 mM sodium orthovanadate, 2 mM DTT, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) and twice in 1 ml of kinase assay buffer (20 mM HEPES, pH 7.5, 20 mM sodium ␤-glycerophosphate, 200 mM NaCl, 2 mM DTT, 10 mM MgCl 2 , 10 mM NaF, 0.1 mM sodium orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, and 0.05% BRIJ 35). p38 MAPK was assayed using 1 g of recombinant MAPKAPK-2 as substrate in kinase assay buffer with 20 M ATP and 4 Ci of [␥-32 P]ATP in an assay volume of 30 l. Samples were incubated at room temperature for 20 min with agitation, and the reaction was stopped by the addition of 4ϫ SDS-polyacrylamide gel electrophoresis sample buffer. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis (31). Gels were stained with Coomassie Brilliant Blue R-250, and phosphorylation of the substrate was measured on a phosphorimager (Fuji FLA-2000; Fuji, Tokyo, Japan).
MAPKAPK-2 immunoprecipitation was performed in an identical fashion, but with 5 g of sheep anti-rabbit MAPKAPK-2 antibody and protein G beads. Samples were assayed for kinase activity as described above using 1 g of recombinant human hsp27 as substrate.
Mono-Q Anion Exchange Chromatography and JNK Assays-10 8 monocytes were lysed in 1.0 ml of Buffer A (20 mM Tris-HCl, pH 8.5, 20 mM sodium ␤-glycerophosphate, 10 mM NaF, 2 mM DTT, 0.1 mM sodium orthovanadate, 0.02% sodium azide, 1 mM EDTA, and 1 mM EGTA) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 3 g ml Ϫ1 aprotinin, and 10 M E64 for 10 min on ice. The extract was clarified and filtered (0.2 m), and 100 l (approximately 500 g of protein) were applied to a 100 l Mono Q column equilibrated with Buffer A containing 0.05% BRIJ 35. The column was washed with 5 column volumes of Buffer A, and protein was eluted with a 3-ml gradient of Buffer A containing 0 -0.5 M NaCl. 100-l fractions were collected, and 10 l of each were assayed for JNK activity by mixing with 10 l of glutathione S-transferase-c-Jun (1-135) (300 g ml Ϫ1 ) and 10 l of assay buffer (150 mM Tris-HCl, pH 7.4, 30 mM MgCl 2 , 60 M ATP, and 0.4 Ci l Ϫ1 [␥-32 P]ATP) and incubated for 20 min at room temperature. The reaction was stopped with 10 l of 4ϫ SDS-polyacrylamide gel electrophoresis sample buffer, and the samples were electrophoresed. Phosphorylation of substrate was assessed as for p38 MAPK and MAPKAPK-2.
Nuclear Run-On Assay-Monocytes (3 ϫ 10 7 cells/assay point) were washed once with phosphate-buffered saline and resuspended in icecold lysis buffer (0.3 M sucrose, 0.5% Nonidet P-40, 1 mM DTT, 1.5 mM MgCl 2 , 3 mM CaCl 2 , and 10 mM Tris-HCl, pH 7.5) and disrupted in a loosely fitting Dounce homogenizer. The lysate was layered onto a 30% sucrose cushion and centrifuged at 3,000 revolutions/min for 10 min at 4°C. The nuclear pellet was resuspended in 200 l of 50 mM Tris-HCl, pH 8.3, 5 mM MgCl 2 , 0.1 mM EDTA, and 40% glycerol and stored in liquid nitrogen. Thawed nuclei were added to 200 l of reaction mixture (10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 300 mM KCl, 5 mM DTT, 40 units of RNasin, 1 mM each of ATP, CTP, and GTP, and 125 Ci of [␣-32 P] UTP (800 Ci mmol Ϫ1 )) and incubated at 30°C for 30 min. Reactions were stopped by the addition of 20 l of 7.2 mg ml Ϫ1 DNase I and an additional 10-min incubation at 30°C. 420 l of 100 mM Tris, pH 7.4, 50 mM EDTA, 2% SDS, 100 g ml Ϫ1 yeast tRNA, and 400 g ml Ϫ1 proteinase K were added, followed by incubation at 37°C for 45 min. Nuclear RNA was isolated by phenol/chloroform extraction and precipitated from 840 l of the aqueous phase with 600 l of isopropanol and 200 l of 7.5 M ammonium acetate. RNA was then resuspended in 100 l of 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, made 0.2 M with respect to NaOH, and incubated on ice for 10 min. The solution was neutralized with HEPES and precipitated with ethanol. RNA was dissolved in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and hybridized to slot blots (plasmids linearized with EcoRI and UV-cross-linked to Hybond N membrane) in 50% formamide, 5ϫ saline sodium phosphate EDTA, 2.5ϫ Denhardt's solution, 0.1% SDS, and 0.2 mg ml Ϫ1 salmon sperm DNA. Blots were washed twice for 10 min in 2ϫ SSC and 0.1% SDS at 37°C, washed for 30 min in 2ϫ SSC and 0.1% SDS at 50°C, and then washed twice for 10 min in 2ϫ SSC at 37°C. Blots were then washed for 30 min in 2ϫ SSC and 10 g ml Ϫ1 RNase A at 37°C, followed by two washes in 2ϫ SSC and 0.1% SDS at 37°C. Signals were quantified in a phosphorimager.
Western Blot-Cell lysates (total protein, 50 g) were electrophoresed on 10% SDS-polyacrylamide gels (31) and blotted on polyvinylidene difluoride membrane. Membranes were treated according to the instructions of the Enhanced Chemifluorescence kit.

Induction of COX-2 Protein and mRNA by LPS Is
Inhibited by the p38 MAPK Inhibitor SB 203580 -Monocytes were untreated or pretreated for 1 h with increasing concentrations of SB 203580. They were then stimulated for 4 h with LPS (with the inhibitor remaining present on the pretreated cells). Cells were then immunoblotted for COX-2 protein (Fig. 1A), or RNA was extracted and Northern blotted for COX-2 mRNA (Fig. 1B). SB 203580 inhibited both protein and mRNA in a similar p38 MAPK Regulates COX-2 mRNA Stability and Transcription dose-dependent fashion. Significant inhibition was seen at 0.1 M (33% for protein and 50% for mRNA); this was stronger at 1 M (55% and 72%, respectively), with some additional increase at 10 M. The results are representative of three separate experiments with different batches of cells.
TNF Protein and mRNA Induced by LPS Are Both Inhibited by SB 203580 -Monocytes were treated for 1 h with SB 203580 or left untreated and further treated for 16 h with LPS or left untreated. Secreted TNF protein, which was measured by an enzyme-linked immunosorbent assay, was inhibited in a dosedependent fashion by SB 203580 (42% at 0.1 M and 67% at 1 M). TNF mRNA showed a sensitivity to the inhibitor that was similar to that seen for the protein (Fig. 1C). In this experiment, LPS stimulation increased the levels of GAPDH mRNA. LPS is a very strong monocyte activator, and some batches of cells show up-regulation of housekeeping genes. TNF protein and mRNA showed similar sensitivities to the inhibitor at a concentration of 2 M (62% and 65%, respectively) in two separate experiments with different batches of cells using a 4-h LPS stimulation (data not shown). Fig. 2A shows the activity of p38 MAPK immunoprecipitated from lysates made from cells at various times after the addition of LPS. Activity increased 6-fold by 10 min and then returned to resting levels after 1 h. It was important to compare the degree of inhibition of p38 MAPK activity with COX-2 mRNA induction. Inhibition of p38 MAPK in monocytes by treatment with SB 203580 (which is a reversible inhibitor) was measured by immunoprecipitating its substrate, MAPKAPK-2, from cell lysates and assaying the latter's activity on hsp27. Monocytes were pretreated with the inhibitor for 20 min or left untreated, and cells were then treated with LPS for 10 min (Fig. 2B, left panel). Monocytes were also treated with LPS for 4 h and either treated with the inhibitor for the final 30 min of stimulation or left untreated (Fig. 2B, right panel). MAP-KAPK-2 was immunoprecipitated from the lysates. At both time points, i.e. at the peak of p38 activation at 10 min and after it had returned to resting levels at 4 h, the MAPKAPK-2 activity was reduced by about 60% in cells treated with 0.1 M and by 80% in cells treated with 1 M. The degree of inhibition of the MAPKAPK-2 correlated well with the effect on COX-2 gene expression (Fig. 1, A and B).

Inhibition of p38 MAPK by SB 203580 in LPS-treated Monocytes-
Effect of SB 203580 on JNK Activity in LPS-treated Monocytes-Certain forms of JNK, particularly the ␤ splice variants of JNK-2, are sensitive to micromolar concentrations of SB 203580 (29,30). We chromatographed lysates of monocytes that had been treated with LPS for 20 min on a Mono-Q anion exchanger and measured JNK activity in the fractions eluted with a 0 -0.5 M NaCl gradient (Fig. 3A). As is characteristic of the enzyme in lysates of activated cells (29,35), two major peaks were eluted. These were tested for sensitivity to SB 203580 (note that p38 MAPK itself elutes after the JNK peaks at about 0.4 M NaCl (2, 3)). The activity of the first peak (which contains the short ϳ46-kDa JNKs) was unaffected by the inhibitor at concentrations up to 10 M (Fig. 3B, left panel). The activity of the second peak (which contains the full-length ϳ54-kDa JNKs) was unaffected by 0.1 M but was significantly inhibited (24%) at 2 M (Fig. 3B, right panel). The data shown in Fig. 3B are means of three experiments on three different batches of monocytes. Because the peaks contain similar amounts of JNK activity, we estimate that total JNK activity might be inhibited by 10 -15% in cells treated with 2 M SB 203580. The inhibition of COX-2 expression by SB 203580 is therefore consistent with its effect on p38 MAPK and not on JNK.
Transcription of COX-2 Is Inhibited by SB 203580 -The effect of p38 MAPK blockade on both the protein and mRNA levels of COX-2 could be due to decreased transcription and/or reduced mRNA stability. We tested for an effect on transcription by performing nuclear run-on assays. Monocytes were left untreated or pretreated for 1 h with 2 M SB 203580 and then further incubated for 4 h in the presence or absence of LPS. Nuclei were isolated as described in "Experimental Procedures," and COX-2 transcription was measured by nuclear run-on assay. Fig. 4A shows a representative experiment. COX-2 transcription was stimulated 12-fold, and this was inhibited by 74% in nuclei from cells treated with 2 M SB 203580. Data from three independent experiments are combined in Fig. 4B, which shows an average 5-fold stimulation of COX-2 transcription by LPS with a mean inhibition of 60% by 2 M SB 203580. The inhibitor had a similar effect when the run-on assay was performed after only a 30-min (rather than a 4-h) stimulation with LPS (data not shown). These results strongly suggested that inhibiting p38 MAPK interfered with the transcription of COX-2.
Inhibition of p38 MAPK Destabilizes COX-2 mRNA-To examine the stability of COX-2 mRNA in human monocytes, actinomycin D was added to cells after 4 h of LPS treatment, and COX-2 mRNA levels were measured from the time of addition up to 90 min (Fig. 5A). The predominant 4.6-kb and the minor 2.8-kb transcripts decayed at a similar slow rate (t 1/2 ϭ 110 min). Simultaneous addition of SB 203580 (2 M) and actinomycin D to the cells after a 4-h stimulation with LPS resulted in a rapid decrease (t 1/2 ϭ 30 min) in COX-2 mRNA levels (Fig. 5A). In cells that were not treated with actinomycin D or the p38 MAPK inhibitor, COX-2 mRNA levels were roughly constant for 1 h after a 4-h LPS stimulation (Fig. 5B), showing that a steady state had been reached. The decay observed in cells treated only with actinomycin D indicated that in LPS-treated monocytes, COX-2 mRNA is continually being degraded and transcribed. When SB 203580 (2 M) alone was added to cells treated with LPS for 4 h, there was a rapid decay of COX-2 mRNA that occurred at a similar rate to that for cells treated with the inhibitor and actinomycin D (Fig. 5B). This p38 MAPK Regulates COX-2 mRNA Stability and Transcription indicated that transcription was not needed for SB 203580 to destabilize COX-2 mRNA. DISCUSSION We found that the p38 MAPK inhibitor prevented the LPS induction of both COX-2 and TNF protein, and that the degree of inhibition of protein and mRNA for both were very similar. We concluded that there was little evidence for p38 MAPK regulating translation for the expression of both these gene products in primary monocytes. Our additional studies were restricted to COX-2, and these indicated that p38 MAPK is required for optimal transcription of the gene, and that it is needed for stabilization of the mRNA. The importance of this second finding is that it suggests a novel function for p38 MAPK that has not previously been considered.
These conclusions are dependent on the inhibitor being specific for this protein kinase. The original description of SB 203580 suggested that it might be a highly specific inhibitor of p38 MAPK (11). Its IC 50 was about 0.5 M, and it did not affect the activity of a wide range of protein kinases (including JNK and extracellular signal-regulated kinase) and phosphatases up to a concentration of 100 M (11). However, two recent reports stress that caution is needed in the use of SB 203580 (29,30). In the first, the ␤ splice variants of JNK-2 were shown to be completely inhibited by 10 -20 M SB 203580 (30). The ␣ splice variants of JNK-2 and JNK-1 were inhibited about 50%, whereas the ␤ forms of JNK-1 were resistant to the drug (30). This raised the possibility that SB 203580 may significantly inhibit JNKs in cells, depending upon the isoforms expressed. An example of this is a recent report that certain chromatographic forms of cardiac myocyte JNKs are strongly inhibited by 10 M SB 203580 (29).
To establish that the effects of SB 203580 on COX-2 gene expression were due to the inhibition of p38 MAPK, we compared the suppression of MAPKAPK-2 activity with the suppression of COX-2 protein and mRNA. We also investigated the sensitivity of human monocyte JNKs activated by LPS to the inhibitor. At 0.1 M SB203580, there was about a 50% inhibition of both COX-2 mRNA and MAPKAPK-2 activity in monocytes, which is consistent with the effects on COX-2 gene expression being due to p38 MAPK blockade.
The LPS-activated JNKs in human monocytes were relatively insensitive to the inhibitor. The second of the two chromatographically distinct forms was inhibited about 10 -20% at 1-2 M SB 203580, whereas the first peak was unaffected. We therefore concluded that the effects of SB 203580 at concentrations up to 1-2 M were unlikely to be due to the inhibition of JNK isoforms, and the effects on COX-2 gene expression were probably due to the inhibition of p38 MAPK.
Our investigation of LPS-induced COX-2 in monocytes was prompted partly by the discrepancy between the finding that SB 203580 inhibited COX-2 mRNA induced by IL-1 in fibroblasts (27) and the suggestion that it might be acting as a translational inhibitor of TNF production in monocytes (25). Young et al. (25) reported a 50% suppression of TNF mRNA in monocytes treated with LPS in the presence of 5 M SKF 86002 (a pyridinyl imidazole very similar to SB 203580) but a 90% inhibition of secreted TNF protein. Further work was carried out in the human monocytic cell line THP 1 (26). In this cell line, TNF protein induced by LPS was inhibited by SKF 86002, whereas mRNA induction was unaffected. Furthermore, TNF mRNA accumulated in a pre-ribosomal compartment (26). It may be that different mechanisms predominate in the immortalized cell line and primary monocytes. Working with SB 203580 at low doses, we have found no discrepancy between the suppression of TNF protein and mRNA. It is possible that p38 MAPK may regulate TNF and COX-2 by similar mechanisms.
We found that p38 MAPK activity is required for the full induction of COX-2 transcription. Analysis of the COX-2 promoter by means of transfection of reporter constructs has implicated several transcription factors in its regulation. These experiments have generally been carried out on transformed cell lines using a variety of stimuli (e.g. phorbol ester, IL-1, TNF, LPS, and growth factors). The transcription factors implicated include NFB (36), NF-IL-6 (36 -38), c-jun (39), signal transducer and activator of transcription 5 (40), and NF-1 (41). A number of reports have emphasized the importance of NFB in the induction of COX-2 in response to LPS in cells of monocyte-macrophage lineage (42)(43)(44)(45). The relative importance of different transcription factors for COX-2 expression in primary monocytes is not known.
It has recently been suggested that the inhibition of p38 . RNA was isolated at the times shown and Northern blotted for COX-2 mRNA and GAPDH mRNA as described under "Experimental Procedures." The graph shows COX-2 mRNA levels normalized to GAPDH mRNA levels for three separate experiments with different batches of cells. Error bars are not shown where they are smaller than the symbol. B, monocytes were left untreated or treated with LPS for 4 h. They were then left untreated (f), or 2 M SB 203580 was added (Ⅺ). RNA was isolated at the times shown and Northern blotted as described in A. The graph shows COX-2 mRNA levels normalized to GAPDH mRNA levels for two separate experiments.
p38 MAPK Regulates COX-2 mRNA Stability and Transcription MAPK by SB 203580 blocks the activation of NFB reporter constructs in cells by a mechanism unrelated to either the phosphorylation and degradation of IB or nuclear translocation and DNA binding of NFB (46 -48). However, in these studies, the inhibitor was used at concentrations substantially above its reported IC 50 , and the observed effects on NFB-dependent transcription were only partial. For example, the response of NFB-dependent reporter constructs to TNF was diminished less than 50% by 10 M SB203580 (47). Because we observe stronger effects on COX-2 expression at much lower concentrations of the inhibitor, it seems unlikely that the inhibition of NFB activity accounts for our findings. However, it is difficult to examine this proposition directly, because the study of events downstream of DNA binding requires the transfection of primary monocytes.
The human COX-2 promoter also contains potential binding sites for members of the cAMP response element binding protein activating transcription factor, Ets/ternary complex factor, and myocyte enhancer factor 2 families; activating transcription factor 2 (4), Ets family members (49,50), and myocyte enhancer factor 2C (17) can all be phosphorylated and activated by p38 MAPK. cAMP-responsive element binding protein (13) is phosphorylated and activated by MAPKAPK-2, which is directly downstream of p38 MAPK. These factors all represent possible targets for the transcriptional regulation of the human COX-2 gene by p38 MAPK.
We have found strong evidence that p38 MAPK stabilizes COX-2 mRNA. Although COX-2 mRNA stability was studied in stimulated cells, it is not clear whether LPS actually regulates this, because p38 MAPK activity had returned to resting levels after 4 h of stimulation. It appears that a basal level of p38 MAPK activity plays a housekeeping role in maintaining COX-2 mRNA stability. Destabilization of COX-2 mRNA is also caused by dexamethasone (51). This effect was described in IL-1-stimulated endothelial cells and inhibited by actinomycin D. Thus, the effects of dexamethasone and SB 203580 differ.
Sequences that determine the stability of an mRNA have been found in the 5Ј untranslated region, the coding region, and the 3Ј untranslated region (52). The 3Ј untranslated region of COX-2 contains 22 AUUUA motifs that are recognized to be important determinants of mRNA instability (51). Both proximal and distal regions have been identified as important in regulating the stability of transfected reporter constructs bearing the 3Ј untranslated region in resting cells (53). It is possible that the instability caused by the p38 MAPK inhibitor is mediated through the AU-rich region proximal to the coding region that is contained in the 4.6-and 2.8-kb transcripts, both of which we have found to be stabilized through this protein kinase.
The results of the present study provide evidence for a new role of p38 MAPK in mRNA stabilization. It will be of interest to determine whether this process occurs in other cell types for other genes and to identify the downstream pathway and molecular mechanisms involved.