No abstract

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 M SB 203580 significantly inhibited the LPS induction of COX-2 and tumor necrosis factor protein and mRNAs. The activity of MAPKactivated protein kinase-2 (a substrate of p38 MAPK) in the cells was commensurately reduced. Some isoforms of c-jun N-terminal kinase (which is also activated by LPS) are sensitive to SB 203580; the inhibitor had little effect on monocyte c-jun N-terminal kinases up to 2 M. 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 M). 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 M 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 (1-5). 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␦) (22-24). 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 ...

No abstract

Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-␣) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3 untranslated region (UTR) of TNF-␣ mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated by trans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-␣ mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-␣, the subcellular location of TNF-␣ mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-␣ mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 ؋ g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-␣ mRNA 3 UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-␣ ARE. These results demonstrate that the TNF-␣ ARE is a target of the mRNA-stabilizing factor HuR.

The mitogen-activated protein kinase p38 pathway was originally identi¢ed as a signalling cascade activated by pro-in£ammatory stimuli and cellular stresses, and playing a critical role in the translational regulation of pro-in£ammatory cytokine synthesis. In almost a decade since this discovery, a great deal has been learned about the role of the p38 pathway in the post-transcriptional regulation of pro-in£ammatory gene expression. However, important questions remain to be answered concerning the speci¢city and mechanism or mechanisms of action of p38. This review describes recent progress and remaining puzzles in the ¢eld of post-transcriptional regulation by p38. ß