Fragile X-related Protein FXR1P Regulates Proinflammatory Cytokine Tumor Necrosis Factor Expression at the Post-transcriptional Level

Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3′-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3′-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation. Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3′-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3′-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation. Tumor necrosis factor α (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; IFN, interferon; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, AU-rich element; GFP, green fluorescent protein; UTR, untranslated region; KO, knock-out; WT, wild type; FACS, fluorescence-activated cell sorter; IPTG, isopropyl 1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; MHC, major histocompatibility complex; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RNP, ribonucleoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; mRNP, messenger ribonucleoprotein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; FXR, fragile X-related; ELISA, enzyme-linked immunosorbent assay; CMV, cytomegalovirus.1The abbreviations used are: TNF, tumor necrosis factor; IFN, interferon; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, AU-rich element; GFP, green fluorescent protein; UTR, untranslated region; KO, knock-out; WT, wild type; FACS, fluorescence-activated cell sorter; IPTG, isopropyl 1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; MHC, major histocompatibility complex; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RNP, ribonucleoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; mRNP, messenger ribonucleoprotein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; FXR, fragile X-related; ELISA, enzyme-linked immunosorbent assay; CMV, cytomegalovirus. is a proinflammatory cytokine produced primarily by macrophages and is crucial for immune responses. The beneficial role of TNF can be perturbed upon its aberrant expression, which is involved in many inflammatory disorders (1Beutler B. J. Investig. Med. 1995; 43: 227-235PubMed Google Scholar). TNF is a major mediator of the systemic inflammatory response syndrome where high TNF levels have been associated with mortality. To protect against the detrimental aspects of TNF, its expression is under strict control. The production of TNF is controlled both transcriptionally and post-transcriptionally by modulation of translational efficacy and mRNA stability (2Zhang T. Kruys V. Huez G. Gueydan C. Biochem. Soc. Trans. 2002; 30: 952-958Crossref PubMed Scopus (184) Google Scholar). The expression of TNF is induced by various agents including bacterial LPS and IFNγ. Induction of TNF expression depends on two signals; the first signal induces transcription, and the second signal releases the normally operating translational repression that controls TNF protein expression. The sequences responsible for the translational repression and LPS inducibility are located in the 3′-UTR of TNF mRNA and consist of multiple repeats of an AUUUA motif (AU-rich element, ARE). Previously, we mapped TNF mRNA for RNA-protein interaction and identified two protein-binding regions in the 3′-UTR (3Hel Z. Skamene E. Radzioch D. Mol. Cell. Biol. 1996; 16: 5579-5590Crossref PubMed Scopus (46) Google Scholar, 4Hel Z. Di Marco S. Radzioch D. Nucleic Acids Res. 1998; 26: 2803-2812Crossref PubMed Scopus (36) Google Scholar). The first protein-binding region was located inside the main ARE of TNF 3′-UTR (bases 1291–1329), and the second ARE region was 147 bases downstream. Both AREs interacted with several macrophage proteins that play an active role in the post-transcriptional regulation of TNF synthesis. We have also identified HuR as an important RNA-binding protein involved in the post-transcriptional regulation of TNF expression in macrophages (5Di Marco S. Hel Z. Lachance C. Furneaux H. Radzioch D. Nucleic Acids Res. 2001; 29: 863-871Crossref PubMed Google Scholar). Along with HuR, several other proteins have been shown to target the ARE of TNF mRNA influencing its post-transcriptional control, including TTP, TIA-1, TIAR, AUF1, and CBF-A (6Dean J.L. Wait R. Mahtani K.R. Sully G. Clark A.R. Saklatvala J. Mol. Cell. Biol. 2001; 21: 721-730Crossref PubMed Scopus (243) Google Scholar, 7Dean J.L. Sully G. Wait R. Rawlinson L. Clark A.R. Saklatvala J. Biochem. J. 2002; 366: 709-719Crossref PubMed Google Scholar, 8DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 9Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 10Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (619) Google Scholar, 11Gueydan 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 (230) Google Scholar, 12Piecyk 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 Google Scholar). this we that RNA-binding is to TNF expression in FXR1P is a of fragile syndrome protein and with the of RNA-binding The proteins RNA-binding a of and an motif The proteins also have nuclear signal and nuclear signal of the proteins suggest that are involved in post-transcriptional mRNA as as a role in B. J.L. Res. 2001; PubMed Scopus Google Scholar). By using macrophages from FXR1 knock-out mice and wild type we that FXR1P is involved in the post-transcriptional regulation of TNF expression. macrophage cell was from the of mice and D. T. M. L. L. Skamene E. J. Biol. PubMed Scopus Google Scholar). FXR1 knock-out mice in and mice by and and to knock-out was by using the following and We have generated macrophage cell from the and of and using E. L. Cell. PubMed Scopus Google Scholar). in this generated using and in with and in to with LPS of Expression was from mRNA from macrophages using and and on bacterial in to and for in containing and screened by using cRNA probe the ARE of TNF The for the second on for the first of and the second The from using and on the in and by and for in this was identified as FXR1P by the for is with was in and in for and in with in for in in for in for and for in containing of cRNA in and to was to an for a protein identified as FXR1P. The region was using and in using and and are The first of the in the the was To was from to the A generated with using as and and was by and following with and was with the of To a that for of the of FXR1P was from The was with and of to the To the from was in of with To TNF 3′-UTR was following the A (bases containing the 3′-UTR of TNF was generated by with from a TNF using with the and To the TNF 3′-UTR was of To the from that with TNF 3′-UTR the of transfected with by the to in of with in a with of with on for and and on for and in of with in a The the of was to the following was using the following the of the Macrophages to with of PBS, and the was with in for by of of with for of was from E. with by J. J.R. Cell. Full Text PDF PubMed Scopus Google a E. was used to translational of FXR1P. in for was induced with for protein was by induced in PMSF, by on and for the protein was on on a and with by a with containing to with containing This resulted in the of FXR1P. against the protein was in containing DTT, and The FXR1P was from E. with protein was by in and using was with with PBS, which was to was also by was by by a FXR1P was under using a against the protein was in containing DTT, and and of cRNA as (3Hel Z. Skamene E. Radzioch D. Mol. Cell. Biol. 1996; 16: 5579-5590Crossref PubMed Scopus (46) Google Scholar). of of recombinant protein with of cRNA probe in a of for by with and The cRNA was to protein by an to a using a on proteins BSA, and the TNF 3′-UTR including (bases (bases (bases 1291–1329), (bases and by C. B. and 3′-UTR (bases as (3Hel Z. Skamene E. Radzioch D. Mol. Cell. Biol. 1996; 16: 5579-5590Crossref PubMed Scopus (46) Google Scholar, B. T. J. Biol. Chem. Full Text PDF PubMed Google the containing was by P. of was with was using with with with TNF, GAPDH, and generated as D. M. L. J. Google Scholar, G. Skamene E. Radzioch D. J. Res. Full Text PDF PubMed Google Scholar). was using the and the and from was with of the following for for and for to the of was using the was by using and of was used to TNF and and and induced with LPS for and to was The on for for and with with The was in and containing and a on on by in a by for The was and was The was for on a and in was using from was on The that was for the was used for with we the cell in DTT, and We used a with a containing and for the We the in a containing and that was by for and the was The was in a for and in of containing The to and for and of the by to generated to an of FXR1P as B. S. D. S. C. D. J. Mol. 1998; PubMed Scopus Google Scholar). was used for was generated against the FXR1P. with of protein by two containing was from using and with by against was with as by the and used for the of proteins by with and for using to by and in in containing with used as and on with with and in The with with BSA, and with by in on using using a with a Macrophages transfected with the on with of PBS, in for with of for from in first and on using Macrophages transfected with for TNF, and of TNF the by macrophages was by as J. Google Scholar). of protein was by with from following the using a of recombinant The expression levels of and proteins by in and in and with of was for with of in and for The of was using a and with and for on a which was and to for TNF mRNA of of the regulation of TNF expression occurs post-transcriptionally the ARE sequences located in the 3′-UTR of its mRNA that are by proteins that the and stability of the To macrophage proteins of the ARE, a protein expression library from a macrophage cell was screened by with cRNA to the region of TNF 3′-UTR the main using and that on using the TNF ARE probe of several was the which is in this The from was bacterial and induced with IPTG, and bacterial containing the recombinant protein was by The recombinant with an of the cRNA probe containing the 3′-UTR of TNF mRNA. of that the RNA-binding protein FXR1P. FXR1P RNA-binding a of and an as in of FXR1P Expression in of from various the of two of the and mRNA was in including the an that is to a of and of expression in macrophages two mRNA This is with and the several and of the Z. D. H. S. P. H. Mol. 1995; PubMed Scopus Google Scholar, R. P. Mol. 2001; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). The expression of the mRNA in macrophages to be by and that TNF expression. FXR1P was in macrophages by The the of FXR1P with an of that TNF expression the of FXR1P. 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To and and was to the We that cell that we from the of mice and controls used in the in this The of cell was by using for and of FXR1P expression in the macrophage cell was by macrophage cell by as shown in using a of against as D. T. M. L. L. Skamene E. J. Biol. PubMed Scopus Google Scholar). Both the and cell macrophage and protein with and for expression for the and of FXR1P on to the and macrophages for to to LPS and by the of We found that both cell in to the shown in both and levels of proteins with LPS for proteins levels induced to a was modulation the of FXR1P following macrophage with LPS with IFNγ. FXR1P was in We the and macrophages to LPS by The of occurs upon of macrophages and to of the that is important for was by using an for the of shown in LPS for induced in both the and macrophage cell of FXR1P on Expression of an we the expression of various that have an ARE in 3′-UTR to FXR1P has an on expression. The levels of the proteins produced from following of macrophages with LPS Our findings that FXR1P resulted in enhanced protein expression of TNF, and Our findings suggest that FXR1P is involved in the regulation of mRNA. was that FXR1P in a of translation. on the of the expression of several proteins in and was that the ablation of FXR1P have a on the To this we of and and shown in protein on as as proteins both in and that was of translational in compared with of TNF Expression in and our on the responsible for the enhanced expression of TNF in the The macrophages with LPS, both and expression of TNF was shown in following the production of TNF protein was in to FXR1P resulted in an the of TNF protein production of following LPS and following LPS and The in TNF protein production and macrophages in The of TNF mRNA was compared the macrophage cell following with LPS for for We found levels of TNF mRNA in macrophages with LPS in from of The in the levels of TNF mRNA and macrophages with LPS and We TNF mRNA levels in and nuclear of macrophages with LPS The also that the of TNF mRNA was in macrophages deficient in FXR1P. the that we on TNF protein production in macrophages FXR1P that the FXR1P role is the of translational control. To that the affected TNF regulation was the post-transcriptional an reporter was The reporter the 3′-UTR of TNF mRNA to This the expression under the control of both the ARE in the 3′-UTR and the as in transfected macrophages by and A wild type macrophage cell transfected with had high expression, which was affected by LPS the macrophage cell was transfected with the expression of was demonstrating the post-transcriptional of the The repression of expression in the macrophages transfected with was following macrophage with The and macrophage cell transfected with the reporter to the of FXR1P ablation on the post-transcriptional regulation of in both cell the of the ARE on expression in that was with with the of expression from the reporter following was enhanced to a in compared with the macrophages in was expression of that was in that a control operating the 3′-UTR of TNF mRNA is in the of FXR1P. To of the control that FXR1P we the of GFP-ARE mRNA in the as in had levels of GFP-ARE mRNA compared with which enhanced with LPS The in to a from the of GFP-ARE which was in compared with the This the that FXR1P ablation of the in The the of mRNA are compared with the in expression by FXR1P ablation that was the protein both in and with the GFP-ARE mRNA in the was a of protein in this compared with the the of protein expression in the was the in the that FXR1P has a on translational regulation and on stability in of the of FXR1P in the of TNF are two of regulation that on TNF expression, the stability and of its To FXR1P has an on the stability of TNF was Macrophages for with LPS, and TNF mRNA levels by various following with as shown in of the TNF mRNA following of LPS and macrophages we have that macrophages had a of TNF mRNA following with LPS our with the reporter an of GFP-ARE mRNA in compared with findings suggest that FXR1P had on the stability of mRNA. the of TNF mRNA of LPS as used in our be to the that the regulated by FXR1P to during the of LPS in a of the of To this we the of TNF mRNA following a with LPS of and the are in the stability of the TNF mRNA is in both and cell the suggest that FXR1P have a on TNF mRNA of the of TNF mRNA following the of suggest that was a in stability in macrophages LPS on in and the be upon of the macrophages with LPS, which the that releases the control on TNF expression. To the other of of the TNF is affected by FXR1P was A is in we from and macrophages by using and RNA-protein with The by using against FXR1P and protein The in FXR1P in that FXR1P is to with the translational The of FXR1P following LPS was and macrophages induced with LPS for and the using from the was and TNF mRNA levels shown in the of TNF mRNA was on in the the TNF mRNA in the macrophages was in associated with the polyribosomes This that FXR1P in an in the of the TNF mRNA. This that FXR1P is active in translational repression of TNF which is in a upon macrophage activation. of with that macrophages deficient in FXR1P have an in regulation and that FXR1P is involved in the translational repression of for this FXR1P was to This was with that of active protein to in To the with recombinant was to macrophage following protein and and was by The was with of the macrophage in the FXR1P was with to shown in following of of the cell FXR1P in the By a the of FXR1P be as in the To of macrophages with FXR1P has expression of TNF protein was FXR1P protein to and macrophages with LPS for shown in of FXR1P to the macrophages resulted in production of TNF The of TNF produced from macrophages was that of control our that FXR1P is involved in the post-transcriptional regulation of TNF in macrophages and as a of TNF protein translation. regulation operating on the ARE has a major on TNF expression. The regulation the and stability of the TNF mRNA. Expression of the TNF protein that occurs upon macrophage depends upon the release from the translational repression and to an on mRNA that this post-transcriptional regulation are proteins that with the this we a TNF identified as FXR1P. FXR1P is a of the which also and The proteins by of two and an and with a of V. P. S. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. V. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, H. M. G. Cell. Full Text PDF PubMed Scopus Google Scholar, H. G. Cell. Full Text PDF PubMed Scopus Google Scholar). The proteins are of that are in the and have been found in association with polyribosomes B. S. D. S. C. D. J. Mol. 1998; PubMed Scopus Google Scholar, S. V. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, M. S. Mol. PubMed Scopus Google Scholar, Mol. 1996; PubMed Scopus Google Scholar, H. G. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar, L. C. N. H. Biochem. 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The of FXR1P be following macrophage the that are involved in TNF post-transcriptional and TIAR, shown to be involved in the repression of TNF 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 (230) Google Scholar, 12Piecyk 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 Google Scholar). macrophages deficient in have TNF protein expression, a in TNF mRNA. This that the repression of TNF is a a regulation in which FXR1P and are association of FXR1P and was by that that with FXR1P and TIA-1, can be in that to also that are R. S. C. Mol. 2002; PubMed Scopus Google Scholar). This a for the translational repression by during in which translational repression was the that by was found associated with polyribosomes H. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, S. M. S. J. Mol. PubMed Scopus Google Scholar). This for on the signal was this to the of the to the The found in that controls the of the polyribosomes was found to be in FXR1P. This that FXR1P have control on of the mRNA with which to the of FXR1P in the control of translational our suggest the that also has a on in This is by the in TNF mRNA in compared with from the in stability of TNF mRNA as by the as as by the reporter FXR1P in control of mRNA with and the of other proteins that TNF mRNA stability as HuR, TTP, AUF1, The of regulation on TNF expression was shown using mice in which the ARE is mice high levels of serum TNF and inflammatory disorders as D. M. G. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). levels of TNF are in several inflammatory of the be many of knock-out mice have high serum TNF levels to an inflammatory E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, Carballo E. Lai W.S. Blackshear P.J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). levels of TNF from the macrophages suggest that a inflammatory mice this be of FXR1P have detrimental on the inflammatory A that FXR1P is involved in in was the that FXR1P is an in systemic a J. S. C. M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). have levels of serum TNF that to the inflammatory M. M. J. Google Scholar). the proteins involved in the regulation of TNF expression the of inflammatory and for our findings that FXR1P is involved in the post-transcriptional regulation of TNF expression its interaction with the ARE located in 3′-UTR of TNF mRNA. Our that the of FXR1P is for the translational repression of This the of the regulation of TNF, where TNF expression is in a by translational This control of FXR1P be upon macrophage activation. findings are of for the regulation of and the of the of We G. for from the FXR1 knock-out mice and We also P. J. and C. B. for

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