Abstract
Using DDRT-PCR, we compared the mRNA content of untreated and TNF-treated mouse embryonic fibroblasts (MEFs). Among differentially represented fragments, we identified and cloned a novel TNF-stimulated gene named Tsg-5. This gene, mapped to mouse chromosome 14, has three exons that can be alternatively spliced giving rise to two mRNA species, one spanning three exons and another that skips the second exon. Analysis of full-length Tsg-5 cDNA revealed a potential start codon within exon 2 encoding an ORF of 40 amino-acids. No homology with known mouse or human sequences, neither at the nucleotide nor at the amino-acid level could be found in public databases. In MEFs, Tsg-5 is induced by tumor necrossis factor-α (TNF) and IL-1β, albeit with distinct kinetics. TNF-induced Tsg-5 expression is NF-κB-dependent as it was inhibited by MG132, lactacystin, Bay 11–7083, and Bay 11–7085. Analysis of Tsg-5 expression in vivo revealed that the gene and its encoded polypeptide are constitutively expressed in the thymus and ovary, whereas, in LPS-treated mice, Tsg-5 mRNA can be detected in the spleen, lung, and brain. Our data suggest that Tsg-5 encodes a new, rare transcript, with a very tight regulation of expression and differential splicing.
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References
Kollias G, Douni E, Kassiotis G, Kontoyiannis D . On the role of tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Immunol Rev 1999; 169: 175–194.
Tracey KJ, Cerami A . Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 1993; 9: 317–343.
Lewis M, Tartaglia LA, Lee A et al. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc Natl Acad Sci USA 1991; 88: 2830–2834.
Hohmann HP, Remy R, Brockhaus M, van Loon AP . Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha). J Biol Chem 1989; 264: 14927–14934.
Banner DW, D'Arcy A, Janes W et al. Crystal structure of the soluble human 55 kd TNF receptor–human TNF beta complex: implications for TNF receptor activation. Cell 1993; 73: 431–445.
Baud V, Karin M . Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 2001; 11: 372–377.
Agaarwall BB, Samanta A, Feldmann M . TNF-alfa. In: Oppenheim JD, Feldmann M (eds). Cytokine Reference. Academic Press: London, 2000, pp 413–434.
Fujita T, Reis LF, Watanabe N, Kimura Y, Taniguchi T, Vilcek J . Induction of the transcription factor IRF-1 and interferon-beta mRNAs by cytokines and activators of second-messenger pathways. Proc Natl Acad Sci USA 1989; 86: 9936–9940.
Reis LF, Ruffner H, Stark G, Aguet M, Weissmann C . Mice devoid of interferon regulatory factor 1 (IRF-1) show normal expression of type I interferon genes. EMBO J 1994; 13: 4798–4806.
Taniguchi T, Ogasawara K, Takaoka A, Tanaka N . IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 2001; 19: 623–655.
Kirchhoff S, Oumard A, Nourbakhsh M, Levi BZ, Hauser H . Interplay between repressing and activating domains defines the transcriptional activity of IRF-1. Eur J Biochem 2000; 267: 6753–6761.
Manzella L, Gualdi R, Perrotti D et al. The interferon regulatory factors 1 and 2 bind to a segment of the human c-myb first intron: possible role in the regulation of c-myb expression. Exp Cell Res 2000; 256: 248–256.
Ohmori Y, Fukumoto S, Hamilton TA . Two structurally distinct kappa B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. J Immunol 1995; 155: 3593–3600.
Kozak M . An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987; 15: 8125–8148.
Rubartelli A, Sitia R . Secretion of mammalian proteins that lack a signal sequence. In: Kuchler K, Rubartelli A, Holland BI (eds). Unusual Secretory Pathways: From Bacteria to Man. R.G. Landes: Austin, 1997, pp 87–104.
Mount SM . A catalogue of splice junction sequences. Nucleic Acids Res 1982; 10: 459–472.
Hsieh CL, Bowcock AM, Farrer LA et al. The serotonin receptor subtype 2 locus HTR2 is on human chromosome 13 near genes for esterase D and retinoblastoma-1 and on mouse chromosome 14. Somat Cell Mol Genet 1990; 16: 567–574.
Sheiness D, Puckett L, Darnell JE . Possible relationship of poly(A) shortening to mRNA turnover. Proc Natl Acad Sci USA 1975; 72: 1077–1081.
Heinlein UA, Lange-Sablitzky R, Schaal H, Wille W . Molecular characterization of the MT-family of dispersed middle-repetitive DNA in rodent genomes. Nucleic Acids Res 1986; 14: 6403–6416.
Bastien L, Bourgaux P . The MT family of mouse DNA is made of short interspersed repeated elements. Gene 1987; 57: 81–88.
Smit AF . Identification of a new, abundant superfamily of mammalian LTR-transposons. Nucleic Acids Res 1993; 21: 1863–1872.
Britten RJ . Cases of ancient mobile element DNA insertions that now affect gene regulation. Mol Phylogenet Evol 1996; 5: 13–17.
Swee MH, Parks WC, Pierce RA . Developmental regulation of elastin production. Expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels. J Biol Chem 1995; 270: 14899–14906.
Zhang M, Pierce RA, Wachi H, Mecham RP, Parks WC . An open reading frame element mediates posttranscriptional regulation of tropoelastin and responsiveness to transforming growth factor beta1. Mol Cell Biol 1999; 19: 7314–7326.
Bauren G, Wieslander L . Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription. Cell 1994; 76: 183–192.
Zhang G, Taneja KL, Singer RH, Green MR . Localization of pre-mRNA splicing in mammalian nuclei. Nature 1994; 372: 809–812.
Pierce JW, Schoenleber R, Jesmok G et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 1997; 272: 21096–21103.
Poppers DM, Schwenger P, Vilcek J . Persistent tumor necrosis factor signaling in normal human fibroblasts prevents the complete resynthesis of I kappa B-alpha. J Biol Chem 2000; 275: 29587–29593.
Sun SC, Ganchi PA, Ballard DW, Greene WC . NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science 1993; 259: 1912–1915.
de Martin R, Vanhove B, Cheng Q et al. Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B. EMBO J 1993; 12: 2773–2779.
Attwood JT, Yung RL, Richardson BC . DNA methylation and the regulation of gene transcription. Cell Mol Life Sci 2002; 59: 241–257.
Baylin SB, Herman JG . DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16: 168–174.
Antequera F, Boyes J, Bird A . High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 1990; 62: 503–514.
Jones PA, Wolkowicz MJ, Rideout III WM et al. De novo methylation of the MyoD1 CpG island during the establishment of immortal cell lines. Proc Natl Acad Sci USA 1990; 87: 6117–6121.
Tate PH, Bird AP . Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev 1993; 3: 226–231.
Santi DV, Norment A, Garrett CE . Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci USA 1984; 81: 6993–6997.
Giroir BP, Brown T, Beutler B . Constitutive synthesis of tumor necrosis factor in the thymus. Proc Natl Acad Sci USA 1992; 89: 4864–4868.
Wolf SS, Cohen A . Expression of cytokines and their receptors by human thymocytes and thymic stromal cells. Immunology 1992; 77: 362–368.
Deman J, Van Meurs M, Claassen E, Humblet C, Boniver J, Defresne MP . In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus. Immunology 1996; 89: 152–157.
Ryffel B, Brockhaus M, Greiner B, Mihatsch MJ, Gudat F . Tumour necrosis factor receptor distribution in human lymphoid tissue. Immunology 1991; 74: 446–452.
Chen HL, Marcinkiewicz JL, Sancho-Tello M, Hunt JS, Terranova PF . Tumor necrosis factor -alpha gene expression in mouse oocytes and follicular cells. Biol Reprod 1993; 48: 707–714.
Simon C, Frances A, Piquette G, Polan ML . Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 1994; 50: 449–457.
Marino MW, Dunn A, Grail D et al. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci USA 1997; 94: 8093–8098.
Rothe J, Lesslauer W, Lotscher H et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 1993; 364: 798–802.
Espey LL . Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod 1994; 50: 233–238.
Terranova PF, Rice VM . Review: cytokine involvement in ovarian processes. Am J Reprod Immunol 1997; 37: 50–63.
Roby KF, Son DS, Terranova PF . Alterations of events related to ovarian function in tumor necrosis factor receptor type I knockout mice. Biol Reprod 1999; 61: 1616–1621.
Wekerle H, Ketelsen UP . Thymic nurse cells—Ia-bearing epithelium involved in T-lymphocyte differentiation? Nature 1980; 283: 402–404.
Houben-Defresne MP, Varlet A, Goffinet G, Boniver J . Thymic nurse cells are the first site of virus replication after inoculation of the radiation leukemia virus. Leuk Res 1982; 6: 231–241.
Papiernik M, Nabarra B, Savino W, Pontoux C, Barbey S . Thymic reticulum in mice. II. Culture and characterization of nonepithelial phagocytic cells of the thymic reticulum: their role in the syngeneic stimulation of thymic medullary lymphocytes. Eur J Immunol 1983; 13: 147–155.
Liang P, Pardee AB . Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992; 257: 967–971.
Silva AM, Pires EG, Abrantes EF, Ferreira LR, Gazzinelli RT, Reis LF . Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes. Braz J Med Biol Res 1999; 32: 845–852.
Silva AM, Reis LF . Sodium salicylate induces the expression of the immunophilin FKBP51 and biglycan genes and inhibits p34cdc2 mRNA both in vitro and in vivo. J Biol Chem 2000; 275: 36388–36393.
Muller U, Steinhoff U, Reis LF et al. Functional role of type I and type II interferons in antiviral defense. Science 1994; 264: 1918–1921.
Ausubel FM, Brent R, Kingston RE et al. Screening of Recombinant DNA Libraries. John Wiley & Sons, Inc.: New York, 1996.
Church GM, Gilbert W . Genomic sequencing. Proc Natl Acad Sci USA 1984; 81: 1991–1995.
Villa-Verde DM, Lagrota-Candido JM, Vannier-Santos MA, Chammas R, Brentani RR, Savino W . Extracellular matrix components of the mouse thymus microenvironment. IV. Modulation of thymic nurse cells by extracellular matrix ligands and receptors. Eur J Immunol 1994; 24: 659–664.
Acknowledgements
We thank Anna Christina de Matos Salim and Elizangela Monteiro for performing all sequences reported in this work and Drs Mike Marino and João Santana da Silva for TNF−/− and TNFRSF1A−/− mice, respectively. We also thank Dr Patricia Bozza for her criticisms during an early phase of these experiments, Carlos Ferreira, Miyuki F da Silva, and Suely Nonogaki for helping with tissue sections, Drs Ana Paula Lepique, Anamaria A Camargo, and Jan Vilcek for critically reading the manuscript, and all members of our labs for their suggestions and discussions. EFA was supported by a predoctoral fellowship from CNPq/Ministry of Science and Technology and is now a postdoctoral fellow from FAPESP. FFC is a predoctoral fellow from FAPESP. This work was supported in part by grants from FAPESP (LFLR) and CNPq, Ministry of Science and Technology.
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Abrantes, E., Pires, E., Carvalho, A. et al. Identification, structural characterization, and tissue distribution of Tsg-5: a new TNF-stimulated gene. Genes Immun 4, 298–311 (2003). https://doi.org/10.1038/sj.gene.6363949
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DOI: https://doi.org/10.1038/sj.gene.6363949