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p38α Stabilizes Interleukin-6 mRNA via Multiple AU-richElements

2007; Elsevier BV; Volume: 283; Issue: 4 Linguagem: Inglês

10.1074/jbc.m707573200

1083-351X

Wenpu Zhao, Min Liu, Keith L. Kirkwood,

NF-κB Signaling Pathways

AU-rich elements (AREs) in the 3′-untranslated region (UTR) ofunstable mRNA dictate their degradation or mediate translational repression.Cell signaling through p38α MAPK is necessary for post-transcriptionalregulation of many pro-inflammatory cytokines. Here, the cis-actingelements of interleukin-6 (IL-6) 3′-UTR mRNA that required p38αsignaling for mRNA stability and translation were identified. Using mouseembryonic fibroblasts (MEFs) derived from p38α+/+ andp38α–/– mice, we observed that p38α isobligatory for the IL-1-induced IL-6 biosynthesis. IL-6 mRNA stability ispromoted by p38α via 3′-UTR. To understand the mechanism ofcis-elements regulated by p38α at post-transcriptional level,truncation of 3′-UTR and the full-length 3′-UTR with individualAUUUA motif mutation placed in gene reporter system was employed.Mutation-based screen performed in p38α+/+ andp38α–/– mouse embryonic fibroblast cells revealedthat ARE1, ARE2, and ARE5 in IL-6 3′-UTR were targeted by p38α,and truncation-based screen showed that IL-6 3′-UTR-(56–173) wastargeted by p38α to stable mRNA. RNA secondary structure analysisindicated that modulated reporter gene expression was consistent withpredicted secondary structure changes. AU-rich elements (AREs) in the 3′-untranslated region (UTR) ofunstable mRNA dictate their degradation or mediate translational repression.Cell signaling through p38α MAPK is necessary for post-transcriptionalregulation of many pro-inflammatory cytokines. Here, the cis-actingelements of interleukin-6 (IL-6) 3′-UTR mRNA that required p38αsignaling for mRNA stability and translation were identified. Using mouseembryonic fibroblasts (MEFs) derived from p38α+/+ andp38α–/– mice, we observed that p38α isobligatory for the IL-1-induced IL-6 biosynthesis. IL-6 mRNA stability ispromoted by p38α via 3′-UTR. To understand the mechanism ofcis-elements regulated by p38α at post-transcriptional level,truncation of 3′-UTR and the full-length 3′-UTR with individualAUUUA motif mutation placed in gene reporter system was employed.Mutation-based screen performed in p38α+/+ andp38α–/– mouse embryonic fibroblast cells revealedthat ARE1, ARE2, and ARE5 in IL-6 3′-UTR were targeted by p38α,and truncation-based screen showed that IL-6 3′-UTR-(56–173) wastargeted by p38α to stable mRNA. RNA secondary structure analysisindicated that modulated reporter gene expression was consistent withpredicted secondary structure changes. Interleukin-6(IL-6) 2The abbreviations used are: IL-6interleukin-6TNFtumor necrosisfactorLPSlipopolysaccharideAREAU-rich elementUTRuntranslatedregionMAPKmitogen-activated protein kinaseMK2MAPK-activated proteinkinase 2MEFmouse embryonic fibroblastGAPDHglyceraldehyde-3-phosphatedehydrogenase. 2The abbreviations used are: IL-6interleukin-6TNFtumor necrosisfactorLPSlipopolysaccharideAREAU-rich elementUTRuntranslatedregionMAPKmitogen-activated protein kinaseMK2MAPK-activated proteinkinase 2MEFmouse embryonic fibroblastGAPDHglyceraldehyde-3-phosphatedehydrogenase. is amultifunctional cytokine produced by lymphocytes, macrophages, fibroblasts,synovial cells, endothelial cells, glia cells, and keratinocytes(1Ray A. Tatter S.B. Santhanam U. Helfgott D.C. May L.T. Sehgal P.B. Ann. N. Y. Acad.Sci. 1989; 557 (discussion 361–352): 353-361Crossref PubMed Scopus (76) Google Scholar). IL-6 expression wasinduced by a variety of stimuli, including interleukin-1(IL-1), tumor necrosisfactor (TNF), platelet-derived growth factor, and lipopolysaccharide (LPS)(1Ray A. Tatter S.B. Santhanam U. Helfgott D.C. May L.T. Sehgal P.B. Ann. N. Y. Acad.Sci. 1989; 557 (discussion 361–352): 353-361Crossref PubMed Scopus (76) Google Scholar). Evidence exists for therole of IL-6 in various diseases, including inflammation and cancer. Forexample, constitutive overexpression of IL-6 by synovial tissues of rheumatoidpatients has been reported (2Hirano T. Matsuda T. Turner M. Miyasaka N. Buchan G. Tang B. Sato K. Shimizu M. Maini R. Feldmann M. et al.Eur. J. Immunol. 1988; 18: 1797-1801Crossref PubMed Scopus (666) Google Scholar)and the role of IL-6 in human malignancy is most clearly established inmultiple myeloma (3Kawano M. Hirano T. Matsuda T. Taga T. Horii Y. Iwato K. Asaoku H. Tang B. Tanabe O. Tanaka H. et al.Nature. 1988; 332: 83-85Crossref PubMed Scopus (1454) Google Scholar) wheremonoclonal antibodies directed against IL-6 enhance the effectiveness ofchemotherapy in this disease. There is also evidence that IL-6 acts as anautocrine growth factor in a number of human epithelial malignancies,including renal, lung, and prostate cancer(4Okamoto M. Lee C. Oyasu R. Cancer Res. 1997; 57: 141-146PubMed Google Scholar, 5Tachibana M. Miyakawa A. Nakashima J. Murai M. Nakamura K. Kubo A. Hata J. Cell TissueRes. 2000; 301: 353-367Crossref PubMed Scopus (19) Google Scholar, 6Fu J. Zheng J. Fang W. Wu B. Chin. Med. J. (Engl). 1998; 111: 265-268PubMed Google Scholar).To clarify the mechanism involved in the abnormal expression of IL-6, it isimperative to investigate the mechanism of the IL-6 gene expression underphysiological conditions. interleukin-6 tumor necrosisfactor lipopolysaccharide AU-rich element untranslatedregion mitogen-activated protein kinase MAPK-activated proteinkinase 2 mouse embryonic fibroblast glyceraldehyde-3-phosphatedehydrogenase. interleukin-6 tumor necrosisfactor lipopolysaccharide AU-rich element untranslatedregion mitogen-activated protein kinase MAPK-activated proteinkinase 2 mouse embryonic fibroblast glyceraldehyde-3-phosphatedehydrogenase. The production of IL-6 is under transcriptional and post-transcriptionalcontrol (7Kishimoto T. Annu. Rev.Immunol. 2005; 23: 1-21Crossref PubMed Scopus (785) Google Scholar). Thetranscriptional regulation of IL-6 expression has been thoroughlyinvestigated. IL-1-responsive element was mapped within both the murine andhuman IL-6 promoter region, whereas regulation at the post-transcriptionallevel has not been as thoroughly investigated(8Isshiki H. Akira S. Tanabe O. Nakajima T. Shimamoto T. Hirano T. Kishimoto T. Mol. Cell.Biol. 1990; 10: 2757-2764Crossref PubMed Scopus (242) Google Scholar,9Clark A. ArthritisRes. 2000; 2: 172-174Google Scholar). Post-transcriptionalmechanisms controlling pre-mRNA splicing and maturation, as well as mRNAtransport, turnover, and translation, critically influence gene expressionprograms in mammalian cells. Central to the post-transcriptional regulatoryevents is the interaction of RNA with RNA-binding proteins that influencetheir splicing, localization, stability, and association with the translationmachinery(10Derrigo M. Cestelli A. Savettieri G. Di Liegro I. Int. J. Mol. Med. 2000; 5: 111-123PubMed Google Scholar, 11Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell. Biol. 2002; 3: 195-205Crossref PubMed Scopus (1119) Google Scholar, 12Hollams E.M. Giles K.M. Thomson A.M. Leedman P.J. Neurochem. Res. 2002; 27: 957-980Crossref PubMed Scopus (171) Google Scholar).Many ribonucleoprotein complexes that govern mRNA stability and translation inresponse to various stimuli (e.g. developmental, stress-inducing,immune, and proliferative) are composed of transcripts that bear adenine anduridine (AU)-rich elements (AREs) in the 3′-untranslated region(-3′-UTR) and the RNA-binding proteins (ARE-RNA-binding proteins)(13Bevilacqua A. Ceriani M.C. Capaccioli S. Nicolin A. J. Cell. Physiol. 2003; 195: 356-372Crossref PubMed Scopus (271) Google Scholar,14Zhang T. Kruys V. Huez G. Gueydan C. Biochem. Soc. Trans. 2002; 30: 952-958Crossref PubMed Google Scholar). Many ARE-RNA-bindingproteins have been described that regulate the stability of target mRNAs,their translation, or both processes: AU-binding factor 1, tristetraprolin, Khomology splicing-regulatory protein, butyrate response factor-1, the Huprotein family (HuR, HuB, HuC, and HuD), T-cell-restricted intracellularantigen-1, and the T-cell-restricted intracellular antigen-1-related proteinTIAR(15Antic D. Keene J.D. Am. J.Hum. Genet. 1997; 61: 273-278Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 16Brennan C.M. Steitz J.A. Cell Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (878) Google Scholar, 17Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 18Gueydan C. Droogmans L. Chalon P. Huez G. Caput D. Kruys V. J. Biol. Chem. 1999; 274: 2322-2326Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 19Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar, 20Min H. Turck C.W. Nikolic J.M. Black D.L. Genes Dev. 1997; 11: 1023-1036Crossref PubMed Scopus (279) Google Scholar, 21Stoecklin G. Colombi M. Raineri I. Leuenberger S. Mallaun M. Schmidlin M. Gross B. Lu M. Kitamura T. Moroni C. EMBO J. 2002; 21: 4709-4718Crossref PubMed Scopus (180) Google Scholar, 22Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (496) Google Scholar). In recently years, significant information has accumulated relative to therole of AREs of pro-inflammatory gene as the targets of the mitogen-activatedprotein kinase (MAPK) p38 pathway, and many of the effects appear to bemediated by its substrate, MAPK-activated protein kinase 2 (MK2)(23Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. et al.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3133) Google Scholar,24Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. CellBiol. 1999; 1: 94-97Crossref PubMed Scopus (686) Google Scholar). It has been shown thatp38 MAPK/MK2 cascade is involved in regulating mRNA stability via3′-UTRs of TNF, IL-8, granulocyte macrophage-colony stimulating factor,Cox-2 and vascular epidermal growth factor mRNA(25Lee J.C. Kassis S. Kumar S. Badger A. Adams J.L. Pharmacol. Ther. 1999; 82: 389-397Crossref PubMed Scopus (333) Google Scholar, 26Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 27Pietersma A. Tilly B.C. Gaestel M. de Jong N. Lee J.C. Koster J.F. Sluiter W. Biochem.Biophys. Res. Commun. 1997; 230: 44-48Crossref PubMed Scopus (182) Google Scholar).In the MK2 knockout mouse strain, LPS-induced expression of IL-6 was blockedat both protein and mRNA levels, while expression of both TNFα andinterferon γ was blocked at the protein but not the mRNA level(24Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. CellBiol. 1999; 1: 94-97Crossref PubMed Scopus (686) Google Scholar). For IL-1-induced IL-6biosynthesis, the role of p38 is involved in vivo atpost-transcriptional level is poorly understood. Some p38α inhibitor and overexpression data indicate that p38αis involved in the regulation of IL-6 production at the post-transcriptionallevel (26Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). However, nocis-elements in IL-6 3′-UTR targeted by p38α have beenconclusively identified. To avoid some of the potential artifacts associatedwith inhibitor and overexpression experiments, we have performed the analysisof IL-6 production in stable fibroblast cell lines derived fromp38α+/+ and p38α–/– embryos. Wefound that p38α is critical for the IL-1-induced IL-6 production andmRNA stability of IL-6 is promoted by p38α via the IL-6 3′-UTR.Deletion and mutation analysis identified three ARE elements that requirep38α signaling, and IL-6 3′-UTR-(56–173) is critical forp38α to promote mRNA stability. We have further analyzed the secondarystructure of IL-6 3′-UTR and mutants by using a computational approach.Interestingly, the data indicated that p38α targeting IL-6 mRNA requiredthe secondary structure of wild-type 3′-UTR. Cell Culture—Mouse embryo fibroblasts (MEFs) were derivedfrom p38α+/+ and p38α–/– mice.The establishment of MEFs has been previously described(1Ray A. Tatter S.B. Santhanam U. Helfgott D.C. May L.T. Sehgal P.B. Ann. N. Y. Acad.Sci. 1989; 557 (discussion 361–352): 353-361Crossref PubMed Scopus (76) Google Scholar). Cells were grown inDulbecco's minimal essential medium supplemented with 10% fetal calf serum,glutamine (2 mm), penicillin (100 units/ml), streptomycin (100mg/ml), and incubated at 37 °C in 5% CO2. The MEFs weretransfected with Lipofectamine Plus Reagent (Invitrogen) according to themanufacturer's protocol. Reagents—IL-6 enzyme-linked immunosorbent assay kit andrecombinant mouse and recombinant mouse IL-1β and TNFα werepurchased from R&D Systems. Lipopolysaccharide from Escherichiacoli strain 0127:B8 was purchased from Sigma. Dual-Luciferase ReporterAssay System was purchased from Promega. SB203580 was from Calbiochem.Actinomycin D was from Invitrogen. Assays-on-Demand Gene Expression Products(mIL-6 and mGAPDH) and Taq Man Universal PCR Master Mix were fromApplied Biosystems. Plasmids—pcDNA3-p38α and p38AF were gifts from J. Han(Scripps Institute). IL-6 3′-UTR-(1–403) were amplified from themIL-6 cDNA built on pCR2.1 by PCR using primers terminating in XbaIrecognition sequences: forward,5′-CCTCTAGATAGTGCGTTATGCCTAAGCA-3′; reverse,5′-CCTCTAGAGTTTGAAGACAGTCTAAACAT-3′ and were ligated in the uniqueXbaI site of the pGL3 promoter vector (Promega Corp.). Deletion constructs andARE mutants were generated from the full-length 3′-UTR as the templateby PCR. The forward primer for 3′-UTR-(1–70) was the same as thatfor the full length; the reverse primer was5′-CCTCTAGAACAACAGGAT-3′. The forward primer for3′-UTR-(56–173) was5′-CCTCTAGAAATATATCCTGTTGTCAGGTAT-3′; the reverse primer was5′-CCTCTAGAAGTTTACTTAT-3′. The forward primer for3′-UTR-(172–403) was5′-GGACTCTAGACTTTAAGTTAATTTATG-3′; the reverse primer was the sameas for the full length. All ARE mutants were generated by PCR-directedmutagenesis with anchor primers and mutagenic primers. The forward mutagenicprimers were: M1, 5′-TTAAGGGATTGATAATTTAAATAAG-3′; M2,5′-TGATAAGGGAAATAAGTAAACTTAAG-3′; M3,5′-AGTTAAGGGATGATTGATATTTATTATTTTTATG-3′; M4,5′-TGATAGGGATTATTTTTATGAAGTGTCACTT-3′; and M5,5′-GCTAAGGGAAATATGTTTTTAAAGAAATCCTTG-3′. The reversemutagenic primers were: M1,5′-TATCAATCCCTTAAAAATAATTAAAATAG-3′; M2,5′-TTATTTCCCTTATCAATAAATTAAAAAT-3′; M3,5′-ATCATCCCTTAACTTAAAGTTTACTTAT-3′; M4,5′-AATAATCCCTATCAATCATAAATTAACTT-3′; and M5,5′-ATATTTCCCTTAGCAATTCATTGGGT-3′. All plasmids weresequenced to verify authenticity. Luciferase Assay—Luciferase activity was determined using aluciferase assay system, following the manufacturer's protocol (Promega,Madison, WI) Briefly, cell monolayers in 12-well plates were removed byscraping into 200 μl of reporter lysis buffer. Cells were vortexed, andcellular debris was removed by centrifugation (30 s at 12,000 ×g). Luciferase activity was measured using a luminometer (LMaxII 384,Molecular Devices). A Renilla luciferase reporter vector was includedin every experiment for transfection efficiency control. Relative luciferaseactivity was determined and normalized to Renilla luciferaseactivity. Northern Blot—RNA was prepared from actively growing cellsby using the TRIzol reagent (Invitrogen) as specified by the manufacturer. 10μg of RNAs was separated in 1.25% Seakem® gold-agarose, transferredonto a nylon membrane (GeneScreen Plus, Dupont), and cross-linked by UVtreatment (Stratalinker apparatus). The membrane was hybridized in ULTRA Hybsolution (Ambion), using 32P-labeled firefly luciferase probe(nucleotides 491–1049). The mRNA levels were normalized by 28 S RNA. Reverse Transcription-PCR and Quantitative Real-timePCR—IL-6 mRNA expression was analyzed by reverse transcription-PCRand quantitative real-time PCR. First strand cDNA was synthesized from RNA(600 ng) using SuperScript® III reverse transcriptase (Invitrogen). Firststrand cDNA was used for PCR with specific oligonucleotide primers for mIL-6:forward, 5′-ATGAAGTTCCTCTCTGCAAGAGACT-3′; reverse,5′-CACTAGGTTTGCCGAGTAGATCTC-3′. Quantitative real-time PCR withprimers were designed by Applied Biosystems (mIL-6, mm00446190; mGAPDH,mm99999915) using an ABI7500 thermocycler, and the gene expression wasanalyzed by using 7500 System SDS v1.4 software. RNA Secondary Structure and Analysis—RNA secondary structureanalysis was performed using the M-fold program(28Zuker M. MethodsEnzymol. 1989; 180: 262-288Google Scholar). The folding temperaturewas fixed at 37 °C. Statistical Analysis—Student t tests were utilizedto obtain individual p values using GraphPad Prism version 4.03 forWindows, GraphPad Software, San Diego, CA. p38α Mediates IL-1 Receptor-induced IL-6Production—To investigate the in vivo function ofp38α in the mechanism of IL-6 production, MEFs were prepared fromp38α+/+ and p38α–/– mouseembryos and stimulated with IL-1β, Escherichia coli LPS andTNFα for 24 h. IL-6 content from culture supernatant was analyzed byenzyme-linked immunosorbent assay (Fig.1A). Remarkably, p38α–/–MEFs incubated with IL-1β show significantly decreased amounts of IL-6production compared with p38α+/+ MEFs. E. coli LPS,and TNFα did not induce the dramatic differences of IL-6 productionbetween wild-type and p38α–/– MEFs. To confirm that this defect was caused by p38α deficiency rather thangenetic mutations associated with IL-1R signaling, wild-type p38α andkinase-dead p38 (AF) cDNAs were transiently transfected into MEFs, and IL-6production induced by IL-1β measured. Results indicate that wild-typep38α but not p38AF restored the IL-6 production inp38α–/– MEFs(Fig. 1B). Incontrast, overexpression of p38α in p38α+/+ MEFsproduced IL-6 in amounts similar to those of parental control cells. Asexpected, overexpression of kinase-dead p38AF decreased the IL-6 production inp38α+/+ MEFs. Interestingly, IL-6 production inp38α–/– MEFs was also decreased by p38AFoverexpression. These data strongly indicate that p38α is critical forIL-1R-induced IL-6 production. p38α Stabilizes Endogenous IL-6 mRNA—Previousdata have indicated that the stability of IL-6 mRNA is regulated by MK2 via anAU-rich 3′-UTR of the IL-6 mRNA(29Neininger A. Kontoyiannis D. Kotlyarov A. Winzen R. Eckert R. Volk H.D. Holtmann H. Kollias G. Gaestel M. J. Biol. Chem. 2002; 277: 3065-3068Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). To confirm whetherp38α signaling was required for IL-6 mRNA turnover, the stability ofendogenous IL-6 mRNA transcripts were monitored by actinomycin D pulse chase.After 24 h of IL-1β treatment with or without SB203580 (p38αinhibitor), actinomycin D was added to p38α+/+ MEFs, and IL-6mRNA levels were measured at the indicated time points. The addition ofSB203580 resulted in a rapid decrease in IL-6 mRNA level(Fig. 2A). The densitywas measured and shown in Fig.2B. The analysis revealed that SB203580 decreased IL-6mRNA t½ from 3.7 to 1.7 h. Importantly, thestability of endogenous IL-6 mRNA was compared in the p38α+/+and p38α–/– MEFs. After MEFs were stimulated byIL-1β for 24 h, actinomycin D was added, and IL-6 mRNA levels weremeasured by real-time PCR. Fig.2C shows a relative shorter half-life inp38α–/– MEFs (t½ = 1.9h) compare with p38α+/+ cells (t½= 4.6 h) indicating that IL-6 mRNA is stabilized by p38α. p38α Targets IL-6 3′-UTR to Promote mRNAStability—Because AREs in the 3′-UTR of many cytokine mRNAsare responsible for both translational control and mRNA stability, we examinedwhether the ARE-containing 3′-UTR of IL-6 is sufficient to conferp38-dependent stabilization to a reporter mRNA. The full length of3′-UTR of IL-6 contains five AREs with no overlapping pentanucleotideAUUUA core motifs. Instead, the AUUUA motifs are scattered throughout the IL-63′-UTR where both the human and mouse IL-6 3′-UTR exhibit highhomology to each other(30Stoecklin G. Stoeckle P. Lu M. Muehlemann O. Moroni C. Rna. 2001; 7: 1578-1588PubMed Google Scholar). To clarify the functional role, the full-length 3′-UTR was insertedinto the 3′-UTR of the luciferase reporter gene and transientlytransfected into p38α+/+ andp38α–/– MEF cells. The mRNA stability ofluciferase reporter transcript was measured following addition of actinomycinD. As shown in Fig. 3 (A andB), IL6-3′-UTR was sufficient to elicit rapidluciferase mRNA decay in p38α–/– MEFs with ahalf-life of 8.6 h compared with p38α+/+ MEFs(t½ = 13 h). In contrast, luciferase mRNA lackingIL-6 3′-UTR was stable during a 5-h period in p38α+/+and p38α–/– MEFs. These data indicate thatp38α stabilized mRNA via IL-6 3′-UTR. Proximal AREs Contain Elements Targeted by p38α—Toinvestigate the role of individual AUUUA motifs in mediatingpost-transcriptional control regulated by p38α, a series of luciferasereporter-gene constructs, containing the various regions of IL-6 3′-UTRand the motifs mutated from AUUUA to AGGGA, were generated(Fig. 4A) andtransiently expressed in MEFs. The luciferase activity of the full-length3′-UTR and all truncations was compared first in the individual celltype. For the p38α+/+ MEFs, when the full-length3′-UTR-(1–403) was placed in the reporter message, luciferaseactivity decreased more than 30% compared with luciferase alone(Fig. 4B). These dataindicate that the 3′-UTR contains the elements that confer mRNAstability and/or translational efficiency. The pGL3-IL-6ARE-(56–173)truncation reporter includes the two proximal AREs (ARE1 and ARE2), whereasthe three distal AREs (ARE3, ARE4, and ARE5) are included in pGL3-IL-63′-UTR-(172–403). The decreased expression was lost in bothpGL3-IL-6 3′-UTR-(56–173) and pGL3-IL-63′-UTR-(172–403) compared with the full-length3′-UTR-(1–403), suggesting that multiple control elements wereoperative. Whereas in the p38α–/– MEFs, theluciferase activity of the full-length 3′-UTR decreased more than 50%compared with luciferase alone and the significant decreased expression stilloccurred in the presence of both proximal and distal truncations. This resultindicated that p38α was involved when these multiple elements mediatedpost-transcriptional control. When the truncation pGL3-IL-63′-UTR-(1–70), which includes none of the AREs was inserted in thereporter message, luciferase activity in both wild-type andp38α–/– MEFs was comparable with the control.These data indicate that the decreased expression only occurred in thepresence of AUUUA motifs. To confirm the involvement of p38α clearly, a further comparison ofluciferase activity between p38α+/+ andp38α–/– MEFs was analyzed. Again, for thefull-length IL-6 3′-UTR-(1–403), pGL3-IL-63′-UTR-(56–173), and pGL3-IL-6 3′-UTR-(172–403), theluciferase activity in p38α–/– MEFs decreasedsignificantly compared with p38α+/+ cells (p <0.01). As shown in Fig.4D, p38α enhanced luciferase mRNA stability via thefull length of IL-6-3′-UTR. To confirm the cis-elementstargeted by p38α, the luciferase reporter mRNA stability of truncatedreporter constructs was examined in p38α+/+ andp38α–/– MEF. As shown inFig. 5, IL-63′-UTR-(56–173) elicited the rapid luciferase mRNA decay inp38α–/– MEFs (t½ = 3.5h), whereas the mRNA contained with IL-6 3′-UTR-(172–403) appearedto be more stable compared with 3′-UTR-(56–173)(t½ = 6.1 h) indicating that p38α targets the3′-UTR-(56–173) region to stabilize IL-6 mRNA. To estimate the influence of the individual AREs, AUUUA motifs were mutatedindividually and transiently expressed in MEFs. In p38α+/+MEFs, the luciferase activity of Mu3 increased significantly compared withfull-length 3′-UTR (p < 0.05) but still was significantlylower than that of control (p < 0.05). For the Mu5, the luciferaseactivity decreased another 20% compared with full-length 3′-UTR. Thisresult indicated that ARE3 included the major repressive element, whereas ARE5bears an enhancive element. In the p38α–/– MEFs,the luciferase activity of Mu1–4 increased significantly compared withfull-length 3′-UTR, whereas Mu5 was comparable. Again, the luciferase activity of all mutants betweenp38α+/+ and p38α–/– MEFs wascompared. After the individual ARE motifs mutated, the significant differencedue to the p38α deficiency was lost for M1, M2, and M5, whereas theluciferase activity of M3 and M4 were still significantly lower inp38α–/– MEFs compared withp38α+/+ cells suggesting that ARE1, -2, and -5 were theelements targeted by p38α. Secondary structure prediction of IL-6 3′-UTR and mutants. AlthoughRNA-binding proteins bind RNA in a sequence-specific manner, previous studieshave indicated that RNA secondary structure plays a critical role in defining3′-UTR RNA-protein interaction. Binding sites are often located insingle-stranded RNA region(31Hiller M. Pudimat R. Busch A. Backofen R. Nucleic Acids Res. 2006; 34: e117Crossref PubMed Scopus (117) Google Scholar). Using M-fold, thesecondary structures of IL-6 3′-UTR and mutants were predicted(Fig. 6). For wild-type3′-UTR, ARE2 and ARE5 comprised the sequence to form the external loopstructures (LoopA and LoopD), whereas ARE3 and ARE4 formed the internal loopstructures (LoopB and LoopC). ARE1 formed the stem adjacent to the externalLoopA. Mutation of ARE1 and ARE2 abolished external LoopA, and mutation of ARE5abolished external LoopD, whereas mutation of ARE3 and ARE4 had a relativelyminor effect on the secondary structure. Consistent with minor changes insecondary structure, mutation of ARE3 and ARE4 to luciferase reporter geneassay did not abolish the effect of p38α. This indicates that thesecondary structure of wild-type 3′-UTR is necessary for the p38αtargeting. TNFα remains the best demonstrated cytokine regulated by thep38α MAPK pathway at post-transcriptional level. The tight control ofTNFα expression is achieved by regulating mRNA stability, translationalinitiation, and polyadenylation. During this process, trans-actingfactors that bind to TNFα 3′-UTR, including AREs are essential. Incontrast, limited information is available regarding the nature of IL-6 mRNAstability and regulation by MAPK p38α. Using a p38α-deficient cellline, strict criteria regarding signaling intermediate requirements for IL-6expression and mRNA stability could be applied. According to previousp38α inhibitor experiments, we predicted that IL-6 expression wasdown-regulated in p38α–/– MEFs atpost-transcriptional level. Consistent with our expectation, IL-6 mRNA decayincreased in p38α–/– MEFs compared withp38α+/+ cells. In the present study, MEF cells treated with IL-1 resulted inphosphorylation of p38α to produce IL-6. Fibroblast IL-6 production wasinhibited by SB203580 (26Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Todetermine p38α is critical for the IL-1-induced IL-6 production by usingp38α–/– cells, the normal expression of IL-1receptor in p38α–/– cells is required. It hasbeen reported that p38α–/– ES cells also gaverise to a population of IL-1R-positive cells after the in vitrodifferentiation process (32Allen M. Svensson L. Roach M. Hambor J. McNeish J. Gabel C.A. J. Exp. Med. 2000; 191: 859-870Crossref PubMed Scopus (251) Google Scholar).The percentage of cells that express IL-1R was comparable inp38α+/+ and p38α–/– cultures.Our data show that reconstitution of p38α cDNA inp38α–/– cells restored the IL-1β-inducedIL-6 production consistent with that the decrease of IL-6 production is due tothe p38α deficiency rather than the defect of IL-1R. p38α iscritical but not essential for the IL-6 production. Therefore, failure toincrease IL-6 production by overexpression p38α in wide-type cells wasnot unexpected. It is possible that other proteins are limiting theinvolvement of p38α. For example, MAPK/extracellular signal-regulatedkinase kinase kinase 3 (MEKK3), which is the potent activator of MAPKs, isessential for the IL-1-induced IL-6 production(33Huang Q. Yang J. Lin Y. Walker C. Cheng J. Liu Z.G. Su B. Nat. Immunol. 2004; 5: 98-103Crossref PubMed Scopus (228) Google Scholar). However, it was somewhatsurprising that p38AF decreased the IL-6 production in bothp38α+/+ and p38α–/– cells. Fourmembers of the p38 MAPK family have been cloned: p38α,-β,-γ,and -δ (34Ono K. Han J. CellSignal. 2000; 12: 1-13Google Scholar,35Zarubin T. Han J. CellRes. 2005; 15: 11-18Google Scholar). p38β shares ∼74%sequence identity with p38α and contains the TGY dual phosphorylationmotif observed in p38α(36Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol.Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). p38β is activatedby IL-1 stimulation, but 4-folder lower than p38α(37Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). In addition, MK2 is thesubstrate of both p38α and p38β(37Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Therefore, we predictthat p38AF decreased the activation of MK2 by the co-repression ofp38β. In our present study, no significant effects of p38α deficiency onIL-6 proximal promoter reporter activity were demonstrated (data not shown).This result is consistent with previous reports from our group, where p38inhibitors had minimal effects on IL-6 proximal promoter in MC3T3-E1osteoblastic-like cells (38Patil C. Zhu X. Rossa Jr., C. Kim Y.J. Kirkwood K.L. Immunol. Invest. 2004; 33: 213-233Crossref PubMed Scopus (64) Google Scholar).Others have reported that SB203580 did not effect the activation of the IL-6promoter by IL-1β in human fibroblast-like synoviocytes(26Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Collectively, theseresults indicate that the effect of p38α deficiency on IL-6 geneexpression results from the down-regulation of post-transcriptional level,however, we cannot rule out any role of transcriptional activation in thesepresent studies. SB203580 significantly reduced the mRNA stability of endogenous IL-6. Theresults were also confirmed in vivo by comparing half-life of IL-6mRNA between p38α+/+ and p38α–/–MEFs. To analyze the regulation of IL-6 mRNA stability, the luciferasereporter gene system was employed. We found that p38α stabilized themRNA reporter construct carrying the IL-6 3′-UTR. All these datasupported the conclusion that p38α signaling increases IL-6 mRNAstability via 3′-UTR. The degradation of endogenous IL-6 mRNA and mRNAreporter construct containing the IL-6 3′-UTR is only marginallyaffected in p38α+/+ MEFs. This is probably due to the lessclassic ARE and perhaps weaker IL-6 ARE elements of the IL-6 3′-UTRcompared with TNFα and other cytokines. TNFα, granulocytemacrophage-colony stimulating factor, and IL-3 AREs are typical class II AREswith a core AUUUA motif cluster, which mediated rapid mRNA decay(30Stoecklin G. Stoeckle P. Lu M. Muehlemann O. Moroni C. Rna. 2001; 7: 1578-1588PubMed Google Scholar). The AREs ofc-myc and c-fos are prototypes of class I, containing one tothree scattered copies of the AUUUA motif, whereas other cytokine transcripts,including IL-2, IL-4, and IL-6 contain class I-like AREs that arenon-clustered (30Stoecklin G. Stoeckle P. Lu M. Muehlemann O. Moroni C. Rna. 2001; 7: 1578-1588PubMed Google Scholar). Thesenon-clustered motifs are considered weaker AREs. The differences in cellstypes and stimulus may also account for this minimal degradation. In thepresent study, we observed only modest changes with IL-6 ARE reporter activityfollowing stimulation with IL-1β (data not shown). These data areconsistent with the concept that the IL-6 ARE is weaker compared with otherAREs or alternatively there are differences in the MEF cells lines used inthese studies. A paradigm that has been helpful in understanding the regulation ofpost-transcriptional control was that RNA-binding protein binds to specificcis-acting elements in target transcript and activate3′-to-5′-exonucleolytic decay or/and influence the translationalcontrol. Some RNA-binding proteins, like tristetraprolin, AU-binding factor 1,and K homology splicing-regulatory protein, promote mRNA decay, whereasothers, like members of the Hu family, prevent mRNA degradation(15Antic D. Keene J.D. Am. J.Hum. Genet. 1997; 61: 273-278Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 16Brennan C.M. Steitz J.A. Cell Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (878) Google Scholar, 17Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar,19Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar,20Min H. Turck C.W. Nikolic J.M. Black D.L. Genes Dev. 1997; 11: 1023-1036Crossref PubMed Scopus (279) Google Scholar). Deletion or mutation ofAUUUA motifs could alter the components of mRNA-trans factor complexand affect the luciferase activity of reporter gene containing the IL-63′-UTR with mutated AUUUA motifs. Using this approach, we identifiedARE1, ARE2, and ARE5 as the cis-elements regulated by p38α inIL-6-3′-UTR. Interestingly, mutation of ARE1 and ARE2 resulted in theenhanced expression due to the deficiency of p38α, whereas mutation ofARE5 decreased the luciferase expression in the presence of p38α. Thesedata are consistent with the idea that p38α is required for the ARE1 andARE2 to repress expression and for ARE5 to enhance expression. Our data indicated that distinct cis-elements of 3′-UTRexert opposing influence on the post-transcriptional fate of IL-6 mRNA. Asimilar bi-functional role for the 3′-UTR has been described for othermRNAs, including RNase-L (39Li X.L. Andersen J.B. Ezelle H.J. Wilson G.M. Hassel B.A. J. Biol. Chem. 2007; 282: 7950-7960Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).In that study, eight AREs were identified in the RNase-L 3′-UTR, anddeletion analysis identified AREs 7 and 8 served as a strong positiveregulatory function, whereas AREs 2 and 4 are critical for the negativeregulatory function. Also, HuR and AU-binding factor 1 were found to bind top21 and cyclin D1 mRNAs in the nucleus, and independently operate oppositefunction in the cytoplasm depending on the physiological context(40Lal A. Mazan-Mamczarz K. Kawai T. Yang X. Martindale J.L. Gorospe M. EMBO J. 2004; 23: 3092-3102Crossref PubMed Scopus (395) Google Scholar). RNA-binding proteins recognize RNA targets in a sequence-specific manner,and the secondary structure context of the binding site also affects thebinding affinity (31Hiller M. Pudimat R. Busch A. Backofen R. Nucleic Acids Res. 2006; 34: e117Crossref PubMed Scopus (117) Google Scholar). Bindingsites are often located in single-stranded RNA regions. For example, HuRprotein influences mRNA stability by binding to the motif NNTTNNTTT where HuRaffinity correlates with the single-strandedness of this binding motif(41Meisner N.C. Hackermuller J. Uhl V. Aszodi A. Jaritz M. Auer M. Chembiochem. 2004; 5: 1432-1447Crossref PubMed Scopus (109) Google Scholar,42Hackermuller J. Meisner N.C. Auer M. Jaritz M. Stadler P.F. Gene. 2005; 345: 3-12Crossref PubMed Scopus (43) Google Scholar). IL-63′-UTR-(56–173) displays a typical “stem-loop” orhairpin structure. This structure might be critical for IL-63′-UTR-(56–173) inducing rapid mRNA decay. Recently studies have shown that microRNA targeting of ARE appeared to bean essential step in ARE-mediated mRNA degradation(43Jing Q. Huang S. Guth S. Zarubin T. Motoyama A. Chen J. Di Padova F. Lin S.C. Gram H. Han J. Cell. 2005; 120: 623-634Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). The ability of microRNAto target mRNA is directed by the paring of microRNA to mRNA. It is anintriguing possibility that microRNA is implicated for p38α to regulatethe repressive elements in IL-6 3′-UTR. Cell signaling through p38α MAPK is necessary forpost-transcriptional regulation of many pro-inflammatory cytokines. In thisstudy, the cis-acting elements of IL-6 3′-UTR mRNA thatrequired p38α signaling for mRNA stability and translation wereidentified. AUUUA motif mutation analysis using gene reporter systemsperformed in p38α+/+ and p38α–/–MEF cells revealed that ARE1, ARE2, and ARE5 in IL-6 3′-UTR weretargeted by p38α, and IL-6 3′-UTR-(56–173) is critical forp38 to promote mRNA stability. The predicted RNA secondary structure of theseARE reporter mutants was consistent with the ability to alter reporterexpression data, suggesting that the molecular targets of p38 MAPK thatinteract with ARE mutants are dependent upon IL-6 3′-UTR secondarystructure.