- Original Articles
- Open Access
Engagement of Tumor Necrosis Factor mRNA by an Endotoxin-Inducible Cytoplasmic Protein
© Molecular Medicine 1996
- Published: 1 July 1996
The Erratum to this article has been published in Molecular Medicine 1996 2:BF03401661
Tumor necrosis factor (TNF) production by macrophages plays an important role in the host response to infection. TNF-α gene expression in RAW 264.7 macrophages is predominantly regulated at the translational level. A key element in this regulation is an AU-rich (AUR) sequence located in the 3′ untranslated region (UTR) of TNF mRNA. In unstimulated macrophages, the translation of TNF mRNA is inhibited via this AUR sequence. Upon stimulation with LPS, this repression is overcome and translation occurs. In this study, we attempted to identify cellular proteins that interact with the AUR sequence and thereby regulate TNF mRNA translation.
Materials and Methods
RNA probes corresponding to portions of TNF mRNA 3′ UTR were synthesized. These labeled RNAs were incubated with cytoplasmic extracts of either unstimulated or lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The RNA/protein complexes formed were analyzed by gel retardation. Ultraviolet (UV) cross-linking experiments were performed to determine the molecular weight of the proteins involved in the complexes.
TNF mRNA AUR sequence formed two complexes (1 and 2) of distinct electrophoretic mobilities. While the formation of complex 1 was independent of the activation state of the macrophages from which the extracts were obtained, complex 2 was detected only using cytoplasmic extracts from LPS-stimulated macrophages. Upon UV cross-linking, two proteins, of 50 and 80 kD, respectively, were capable of binding the UAR sequence. The 50-kD protein is likely to be part of the LPS-inducible complex 2, since its binding ability was enhanced upon LPS stimulation. Interestingly, complex 2 formation was also triggered by Sendaï virus infection, another potent activator of TNF mRNA translation in RAW 264.7 macrophages. In contrast, complex 2 was not detected with cytoplasmic extracts obtained from B and T cell lines which are unable to produce TNF in response to LPS. Protein tyrosine phosphorylation is required for LPS-induced TNF mRNA translation. Remarkably, the protein tyrosine phosphorylation inhibitor herbimycin A abolished LPS-induced complex 2 formation. Complex 2 was already detectable after 0.5 hr of LPS treatment and was triggered by a minimal LPS dose of 10 pg/ml.
The tight correlation between TNF production and the formation of an LPS-inducible cytoplasmic complex suggests that this complex plays a role in the translational regulation of TNF mRNA.
Post-transcriptional regulatory mechanisms have been shown to play a major role in gene expression in eukaryotic cells (1–4). Several messenger RNAs (mRNAs) encoding cytokines, growth factors or oncoproteins contain a conserved AU-rich (AUR) sequence in their 3′ untranslated regions (UTR) (5). This sequence is composed of several repeats of the pentanucleotide AUUUA. Depending on the cell system, this sequence can either decrease mRNA stability (6,7) or inhibit its translation (8).
It has been recently shown that the minimal motif that mediates mRNA degradation is the UUAUUUAUU nonanucleotide (9,10). Several proteins that bind AUR sequences within cytokine or protooncogene mRNAs have been identified using cell systems where AUR sequences have been shown to have a destabilizing effect (11–14). The exact function of these proteins has not yet been characterized.
The translation inhibition of mRNAs by the AUR sequences has been observed in reticulocyte lysates, Xenopus oocytes (15–17) and somatic cells (18). Three octanucleotides UUAUUUAU present in the 3′ UTR inhibit mRNA translation to the same extent as the natural AUR sequence. The presence of a single octanucleotide in the 3′ UTR is, however, sufficient to decrease significantly translation efficiency (17). The mechanism by which AUR sequences exert their translation inhibitory effect has not been elucidated. We previously demonstrated that while the interferon-β (IFNβ) mRNA AUR sequence inhibits translation independent of its position within the 3′ UTR, when it is transferred upstream of the initiation codon, the inhibitory effect is lost (16).
In RAW 264.7 macrophages, tumor necrosis factor α (TNFα) gene expression is predominantly regulated at the translational level. In unstimulated macrophages, translation of TNF mRNA is repressed by the AUR sequence within the 3′ UTR. Lipopolysaccharides (LPS) treatment overcomes this repression (19,20). Since the AUR sequence has no effect on TNF mRNA stability in this system (21), RAW 264.7 macrophages may be useful for identifying factors involved in mRNA translational inhibition mediated by AUR sequences.
In this study, we performed electrophoretic mobility shift assays (EMSA) using labeled RNA probes corresponding to portions of the TNF 3′ UTR with RAW 264.7 cytoplasmic extracts. This technique allows the detection of multimeric RNA/protein(s) complexes. The results show that TNF mRNA AUR sequence can form two complexes (1 and 2) of different electrophoretic mobilities when incubated with macrophage cytoplasmic extracts. Complex 1, of lower electrophoretic mobility, forms when extracts from both unstimulated and stimulated macrophages are used. Complex 2, of higher mobility, is only detected with extracts from TNF-producing macrophages. The time course of complex 2 formation tightly correlates with that of TNF production by RAW macrophages.
Enzymes used for construction of the different DNA plasmids and for in vitro transcription were purchased from Gibco-BRL (Gaithersburg, MD, U.S.A.). RNAse T1 was purchased from Boehringer (Indianapolis, IN, U.S.A.). RNAse A and LPS (Escherichia coli strain 0.127:B8) were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Herbimycin A was purchased from Gibco-BRL. The Sendai virus was obtained from SPAFAS Inc. (Sendaï virus-cat# 900726-Cantell-SPAFAS Inc., CT, U.S.A.) and has been demonstrated free of detectable amount of endotoxin contamination (22).
In Vitro Transcription
The SP643′TNF, SP643′TNFAU− plasmids were linearized with EcoRI before in vitro transcription. The RNA probes 3′TNFAU+ and 3′TNFAU− were synthesized by in vitro transcription of the different linearized constructs with the SP6 RNA polymerase (45 units/reaction) in the presence of 660 µM ATP, CTP, GTP, 66 µM UTP, and [α-32P] UTP (80 µCi; 800 Ci/mmole). The plasmid encoding the AU+ probe was linearized with BamHI and the AU+ probe was transcribed with the T3 RNA polymerase according to the same protocol.
RAW 264.7 mouse macrophages, originally from the American Type Culture Collection (ATCC) were passaged in Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented with 5% fetal bovine serum (FBS myoclone super plus, Gibco BRL) and 1% penicillin/streptomycin solution. 3B4.15 T cell hybridoma (originally described in Ref. 24) and A20 lymphoma B cell line (from ATCC) were cultured in RPMI 1640 medium (Gibco BRL) supplemented with 5% FBS, 10 mM HEPES, 2 mM glutamine, 50 µM β-mer-captoethanol, 1% penicillin/streptomycin. LPS was added at a final concentration of 10 ng/ml in all experiments unless specified.
Preparation of Macrophage Extracts
Postribosomal extracts were prepared according to the method described by Dignam et al. (25) with some modifications. All operations were performed at 0–4°C. Subconfluent cell cultures were rinsed once with ice-cold phosphate-buffered saline (PBS) and harvested by scraping. Cells were centrifuged at 2000 rpm, resuspended in buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.5 mM DTT, 100 nM okadaic acid, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 20 × 106 cells/100 µl and lysed by 20 strokes of Dounce homogenizer (B type pestle). Homogenates were centrifuged at 400 × g for 10 min to pellet the nuclei and the supernatants were subsequently ultracentrifuged at 100,000 × g for 90 min (Beckman rotor SW50.1). Supernatants were collected and are referred as S100 extracts in the results. Protein concentration in S100 extracts was determined by the bicinchoninic acid method (PIERCE, Rockford, IL, U.S.A.), and samples were immediately frozen at −80°C.
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays (EMSAs) were performed according to Konarska and Sharp (26), and Leibold and Munro (27). The same number of counts per minute (50,000 cpm/reaction) for each probe was used in the binding reaction. For the 3′ TNF AU+ probe, 50,000 cpm correspond to 7.6 fmoles of RNA, for the 3′ TNF AU− to 11.6 fmoles and for the AU+ probe to 20 fmoles. 32P-labeled RNA probe (50,000 cpm/µl) was heated at 70°C for 15 min, then cooled to room temperature for 3 min. One microliter of radiolabeled RNA was incubated for 30 min on ice with 20 µg of S100 protein extract in binding buffer (20 mM HEPES, pH 7.6; 3 mM MgCl2; 40 mM KCl; 2 mM DTT; 5% glycerol) in a total volume of 15 µl. RNAse T1 (6 units) was added to the samples and the mixtures were further incubated for 10 min at room temperature. Heparin was then added at a final concentration of 5 mg/ml. Three microliters of loading buffer (90% glycerol, 0.025% bromophenol blue) were added to the samples which were then resolved by electrophoresis on a 1 × TBE nondenaturing 4% Polyacrylamide gel at 4 mA for 14 hr at 4°C. Gels were dried and autoradiographed with Kodak films.
UV Cross-Linking and SDS Polyacrylamide Gel Electrophoresis
The first steps of the experiment were identical to the EMSA described above. After the addition of heparin, the binding mixes were irradiated with ultraviolet (UV) light for 10 min at 4°C, using a Sylvania G15T8 germicidal light placed 15 cm above the samples. Twenty units of RNAse T1 and 10 µg of RNAse A were added to the samples, and the mixtures were further incubated at 37°C for 30 min to degrade unbound RNA. An equal volume of loading buffer (1% SDS; 1% β-mercaptoethanol; 10% glycerol; 0.05 M Tris, pH 6.5, 0.025% bromophenol blue) was added to the samples before heating to 100°C for 4 min. The samples were analyzed by electrophoresis on a 20% Polyacrylamide denaturing gel containing 0.1% SDS. 32P-labeled proteins were visualized by autoradiography with Kodak films.
TNF production was measured in cell supernatants by sandwich enzyme-linked immunosorbent assay (ELISA) using a polyclonal rabbit anti-mouse TNF antibody for coating and the same polyclonal antibody that was biotinylated for detection (generously provided by Dr. Wim Buurman. University of Limburg, Maastricht, The Netherlands).
Postribosomal Supernatant (S100) from RAW 264.7 Macrophages Contains Constitutive and Inducible Proteins Which Bind the TNF mRNA AUR Sequence
Characterization of the Proteins Involved in Complexes 1 and 2 by UV Cross-Linking
Infection of RAW Cells with the Sendaï Virus Induces Complex 2 Formation
Complex 2 Does Not Form with S100 Extract from Cells That Do Not Produce TNF in Response to LPS
Tyrosine Kinase Inhibitor Herbimycin A Inhibits LPS-induced Complex 2 Formation
Kinetics and LPS Dose-Dependence of Complex 2 Formation
Complex 2 formation and TNF production were analyzed after stimulation of RAW 264.7 macrophages with increasing concentrations of LPS for 2 hr. LPS concentrations as low as 10 pg/ml were sufficient to induce both complex 2 formation and TNF production. Treatment of the cells with increasing doses of LPS led to increases in both complex 2 intensity and TNF production (Fig. 8B).
We report the identification of two protein complexes (1 and 2) that specifically involve the AUR sequence of TNFα mRNA. Remarkably, formation of complex 2 is dependent on the activation state of the cells. While it is hardly detectable in EMSA performed with cytoplasmic extracts from unstimulated RAW cells, complex 2 is readily observed with cytoplasmic extracts obtained from LPS-stimulated RAW cells.
Both complexes 1 and 2 specifically involve the TNF mRNA AUR sequence since removal of this sequence abolishes the formation of both complexes. Complex 2 is detected only when the 513 nucleotides probe containing the AUR together with its flanking sequences (3′ TNF AU+) is used, and not with the shorter 72 nt AU+ probe. Competition experiments, however, show that this shorter AU+ RNA effectively impairs the formation of complex 2 by the 3′ TNF AU+ RNA. These observations suggest that the AUR element is recognized by factor(s) necessary for complex 2 formation, but that the stabilization of this complex requires either additional flanking sequences or additional proteins that interact with these sequences. In this regard, it has been shown that the AUR sequence is necessary but not sufficient to confer LPS inducibility to a CAT reporter gene and that the presence of additional flanking sequences from TNF mRNA 3′ UTR are also required (19,20).
UV cross-linking and label transfer experiments demonstrate that S100 extracts of RAW macrophages contain two proteins of 50 and 80 kD which bind TNF mRNA AUR sequence. While the amount of 80-kD binding protein decreases upon stimulation of the cells with LPS, the amount of 50-kD binding protein increases. Although the association of the 50- and 80-kD proteins to one or the other complexes remains speculative, these data suggest that the 50-kD protein might be the component of complex 2 involved in the recognition of the RNA target sequence.
The biological significance of complex 2 formation with respect to TNF mRNA translational activation is supported by the following findings: (i) two unrelated inducers of TNF mRNA translation (LPS and Sendai virus) can promote complex 2 formation; (ii) complex 2 is not observed upon LPS stimulation of A20 B cells or 3B4.15 T cells which do not produce TNF in response to LPS; (iii) preincubation of cells with the tyrosine phosphorylation inhibitor herbimycin A, which is known to abrogate LPS-induced TNF mRNA translational activation (22), abolishes complex 2 formation induced by LPS; (iv) the kinetics of complex 2 formation closely parallels the induction of TNF upon LPS stimulation; and (v) very low doses of LPS, sufficient to trigger TNF production in RAW cells, also promote complex 2 formation.
A recent report shows that in unstimulated macrophages TNF mRNA remains unbound to the translational machinery or is sequestered in a 43S preinitiation complex. Upon stimulation with LPS, TNF mRNA shifts into the polyribosomes (31). These data suggest that TNF mRNA translation is controlled at an early step of mobilization or initiation. Based on these observations, we speculate that complex 2 formation is a prerequisite step for the targeting of TNF mRNA to the translation machinery since the complex 2 is observed with cytosolic extract and not with a salt wash of the ribosomal pellet (data not shown). This event is dependent on tyrosine phosphorylation since complex 2 formation is inhibited by the protein-tyrosine kinase inhibitor herbimycin A.
Several cytoplasmic proteins binding to AUR sequences have been identified so far (11,12,14,32). These proteins were, however, only found in cells where the destabilizing effect of the AUR sequences has been observed. While some of these factors bind constitutively to the sequence, others are induced by certain stimuli. Indeed, Bohjanen et al. identified two other proteins of 30 and 43 kD which bind to the AUR sequence upon activation of T lymphocytes with anti-CD3 antibodies (14,33). Based on apparent molecular weight and subcellular localisation, the 80- and 50-kD proteins identified in this study seem not to be related to any of these previously identified proteins.
The precise characterization of the proteins involved in the LPS-inducible complex and the cloning of the corresponding genes will provide more insight into the understanding of TNF mRNA translational regulation.
We thank Oberdan Leo, Patrice Dubois, and Serge Barcy for helpful discussion; Zoulikka Amraoui for help with the TNF ELISA; Véronique Willeaume for providing us the AU+ DNA construct; and Fabienne Andris for the 3B4.15 cell line. This work was supported by the “Fonds pour la Recherche Scientifique Médicale” (Belgium, Grant 3.4586.93), the “Fonds de la Recherche Fondamentale Collective” (Grant 2.4572.93), the “Fonds Dehousse,” the Belgian sport association against cancer and by a grant from “Actions de Recherches Concertées” (Contract 94/99–181). C. Gueydan and L. Houzet are supported by the “Fonds des bourses de l’U.L.B.”
- Jackson RJ. (1993) Cytoplasmic regulation of mRNA function: The importance of the 3′ untranslated region. Cell 74: 9–14.View ArticlePubMedGoogle Scholar
- Atwater JA, Wisdom R, Verma IM. (1990) Regulated mRNA stability. Annu. Rev. Genet. 24: 519–541.View ArticlePubMedGoogle Scholar
- Morris D. (1995) Growth control of translation in mammalian cells. Prog. Nucleic. Acid. Res. Mol. Biol. 51: 339–362.View ArticlePubMedGoogle Scholar
- Beelman CA, Parker R. (1995) Degradation of mRNA in eukaryotes. Cell 81: 179–183.View ArticlePubMedGoogle Scholar
- Caput D, Beutler B, Hartog K, Thayer R, Brown Shimer S, Cerami A. (1986) Identification of a common nucleotide sequence in the 3′-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. U.S.A. 83: 1670–1674.View ArticlePubMedPubMed CentralGoogle Scholar
- Shaw G, Kamen R. (1986) A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659–667.View ArticlePubMedGoogle Scholar
- Wilson T, Treisman R. (1988) Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3′ AU-rich sequences. Nature 336: 396–399.View ArticlePubMedGoogle Scholar
- Kruys V, Huez G. (1994) Translational control of cytokine expression by 3′ UA-rich sequences. Biochimie 76: 862–866.View ArticlePubMedGoogle Scholar
- Lagnado CA, Brown CY, Goodall GJ. (1994) AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: The functional sequence within AU-rich elements may be UUAUUUA(U/A) (U/A). Mol. Cell. Biol. 14: 7984–7995.View ArticlePubMedPubMed CentralGoogle Scholar
- Zubiaga AM, Belasco JG, Greenberg ME. (1995) The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol. Cell. Biol. 15: 2219–2230.View ArticlePubMedPubMed CentralGoogle Scholar
- Maker JS. (1989) Identification of an AUUUA-specific messenger RNA binding protein. Science 246: 664–666.View ArticleGoogle Scholar
- Vakalopoulou E, Schaack J, Shenk T. (1991) A 32-kilodalton protein binds to AU-rich domains in the 3′ untranslated regions of rapidly degraded mRNAs. Mol. Cell. Biol. 11: 3355–3364.View ArticlePubMedPubMed CentralGoogle Scholar
- Brewer G. (1991) An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol. Cell. Biol. 11: 2460–2466.View ArticlePubMedPubMed CentralGoogle Scholar
- Bohjanen PR, Petryniak B, June CH, Thompson CB, Lindsten T. (1991) An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3′ untranslated region of lymphokine mRNA. Mol. Cell. Biol. 11: 3288–3295.View ArticlePubMedPubMed CentralGoogle Scholar
- Kruys V, Wathelet M, Poupart P, et al. (1987) The 3′ untranslated region of the human interferon-beta mRNA has an inhibitory effect on translation. Proc. Natl. Acad. Sci. U.S.A. 84: 6030–6034.View ArticlePubMedPubMed CentralGoogle Scholar
- Kruys VI, Wathelet MG, Huez GA. (1988) Identification of a translation inhibitory element (TIE) in the 3′ untranslated region of the human interferon-beta mRNA. Gene 72: 191–200.View ArticlePubMedGoogle Scholar
- Kruys V, Marinx O, Shaw G, Deschamps J, Huez G. (1989) Translational blockade imposed by cytokine-derived UA-rich sequences. Science 245: 852–855.View ArticlePubMedGoogle Scholar
- Kruys V, Kemmer K, Shakhov A, Jongeneel V, Beutler B. (1992) Constitutive activity of the tumor necrosis factor promoter is canceled by the 3′ untranslated region in non-macrophage cell lines; A trans-dominant factor overcomes this suppressive effect. Proc. Natl. Acad. Sci. U.S.A. 89: 673–677.View ArticlePubMedPubMed CentralGoogle Scholar
- Han J, Brown T, Beutler B. (1990) Endotox-in-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level [published erratum appears in J Exp Med 1990 Mar 1;171: 971–972]. J. Exp. Med. 171: 465–475.View ArticlePubMedGoogle Scholar
- Han J, Beutler B. (1990) The essential role of the UA-rich sequence in endotoxin-induced cachectin/TNF synthesis. Eur. Cytokine Netw. 1: 71–75.PubMedGoogle Scholar
- Han JH, Beutler B, Huez G. (1991) Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages. Biochim. Biophys. Acta 1090: 22–28.View ArticlePubMedGoogle Scholar
- Willeaume V, Kruys V, Mijatovic T, Huez G. (in press) TNF-α production induced by viruses and by LPS in macrophages: Similarities and differences. J. Inflamm.Google Scholar
- Beutler B, Brown T. (1991) A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines. J. Clin. Invest. 87: 1336–1344.View ArticlePubMedPubMed CentralGoogle Scholar
- Andris F, Leo O, Van Mechelen M, Urbain J, Slaoui M. (1989) CD4-Ia interactions can occur in the absence of T-cell receptor/anti-gen-Ia recognition. Immunology 68: 1–6.PubMedPubMed CentralGoogle Scholar
- Dignam JD, Lebovitz RM, Roeder RG. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11: 1475–1489.View ArticlePubMedPubMed CentralGoogle Scholar
- Konarska MM, Sharp PA. (1986) Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell 46: 845–855.View ArticlePubMedGoogle Scholar
- Leibold EA, Munro HN. (1988) Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′ untranslated region of ferritin heavy- and light-subunit mRNAs. Proc. Natl. Acad. Sci. U.S.A. 85: 2171–2175.View ArticlePubMedPubMed CentralGoogle Scholar
- Aderka D, Holtmann H, Toker L, Hahn T, Wallach D. (1986) Tumor necrosis factor induction by Sendai virus. J. Immunol. 136: 2938–2942.PubMedGoogle Scholar
- Weinstein SL, June CH, DeFranco AL. (1993) Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14. J. Immunol. 151: 3829–3838.PubMedGoogle Scholar
- Weinstein SL, Gold MR, DeFranco AL. (1991) Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc. Natl. Acad. Sci. U.S.A. 88: 4148–4152.View ArticlePubMedPubMed CentralGoogle Scholar
- Prichett W, Hand A, Shields J, Dunnington D. (1995) Mechanism of action of bicyclic imidazoles defines a translational regulatory pathway for tumor necrosis factor alpha. J. Inflamm. 45: 97–105.PubMedGoogle Scholar
- Zhang W, Wagner BJ, Ehrenman K, et al. (1993) Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol. Cell. Biol. 13: 7652–7665.View ArticlePubMedPubMed CentralGoogle Scholar
- Bohjanen PR, Petryniak B, June CH, Thompson CB, Lindsten T. (1992) AU RNA-binding factors differ in their binding specificities and affinities. J. Biol. Chem. 267: 6302–6309.PubMedGoogle Scholar