The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.The tandem zinc finger protein tristetraprolin (TTP; also known as Nup475, Tis11, or Zfp36) (23,26,40,46,62) is expressed in activated monocytic cells (13, 47) and T lymphocytes (49, 51). It functions to regulate the expression of tumor necrosis factor ␣ (TNF-␣) by binding to a conserved adenosine/uridine-rich element (ARE) within the 3Ј-untranslated region of TNF-␣ mRNA (13,31,32,36,47). TTP promotes both mRNA deadenylation and 3Ј to 5Ј degradation of the mRNA body (35, 37-39), consistent with its ability to recruit several factors involved in these processes (14,25,39,45). The pivotal role of TTP in the regulation of TNF-␣ is illustrated by the proinflammatory phenotype of a TTP Ϫ/Ϫ mouse strain, in which chronic overexpression of TNF-␣ by macrophages results in severe polyarthritis and cachexia (11,13,57). TTP has also been implicated in the posttranscriptional regulation of granulocyte-macrophage colony-stimulating factor (12), interleukin-2 (51), cyclooxygenase 2 (COX-2) (50), and inducible nitric oxide synthase (24). It may also regulate its own expression by binding to an ARE in the 3Ј untranslated region of TTP mRNA (60). The minimum binding site of TTP is the nonameric sequence UUAUUUAUU (2,3,38,65), and it is likely that additional posttranscriptional targets of TTP containing this sequence remain to be identified.The p38 mitogen-activated protein kinase (MAPK) and its downstream kinase MK2 play a central role in the posttranscriptional regulation of inflammatory gene expression in myeloid and other cells (5, 16, 20-22, 33, 34, 54). We and others have therefore investigated interactions of the p38 MAPK pathway with TTP. In a mouse macrophage-like...

Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as tumor necrosis factor ␣, granulocyte/macrophage colony-stimulating factor, and cyclooxygenase 2. Here we investigate the regulation of TTP expression in the mouse macrophage cell line RAW264.7. We show that TTP mRNA is expressed in a biphasic manner following stimulation of cells with lipopolysaccharide and that the second phase of expression, like the first, is dependent on mitogen-activated protein kinase (MAPK) p38. MAPK p38 acts through a downstream kinase to stabilize TTP mRNA, and this stabilization is mediated by an adenosine/uridine-rich region at the 3-end of the TTP 3-untranslated region. Hence TTP is post-transcriptionally regulated in a similar manner to several proinflammatory genes. We also demonstrate that TTP is able to bind to its own 3-untranslated region and negatively regulate its own expression, forming a feedback loop to limit expression levels.

Anti-inflammatory effects of glucocorticoids (GCs) are partly mediated by up-regulation of DUSP1 (dual specificity phosphatase 1), which dephosphorylates and inactivates mitogen-activated protein kinases. We identified putative GC-responsive regions containing GC receptor (GR) binding site consensus sequences that are well conserved between human and mouse DUSP1 loci in position, orientation, and sequence (at least 11 of 15 positions identical) and lie within regions of extended sequence conservation (minimum 65% identity over at least 100 bp). These were located ϳ29, 28, 24, 4.6, and 1.3 kb upstream of the DUSP1 transcription start site. The homology-based approach successfully identified four cis-acting regions that mediated transcriptional responses to dexamethasone. However, there was surprising interspecies divergence in site usage. This could not be explained by variations of the GR binding sites themselves. Instead, variations in flanking sequences appear to have driven the evolutionary divergence in mechanisms of regulation of mouse and human DUSP1 genes. There was a good correlation between the ability of cis-acting elements to respond to GC in transiently transfected reporter constructs and their ability to recruit GR in the context of intact chromatin. We propose that divergence of gene regulation has involved the loss or gain of binding sites for accessory transcription factors that assist in GR recruitment. Finally, a novel GC-responsive region of the human DUSP1 gene contains a highly unusual element, in which three closely spaced GR half-sites are required for potent transcriptional activation by GC.

The powerful anti-inflammatory effects of glucocorticoids (GCs) have been known for more than sixty years, but their molecular mechanisms are still incompletely understood and hotly debated. The GC receptor (GR) was cloned in 1985 and shown to be a transcription factor. Initially, the anti-inflammatory actions of GCs were explained in terms of genes that were up-regulated by the receptor. However, none of these putative mediators seemed able to account for the spectrum of anti-inflammatory responses to GCs. The discovery of a negative regulatory function of GR then shifted the focus away from GC-induced genes as anti-inflammatory mediators. In recent years, attention has begun to move back toward the idea that the antiinflammatory response to GCs is partially dependent on the positive regulation of gene expression by GR. Classical Model of Glucocorticoid ActionSynthetic GCs 2 inhibit expression of many immune and inflammatory mediators in several cell types. For this reason, they are of great use as immunosuppressants and in the treatment of chronic inflammatory diseases (1, 2), yet they can also give rise to a number of side effects of varying severity (3). Both the therapeutic and undesired effects of GCs are mediated by GR, a member of a large family of transcription factors, the nuclear hormone receptors. GR activates or inhibits gene expression via mechanisms known as transactivation and transrepression. According to the current paradigm, the side effects of GCs are largely dependent on dimerization of GR, binding to palindromic GC-response elements, and activation of expression of genes (for example, regulators of gluconeogenesis). On the other hand, anti-inflammatory effects are thought to be largely due to the dimerization-independent transrepression of NF-B, AP-1, and other transcription factors that contribute to pro-inflammatory gene expression (3-5). Hence, novel GR ligands that selectively promote transrepression rather than transactivation might retain anti-inflammatory effects but cause fewer side effects (6, 7).This model of GC action is based largely on the in vivo and in vitro properties of dimerization-defective GR mutants (8). A re-examination of this model (9, 10) has been prompted by several recent findings. (i) Anti-inflammatory functions of GR are not, as originally thought, independent of dimerization (8, 11). (ii) Activation of gene expression by GCs is not invariably dependent on palindromic GC-response elements or on dimerization of GR (9, 12-16). (iii) A growing number of anti-inflammatory mediators have been shown to be up-regulated by GCs (9, 10). Among these factors are phosphatases that inactivate MAPKs. MAPKs and PhosphatasesIn response to extracellular stimuli, MAPKs become activated via the phosphorylation of threonine and tyrosine residues within short activation motifs. The activated MAPKs then modulate cellular responses by phosphorylating a variety of substrates, including transcription factors and downstream effector kinases. Activation of ERK frequently mediates prolifer...