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We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3′-untranslated region (3′-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3′-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control. We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3′-untranslated region (3′-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3′-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control. mitogen-activated protein kinase stress-activated protein kinase interleukin tumor necrosis factor lipopolysaccharide interferon untranslated region AU-rich element enzyme-linked immunosorbent assay total exudate peritoneal macrophage nucleotide(s) tristetraprolin The role of the stress-activated p38 MAPK/SAPK21 protein kinase cascade (reviewed in Ref. 1Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1402) Google Scholar) in inflammation has been defined several years ago by the anti-inflammatory effect of the p38 MAPKα,β/SAPK2a,b inhibitor SB203580 and related compounds (reviewed in Ref. 2Lee J.C. Kassis S. Kumar S. Badger A. Adams J.L. Pharmacol. Ther. 1999; 82: 389-397Crossref PubMed Scopus (334) Google Scholar). Accordingly, it was expected that several components of this kinase cascade may be essential components for early signaling in the inflammatory response and, hence, targets for an anti-inflammatory therapy. Targeted disruption of p38 MAPKα/SAPK2a in mice results in embryonic lethality and impaired IL-1 signaling in differentiated embryonic stem cells in one study (3Allen M. Svensson L. Roach M. Hambor J. McNeish J. Gabel C.A. J. Exp. Med. 2000; 191: 859-870Crossref PubMed Scopus (252) Google Scholar) and in defective erythropoietin expression in a second study (22Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Deletion of one of the two known specific upstream activators of p38 MAPK/SAPK2, the dual-specific MAPK kinase MKK3, leads to a reduction in IL-12 production (4Lu H.T. Yang D.D. Wysk M. Gatti E. Mellman I. Davis R.J. Flavell R.A. EMBO J. 1999; 18: 1845-1857Crossref PubMed Scopus (328) Google Scholar) and impaired tumor necrosis factor (TNF)-induced cytokine expression (5Wysk M. Yang D.D. Lu H.T. Flavell R.A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3763-3768Crossref PubMed Scopus (138) Google Scholar). Interestingly, mice lacking one of the several kinases downstream to p38 MAPK/SAPK2, the serine/threonine kinase MK2 (also designated as MAPK-activated protein (MAPKAP) kinase 2), show a reduction of the bacterial lipopolysaccharide (LPS)-induced biosynthesis of TNF-α, interferon (IFN)-γ, IL-1, IL-6, and nitric oxide resembling the effect of SB203580 at least in part (6Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (687) Google Scholar). The different phenotypes of the mice mutated in components of the p38 MAPK/SAPK2 cascade analyzed so far demonstrate the complexity of this signaling module, which is far away from being a linear step by step reaction (cf. Ref.1Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1402) Google Scholar). In the last few years it became evident that the 3′-untranslated region (UTR) of mRNA contributes to regulation of gene expression by influencing subcellular localization of mRNA, its translation or degradation (reviewed in Ref. 7Conne B. Stutz A. Vassalli J.D. Nat. Med. 2000; 6: 637-641Crossref PubMed Scopus (467) Google Scholar). AU-rich elements (AREs) in the 3′-UTR have been identified as cis-elements that affect mRNA stability (8Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 9Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar) and translation (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (431) Google Scholar). Recently, it has been shown that the p38 MAPK/SAPK2 cascade is involved in regulating mRNA stability via 3′-UTRs of IL-8, IL-6, c-Fos, GM-CSF mRNAs (11Winzen R. Kracht M. Ritter B. Wilhelm A. Xu N. Shyu A.B. Müller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar), via the 3′-UTR of cyclooxygenase 2 mRNA (12Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar), via the 3′-UTR of vascular endothelial growth factor mRNA (13Pages G. Berra E. Milanini J. Levy A.P. Pouyssegur J. J. Biol. Chem. 2000; 275: 26484-26491Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and also via the 3′-UTR of TNF mRNA (14Brook M. Sully G. Clark A.R. Saklatvala J. FEBS Lett. 2000; 483: 57-61Crossref PubMed Scopus (193) Google Scholar). Furthermore, a significant p38 MAPK/SAPK2-dependent contribution to translational control of the ARE in the 3′-UTR of TNF mRNA has also been demonstrated (15Kontoyiannis 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, 23Mijatovic T. Houzet L. Defrance P. Droogmans L. Huez G. Kruys V. Eur. J. Biochem. 2000; 267: 6004-6012Crossref PubMed Scopus (40) Google Scholar). In the case of 3′-UTR-dependent stability of IL-8 mRNA, a role for the kinase MK2 downstream to p38 MAPK/SAPK2 was evident, since mutants of MK2 interfered with IL-8 mRNA stability (11Winzen R. Kracht M. Ritter B. Wilhelm A. Xu N. Shyu A.B. Müller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar). For the mice lacking MK2 it was not clear whether the relatively complex phenotype resulted from secondary effects of the reduced TNF level after LPS induction or whether MK2 is involved in regulation of the different cytokines in response to LPS in parallel. In this paper, we present evidence that deletion of the ARE in the 3′-UTR of the TNF gene restored LPS-induced TNF production in MK2-deficient mice and could even increase TNF production above the wild type level. We analyzed the effect of lacking MK2 on IL-6 biosynthesis in mice where the TNF production was restored and found that IL-6 biosynthesis is still impaired. From the data obtained, a parallel ARE-dependent regulation of TNF and IL-6 biosynthesis by MK2 is proposed. The MK2−/− and TNFΔARE/− mice used for the crossing were on a mixed 129Sv × C57BL/6 background. Genotyping for MK2 was carried out using a three-primer PCR with the oligonucleotides 5′-cgtgggggtggggtgacatgctggttgac (5′-MK2), 5′-ggtgtcaccttgacatcccggtgag (3′-MK2), and 5′-tgctcgctcgatgcgatgtttcgc (Neo). A fragment length of about 500 bp indicates wild type and 800-bp stands for the deletion. For genotyping of TNF the primers 5′-ccttcctcacagagccagccccctc (sense) and 5′-aattacggttaggctcctgtttcc (antisense) were used for PCR. The fragments obtained are about 500 bp for wild type and 620 bp for ΔARE. Spleen cell culture and stimulation was done as described in Ref. 6Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (687) Google Scholar. For macrophage assays, total exudate peritoneal macrophages (TEPM) were isolated by peritoneal lavage from 10-week-old mice, 3 days after a single peritoneal injection of 1.0 ml of thioglycollate broth (4%, Difco Laboratories). For cytokine measurements, TEPM were plated at a density of 5 × 105 cells/well in 24-well tissue culture tissue culture plates. Following adherence, cultures were stimulated with 1 ml of complete RPMI medium + 5% fetal bovine serum in the presence or absence 1 μg/ml LPS (Salmonella enteriditis, Sigma L-6011) for the indicated time points. For the detection of lymphocyte derived TNF, total splenocytes were isolated from mouse spleens via mechanical dissociation. Following erythrocyte lysis, cells were set onto tissue culture plates to remove the adherent fraction of splenocytes corresponding to myeloid cells. The nonadherent fraction was collected, counted, and plated into 24-well plates at a density of 5 × 106 cells/ml/well in the presence or absence of coated 10 μg/ml agonistic monoclonal anti-CD3 antibody (Clone 145-2C11, Low Endotoxin/PharMingen) to activate T-lymphocytes. Supernatants were collected following centrifugation at the indicated time points. Murine TNF immunoreactivity of cultured supernatants (diluted ¼– 110) was measured using specific ELISA as described previously (15Kontoyiannis 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). Murine IL-6 immunoreactivity of cultured supernatants (diluted ½– 18) was measured using a specific ELISA (Endogen) according to the manufacturer's instructions. Bone marrow-derived macrophages were treated with 5 μg/ml LPS (Escherichia coli, Sigma L-6529) for 1 h and subsequent adding of actinomycin D (10 μg/ml). Total RNA was isolated from 1 × 104 cells at different times after actinomycin D treatment using RNApure (PeqLab). RNAs were separated in 1.25% agarose-formaldehyde gels, transfered to nitrocellulose, and hybridized to 32P-labeled IL-6 or TNF cDNA probes (6Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (687) Google Scholar). The mRNA levels were normalized by stripping and reprobing the membranes with a 514-bp β-actin cDNA probe. Northern blots were analyzed by phosphorimaging. Decay of β-globin mRNA carrying an insertion of the IL-6 3′-UTR (nucleotides 767–1022) was monitored using the tet-offsystem as described previously for IL-8 3′-UTR (11Winzen R. Kracht M. Ritter B. Wilhelm A. Xu N. Shyu A.B. Müller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar). For detection of mRNA levels in spleen cells, mice were injected with LPS (S. typhosa; Sigma L-7895) at 5 mg/kg body weight. After 90 min total RNA was isolated from spleens, and 20 μg of RNA were analyzed as described above. To test whether the post-transcriptional regulation of TNF biosynthesis by MK2 proceeds via the ARE of the TNF gene, we analyzed LPS-induced TNF production in systems where both MK2 and the ARE are deleted. In the case that the ARE is a downstream regulatory element of MK2 signaling for TNF biosynthesis, its deletion should lead to MK2 independence of LPS-induced TNF production. We crossed MK2−/− animals with the mouse strain carrying a 69-base pair deletion of the ARE in the 3′-UTR of the TNF gene TNFΔARE/− (15Kontoyiannis 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). Since offspring carrying the genotype MK2−/−TNFΔARE/ΔARE were not viable, we selected mice with the genotypes MK2+/+TNF+/+, MK2−/−TNF+/+, MK2+/+TNFΔARE/+, and MK2−/−TNFΔARE/+ from the F2 generation for further analysis. Splenocytes from these mice were stimulated with LPSin vitro. As seen from Fig.1A, deletion of the ARE in only one allele leads to an almost complete restoration of LPS-induced TNF biosynthesis in the absence of MK2. When thioglycollate-elicited peritoneal macrophages of the different genotypes were stimulated by LPS in vitro (Fig. 1B), a similar result was obtained: deletion of the ARE leads to a re-establishment of TNF production leading to wild type levels after 3 and 6 h and to levels even significantly above the wild type macrophages 12 h after stimulation. This findings indicate that the ARE is downstream to the protein kinase MK2 at the same genetically defined LPS-signaling pathway and that MK2 directly or indirectly targets this element in the TNF-α mRNA. Interestingly, when a spleen cell population was stimulated by antibodies against the CD3 component of the T-cell receptor, deletion of the ARE even leads to a significant increase of TNF production compared with wild type animals, and this increase was independent of MK2 (Fig. 1C). Since it has already been shown that other hemopoietic cell types such as T-cells overproduce TNF as well (15Kontoyiannis 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), this finding indicates that the MK2/ARE axis is also functioning in these cells to activate TNF production following CD3 or antigen engagement similar to the case of LPS-induced macrophages. So far, it was not clear whether the reduced biosynthesis of cytokines like IL-6, IL-1β, and IFN-γ in MK2−/− animals is an indirect effect due to the lack of production of the pro-inflammatory cytokine TNF. Alterations in the serum level of IL-6 have been observed as a secondary effect,e.g. in mice lacking TNF (16Marino M.W. Dunn A. Grail D. Inglese M. Noguchi Y. Richards E. Jungbluth A. Wada H. Moore M. Williamson B. Basu S. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8093-8098Crossref PubMed Scopus (698) Google Scholar) and IL-1β (17Alheim K. Chai Z. Fantuzzi G. Hasanvan H. Di Malinowsky D. Santo E. Ghezzi P. Dinarello C.A. Bartfai T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2681-2686Crossref PubMed Scopus (89) Google Scholar). To decide whether this is also the case in MK2−/− animals, we analyzed IL-6 production in splenocytes and macrophages of the MK2−/− TNFΔARE/+ animals. Although the absolute values of IL-6 production are lower in the ΔARE/+ spleen cell population probably due to a reduced representation of IL-6 producing cells, a comparable relative reduction of IL-6 levels (to about 30%) as a result of ablation of MK2 is observed in both the MK2−/− TNFΔARE/+ and the MK2−/− TNF+/+ background (Fig.2A). Analysis of LPS-stimulated IL-6 production of thioglycollate-elicited peritoneal macrophages shows similar and even more clear data (Fig.2B). For the same MK2 genotype, there is no significant difference in IL-6 production between the ΔARE/+ background, where TNF production is restored and even increased above wild type levels and the TNF +/+ background. This result clearly indicates that the defect in IL-6 biosynthesis in MK2−/− animals is not a secondary effect of reduced TNF production but independent of TNF. When looking at IL-6 mRNA levels in the different spleen cell cultures analyzed (Fig. 2C), a reduction of IL-6 mRNA, which correlates to the decreased biosynthesis of this cytokine in MK2−/−TNF+/+ and MK2−/−TNFΔARE/+ cells (as shown in Fig.2A), is observed. This indicates that the production of IL-6 is regulated at the level of transcription or stability of mRNA. To further analyze the mechanism by which MK2 determines the level of IL-6 mRNA we blocked transcription in LPS-stimulated macrophages from MK2+/+ and MK2−/− animals by using actinomycin D and analyzed half-life of the mRNA by Northern blotting. For comparison, we also determined the stability of TNF mRNA (Fig.3A). As a control, actin mRNA was detected and used to normalize the IL-6 and TNF mRNA level to equal loading. A quantitative of the mRNA (Fig. a half-life of more than 20 h for IL-6 mRNA in LPS-stimulated MK2+/+ macrophages. Interestingly, in macrophages lacking MK2 its half-life is reduced to about 2 This result the evidence for the of MK2 in regulating mRNA TNF mRNA is about than IL-6 mRNA in MK2+/+ cells. This is probably due to the more ARE in the TNF 3′-UTR compared with the AU-rich in the IL-6 3′-UTR the lack of MK2 in the macrophages leads only to a further of TNF mRNA the half-life from to data indicate that MK2 regulate the stability of different cytokine mRNAs to a different For IL-6 the of mRNA could well the about in IL-6 protein biosynthesis in response to LPS in macrophages lacking MK2. the of TNF mRNA in MK2-deficient macrophages compared with wild type cells the reduction of TNF biosynthesis observed. This finding the that MK2 TNF biosynthesis mainly at the level of mRNA translation (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (431) Google Scholar, 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). Since in the 3′-UTR of cytokine mRNAs are for both translational control (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (431) Google Scholar, V. G. J. Huez G. PubMed Scopus Google Scholar) and mRNA stability (8Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 9Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar), we whether the 3′-UTR of IL-6 is to to a reporter mRNA. It is to that in the 3′-UTR of mouse or IL-6 mRNA there are no of are in an region with of in length of the and mouse gene to To role we the 3′-UTR region of IL-6, several AU-rich into the 3′-UTR of a β-globin and the RNA using the in cells (11Winzen R. Kracht M. Ritter B. Wilhelm A. Xu N. Shyu A.B. Müller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar). After of to the cells transcription of the reporter mRNA is and mRNA be As seen in the reporter shows a relatively half-life in cells with a control that the IL-6 the mRNA, which is in its Ref. N. P. C.Y. Shyu A.B. PubMed Scopus Google Scholar). of a of K. H. F. A. K. M. U. Gaestel M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), as well as of the for p38 MAPK/SAPK2, J. T. B. Davis R.J. Mol. Cell. Biol. PubMed Scopus Google Scholar), lead to a significant of the reporter mRNA about Furthermore, a of (11Winzen R. Kracht M. Ritter B. Wilhelm A. Xu N. Shyu A.B. Müller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar), at least the of the reporter mRNA (Fig. half-life of about min reduced to about of a β-globin reporter RNA carrying the 3′-UTR of IL-6 was in the same data indicate that MK2 regulate IL-6 mRNA stability in an 3′-UTR-dependent We have demonstrated that in the LPS response AU-rich elements are downstream to the protein kinase MK2 and that MK2 TNF-α and IL-6 biosynthesis of TNF proceeds through the ARE in the mRNA, since deletion of this ARE leads to restoration of TNF biosynthesis in the absence of MK2. In cells where TNF biosynthesis is MK2 is still for IL-6 that reduced IL-6 levels are not a secondary effect of the reduction of TNF. The defect of IL-6 correlates with decreased mRNA and it is shown that the absence of MK2 leads to a increased degradation of IL-6 mRNA in LPS-stimulated macrophages. Furthermore, MK2 an mRNA reporter carrying the IL-6 which The data not directly that MK2 is also for stimulation of the IL-6 since it is known that MK2 is to the of the cell K. A. Gaestel M. EMBO J. PubMed Scopus Google Scholar), it that regulation of the IL-6 mRNA level by MK2 is mainly the result of stability of its mRNA in the Interestingly, degradation of TNF mRNA in the absence of MK2 is increased only This is in with the finding that the p38 axis mainly translation of TNF mRNA and not its absolute level or stability (6Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (687) Google Scholar, 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) and that TNF mRNA stability and are not significantly in LPS-stimulated macrophages T. Houzet L. Defrance P. Droogmans L. Huez G. Kruys V. Eur. J. Biochem. 2000; 267: 6004-6012Crossref PubMed Scopus (40) Google Scholar). also indicate on the TNF mRNA is also regulated on the stability and such as tristetraprolin E. PubMed Google Scholar) or J.L. R. Mahtani K.R. Sully G. Clark A.R. Saklatvala J. Mol. Cell. Biol. PubMed Scopus Google Scholar), which or increase its have been such as C. Droogmans L. P. Huez G. D. Kruys V. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and M. S. A.R. N. M. B. S. C. Kruys V. M. P. EMBO J. 2000; PubMed Scopus Google Scholar) specific translational regulation of the TNF mRNA. Deletion of from mouse macrophages not stability but the level of TNF mRNA that is with the M. S. A.R. N. M. B. S. C. Kruys V. M. P. EMBO J. 2000; PubMed Scopus Google Scholar), translational control of TNF. MK2 is an element of a signaling mechanism that regulate biosynthesis of different cytokines via different AU-rich 3′-UTRs in parallel and on different So far, it is not clear whether there is a signaling downstream to MK2 with different protein such as or for regulation of stability and such as and for regulation of To it also that MK2 a so far which directly or indirectly with and which stability translation of the mRNA, on the of the ARE and other
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