Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-␣). Macrophages derived from these mice oversecrete TNF-␣, by a mechanism that involves stabilization of TNF-␣ mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-␣ mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-␣ ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-␣ ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-␣ mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-␣ mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-␣ mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-␣ mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TTP protein was used that was incapable of RNA binding, nor when a mutant ARE was present that did not bind TTP. The ability of TTP to promote transcript deadenylation required Mg 2؉ , but not ATP or prior capping of the RNA substrate. Cotransfection and additivity studies with the poly(A) RNase (PARN) demonstrated that TTP promoted the ability of this enzyme to deadenylate ARE-containing, polyadenylated transcripts, while having no effect on transcripts lacking an ARE. There was no effect of TTP to act synergistically with enzymatically inactive PARN mutants. We conclude that TTP can promote the deadenylation of ARE-containing, polyadenylated substrates by PARN. This interaction may be responsible for the ability of TTP and its family members to promote the deadenylation of such transcripts in intact cells.Steady-state levels of cellular mRNAs are determined by the balance between their biosynthesis and turnover. Different mRNAs can exhibit marked differences in turnover rates within the same cell, and the turnover rates of individual mRNAs can also vary significantly in response to changes in the cellular environment. In mammalian cells, the earliest step in mRNA turnover is thought to be removal of the poly(A) tail, or deadenylation, and this process in turn largely determines the overall decay rate of the mRNA (15,16,31,36).It has been appreciated for many years that cis-acting AUrich elements (AREs), often within the 3Ј-untranslated regions (3Ј-UTR) of the mRNA, can confer decreased stability on the mRNAs that contain them (32).

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor ␣ secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor ␣ and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cellfree assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor ␣. Lipolysaccharide (LPS)1 -induced production of tumor necrosis factor ␣ (TNF␣) by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of TNF␣ synthesis occurs in part by modulation of its mRNA stability. This in turn is dependent upon a so-called class II AU-rich element (ARE) found in the 3Ј-untranslated region of TNF␣ transcripts (1). This ARE has been implicated in the regulation of both TNF␣ mRNA stability and its translation (2, 3). Targeted deletion of the TNF␣ mRNA ARE in mice (⌬ARE mice) results in the overproduction of TNF␣ and the development of a systemic inflammatory syndrome (4). A role for the protein serine/threonine kinase p38 has been suggested in ARE-mediated TNF␣ mRNA processing by numerous studies (5-7), and it was found recently that macrophages from the ⌬ARE mice were relatively insensitive to the p38 inhibitor, SB203580 (4). Conflicting studies suggest that these p38 inhibitors can regulate TNF␣ synthesis at either the mRNA stability or protein translation level (8 -10). Mice lacking the p38 substrate MAPKAPK-2 have been reported to have defective TNF␣ synthesis following an LPS challenge (11). In this case, the regulation appears not to be due to a decrease in either TNF␣ mRNA levels or stability but rather to inhibition of translation, suggesting that the effects of the p38 pathway on mRNA stability and translation may be independent and uncoupled.These and other studies have indicated a role for the p38 signaling pathway in the post-transcriptional regulation of TNF␣ synthesis through a mechanism invol...