Decreased Sensitivity of Tristetraprolin-deficient Cells to p38 Inhibitors Suggests the Involvement of Tristetraprolin in the p38 Signaling Pathway

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor α 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 α 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 cell-free 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 α. Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor α 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 α 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 cell-free 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 α. lipopolysaccharide mitogen-activated protein kinase MAPK-activated protein kinase 2 tumor necrosis factor α interleukin AU-rich element tristetraprolin granulocyte-macrophage colony-stimulating factor bone marrow-derived macrophages TNFα-converting enzyme polyacrylamide gel electrophoresis maltose-binding protein calf intestinal alkaline phosphatase enzyme-linked immunosorbent assay wild type Lipolysaccharide (LPS)1-induced production of tumor necrosis factor α (TNFα) by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of TNFα 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 3′-untranslated region of TNFα transcripts (1Xu N. Chen C.Y. Shyu A.B. Mol. Cell. Biol. 1997; 17: 4611-4621Crossref PubMed Scopus (308) Google Scholar). This ARE has been implicated in the regulation of both TNFα mRNA stability and its translation (2Dean J.L. Wait R. Mahtani K.R. Sully G. Clark A.R. Saklatvala J. Mol. Cell. Biol. 2001; 21: 721-730Crossref PubMed Scopus (250) Google Scholar,3Piecyk M. Wax S. Beck A.R. Kedersha N. Gupta M. Maritim B. Chen S. Gueydan C. Kruys V. Streuli M. Anderson P. EMBO J. 2000; 19: 4154-4163Crossref PubMed Scopus (424) Google Scholar). Targeted deletion of the TNFα mRNA ARE in mice (ΔARE mice) results in the overproduction of TNFα and the development of a systemic inflammatory syndrome (4Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar). A role for the protein serine/threonine kinase p38 has been suggested in ARE-mediated TNFα mRNA processing by numerous studies (5Brook M. Sully G. Clark A.R. Saklatvala J. FEBS Lett. 2000; 483: 57-61Crossref PubMed Scopus (193) Google Scholar, 6Wang S.W. Pawlowski J. Wathen S.T. Kinney S.D. Lichenstein H.S. Manthey C.L. Inflamm. Res. 1999; 48: 533-538Crossref PubMed Scopus (100) Google Scholar, 7Badger A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google Scholar), and it was found recently that macrophages from the ΔARE mice were relatively insensitive to the p38 inhibitor, SB203580 (4Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar). Conflicting studies suggest that these p38 inhibitors can regulate TNFα synthesis at either the mRNA stability or protein translation level (8Prichett W. Hand A. Sheilds J. 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In this case, the regulation appears not to be due to a decrease in either TNFα 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 TNFα synthesis through a mechanism involving the ARE. p38 belongs to the growing family of mitogen-activated protein kinases (MAPK). Stress signals, such as LPS, heat shock, and ultraviolet light can initiate a signaling cascade resulting in the activation, by dual tyrosine/threonine phosphorylation, of p38. The activation of p38 results in the phosphorylation of intracellular substrates, among them MAPKAPK-2 and the activating transcription factor 2 (12Obata T. Brown G.E. Yaffe M.B. Crit. Care Med. 2000; 28: N67-N77Crossref PubMed Scopus (320) Google Scholar, 13Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar). There are five known isoforms of p38 (α, β, β2, γ, and δ) in mammals, which differ in expression patterns, activators, inhibitors, and substrate specificity (14Herlaar E. Brown Z. Mol. Med. Today. 1999; 5: 439-447Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). We have shown previously that the RNA-binding protein tristetraprolin (TTP) promotes TNFα mRNA instability in mouse macrophages through direct interactions with its ARE E. PubMed Google Scholar). TTP in mice results in a inflammatory by and E. Lee M.J. Immunity. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This syndrome is the of increased stability of the mRNAs for TNFα and granulocyte-macrophage colony-stimulating factor and increased secretion of these E. PubMed Google Scholar, E. J. 1997; PubMed Scopus Google Scholar, E. 2000; PubMed Google Scholar). We that TTP can be phosphorylated on at by M.J. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and that are other phosphorylation for or protein kinases in These with the of the inflammatory syndrome by the TTP-deficient mice and the that TTP expression is by of the same stimuli that p38, suggested the that TTP could be part of the signaling cascade through which p38 kinase the stability of In this that bone marrow-derived macrophages from TTP-deficient mice are less sensitive than normal macrophages to the p38 kinase inhibitors SB203580 and which inhibit TNFα secretion from these We also that TTP can be phosphorylated by p38 in a cell-free and that phosphorylation of TTP in macrophages can be inhibited by p38 inhibitors. The of TTP did not the of LPS to p38, and p38 derived from TTP-deficient cells was sensitive to the p38 inhibitors in a cell-free Finally, that phosphorylated TTP in cells bound less to an ARE RNA probe than did These that TTP may an role in the effects of p38 activation and may a for Mice in their and of the have been E. Lee M.J. Immunity. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). marrow-derived macrophages were as E. J. 1997; PubMed Scopus Google and after 2 in TNFα were in at a of with of the p38 inhibitors SB203580 A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google and J.R. B. S. Adams J. Griswold D.E. J. Pharmacol. Exp. Ther. Google and the TNFα-converting enzyme B. C. D. R. J. Med. 1999; PubMed Scopus Google were at a LPS was at or were with LPS for and were for the of TNFα by as by the were the of results from of were stimulated with LPS for the indicated and as E. R. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of protein was by gel electrophoresis to and with phosphorylated p38 or p38 was with the kinase was from and in a cell-free kinase assay of the maltose-binding TTP or maltose-binding TTP fusion as These recombinant were in as of TTP with maltose-binding protein and on as previously J.A. PubMed Scopus Google Scholar). We also and the protein as a The of fusion protein expression and be The for p38 phosphorylation of the fusion were as by the SB203580 or was to inhibit p38 kinase activity at were by by kinase assays in which p38 was were stimulated with LPS for and the cells were as E. R. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), with the following and were from the and the kinase was at for was as the were by by TTP phosphorylation in intact of were with and for in the of The p38 inhibitors SB203580 and were for by LPS for were and as and of were immunoprecipitated a TTP M.J. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). were by by and a and the cell the TTP expression E. J.R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google and the TTP as a the was by the the M.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google after the of of the fusion was by cells to as were and of cells with in was as E. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of or of TTP expression of was in extracts were after the of the as E. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the effect of TTP on its binding to involving cells and with after the of the the was with calf in for 2 at was and the cells were for an 2 cell the was and the cell were in The cells were in of the previously E. J.R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). The extracts were in and in an in a for The were for at at for and the and at Cell extracts from cells with of were in and and were at for with or calf intestinal alkaline phosphatase The activity was by of The were and at for of were and to after which the were and for Cell extracts were in a from cells with or The extracts of protein in of and from independent but was and on was to a of to the were at for 2 after which the same of was to the other This was that the from of at the of the The was that the phosphatase was the in and after the in the other the of the the extracts were and at for of these of protein in of the and were with of for and was by were in with a at a recombinant TTP fusion protein with maltose-binding and RNA gel the RNA probe was derived from of the AU-rich element in the mouse 3′-untranslated This was by the of of the was by the RNA with with was as a and the in was The resulting were from the through the gel extracts from cells with TTP expression were with and the phosphatase was either or after the as of protein in of the and were to of the inhibitors, 2 of RNA and in assays as E. J.R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, E. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We the effect of p38 inhibitors, SB203580 and on the secretion of TNFα by derived from either or p38 inhibitors decreased TNFα synthesis in at than to inhibit TNFα synthesis by in to p38 inhibitors among have been reported previously B. T. M.J. T. J. 2001; PubMed Scopus Google Scholar, D.E. P. W. B. J.E. J.L. J. Pharmacol. Exp. Ther. 1999; Google Scholar). shown in SB203580 inhibited the secretion of TNFα by derived from mice The for this was to the in studies with mouse and macrophages (4Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar). However, the same was in in derived from in TTP was inhibition of TNFα production at of SB203580 was the inhibited the production of TNFα by the with an of to the However, in the the was to the with an of results were the cells were stimulated with a of LPS, and to the p38 inhibitors not The specificity of this of to p38 inhibitors was by a of TNFα the B. C. D. R. J. Med. 1999; PubMed Scopus Google Scholar). in both and to with the for inhibition of TNFα secretion in the and 2 the LPS could p38 to the same extent in the and the cells were stimulated with LPS by for phosphorylated and p38. This in the phosphorylation of p38, which at and was after The of phosphorylation was in and cells suggesting that activation of p38 in the of recombinant TTP could as a substrate for p38, cell-free kinase assays p38, in the of a fusion and recombinant mouse and TTP fusion with maltose-binding protein or as shown in p38 was to both and for both fusion in this cell-free SB203580 and and inhibited this phosphorylation was the substrate was the same amount of recombinant phosphorylation of either fusion protein or was the p38 kinase was from the and the of kinases in the The kinase fusion protein to be phosphorylated in these and could be as a of and This phosphorylation was also by the p38 inhibitors. activation of the p38 kinase pathway could to TTP phosphorylation in intact derived from both and mice with and stimulated them with LPS for in the or of the p38 inhibitors these with TTP protein levels were not S. and P. J. shown in a TTP immunoprecipitated a protein of phosphorylation was increased after LPS 2 with with either SB203580 or decreased the level of TTP phosphorylation by The protein was in the cells its as derived from both and mice were stimulated with LPS for and p38 was immunoprecipitated with a for p38. This was in a cell-free protein kinase recombinant as the in the or of shown in p38 immunoprecipitated from either or cells could to the same extent 2 and and this phosphorylation could be inhibited by and phosphorylated were in the of immunoprecipitated p38 or in the of and the that of kinases or were in the These results suggested that p38 kinase could be by LPS in both the and the macrophages and that in both the kinase was sensitive to the of p38 inhibitors in the cell-free kinase We the effect of TTP on its binding to a ARE We that in of TTP in cells with these be for the phosphorylated protein are in A. In this TTP was the in a cell of the could be by the at with and the decrease in was also by a to a in the gel We on cell extracts of TTP from independent but the protein to the of phosphorylated TTP in these and for other to that the extracts phosphorylated and TTP of after the with the phosphatase in the to both of the and in case, the was the with and in the other after the In this the of the of the the of the of the phosphatase The results of a of these are shown in in which TTP was by an a TTP fusion protein with maltose-binding In case, with to of resulting in the of the or the phosphorylated protein to a of to these of extracts as phosphorylated and These same of from the independent cell extracts were in RNA as a probe the ARE from mouse This was in to a TNFα probe the in the of TTP is in and not to the same on the as the in to TNFα ARE These gel assays were with of that in RNA binding activity could be of was in on the same and a gel was in of an from such is shown in C. The ARE RNA probe as the of the an of from cells with was in the gel was of the with the of a this protein is in extracts from this ARE probe is extracts of protein the phosphorylated and TTP were was a in the of the protein to the ARE probe the This is and in but was at protein by probe the same was probe was by the protein in This is the probe in and and in and results were in the other of the gel for the in as well as the for the five of The from of these were by and to of of for the as independent and the phosphorylated to the by a this the to in were with the to the protein bound probe than the phosphorylated protein A the to and in in a in probe binding by the protein the protein to and the was not In a of the as a of the in by the to in C. from of a were and the were These are in A. these could not be of the it is that the protein probe at of protein than the phosphorylated at the at the and at the A was on the of the protein extracts to probe to its normal at the of the In this case, the results of the were by them as a of the from the probe to in C. In case, the in this were for of and these were by a These results are shown in B. the protein amount of the protein probe than the phosphorylated protein of probe of the was of probe by the protein by the phosphorylated protein Finally, at of of probe by the protein with by the phosphorylated protein of the in these studies was that macrophages derived from mice were less sensitive than normal cells to the effects of p38 inhibitors on TNFα These in turn suggest that TTP is part of the p38 cascade, initiated by LPS in that leads ultimately to stimulated TNFα secretion. In the mouse macrophages of TNFα to of TNFα mRNA E. PubMed Google Scholar, E. J. 1997; PubMed Scopus Google Scholar). This occurs of the of which to the ARE in the TNFα it by a mechanism E. PubMed Google Scholar, E. J.R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). p38 has been implicated previously in the regulated synthesis of inflammatory in the of p38 is to its effects on the regulation of TNFα mRNA stability (5Brook M. Sully G. Clark A.R. Saklatvala J. FEBS Lett. 2000; 483: 57-61Crossref PubMed Scopus (193) Google Scholar, 6Wang S.W. Pawlowski J. Wathen S.T. Kinney S.D. Lichenstein H.S. Manthey C.L. Inflamm. Res. 1999; 48: 533-538Crossref PubMed Scopus (100) Google Scholar, R. M. B. A. Chen C.Y. Shyu A.B. M. Gaestel M. EMBO J. 1999; PubMed Scopus Google Scholar). These and other results have suggested a p38 and TTP in the regulation of TNFα mRNA stability and TNFα secretion. TNFα secretion by macrophages is a sensitive to the of p38 inhibitors, such as of the pyridinyl imidazole the effect of of this class of p38 inhibitors on the secretion of TNFα by derived from and cells to the inhibitors with a decrease in the secretion of to in a cell (5Brook M. Sully G. Clark A.R. Saklatvala J. 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Google Scholar), was inhibited secretion of However, the this was to the in the with a that with the These results suggest that TTP may be a component of the cascade by which p38 TNFα the that p38 activation could be in the of that LPS stimulated the phosphorylation of p38 with a in both and cells and that levels of p38 were in the cell kinase assays with p38 and recombinant and mouse fusion that p38 could the phosphorylation of TTP in a inhibited by both SB203580 and In TTP phosphorylation in intact macrophages was stimulated were to LPS, and this stimulated phosphorylation was by the of the p38 inhibitors. These results suggest that TTP can be phosphorylated in macrophages after activation of p38, not that TTP is phosphorylated by p38 in intact Finally, that the p38 in the cells after to LPS was to the fusion protein in a cell-free kinase a that was sensitive to the p38 This is evidence the of activation or expression of p38 in the of the and cells is that the cells five TNFα after LPS than their as have shown previously E. J. 1997; PubMed Scopus Google Scholar). However, both cell were to which TNFα production by B. C. D. R. J. Med. 1999; PubMed Scopus Google through a mechanism that is not to p38 kinases J. Pharmacol. 1999; PubMed Scopus Google Scholar). This be in the effects of the inhibitors in these is the expression of p38 in macrophages have been shown to the and D. M. Manthey C.L. J. 1999; Google Scholar). These differ in their substrate with p38 of activation, and to p38 inhibitors D. M. Manthey C.L. J. 1999; Google Scholar, S. McDonnell P.C. Hand Lee J.C. Young P.R. 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J. 1993; Google Scholar). The of p38 inhibitors in the of inflammatory has been and inhibitors of this type have been in of inflammatory A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google Scholar, J.R. B. S. Adams J. Griswold D.E. J. Pharmacol. Exp. Ther. Google Scholar, D.E. P. W. B. J.E. J.L. J. Pharmacol. Exp. Ther. 1999; Google Scholar). However, is known to the of the signaling that regulate this The results reported suggest the of TTP from p38. TTP is a that phosphorylation for kinases M.J. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and it can be phosphorylated in and in by of these found that TTP with alkaline phosphatase bound an ARE probe with than did the phosphorylated TTP appears to be in macrophages E. PubMed Google but can the and in M.J. Mol. 1996; 10: Google this is to the of p38 which is to be from the to the by its phosphorylated substrate R. S. R. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). A for a is shown in to this TTP was phosphorylated by p38 or kinase from p38, TTP could its decreased binding to the TNFα mRNA the stability of TNFα mRNA and the synthesis and secretion of this In decreased TTP phosphorylation resulting from the of the p38 inhibitors could TTP binding to the TNFα resulting in the and of the TNFα and in the levels of protein The in the of TTP that in of TTP TNFα mRNA stability and increased TNFα secretion in both the and of p38 inhibitors and This mechanism could the cells were relatively or to the p38 inhibitors. studies are to the interactions TTP and p38 other and the in which these interactions However, this of TTP in the p38 pathway of the regulation of the inflammatory response by p38 and may a for the development of this was were that are to In the W. F. J. J. 2001; PubMed Google Scholar), the that TTP could be phosphorylated by p38 both in a cell-free and in the In G. M. R. C. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google the regulation of mRNA which previously could be by TTP in a assay G. R. C. Mol. Cell. Biol. 2000; PubMed Scopus Google and have shown could be by TTP in a assay E. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The the of the and p38 kinase on the regulation of mRNA stability by in These that TTP was not to be a direct substrate of either the activation of either pathway to the effect of TTP on on the phosphorylation of TTP were Finally, and D. A. E. Gaestel M. R. R. Kollias G. EMBO J. 2001; PubMed Scopus Google that macrophages derived from TTP-deficient mice were sensitive to the p38 their from in of of the cells to the inhibitors, a for the inhibitors, and a of a for this in be to for of these We for the and and for of the