Human Tumor Necrosis Factor-α Gene 3′ Untranslated Region Confers Inducible Toxin Responsiveness to Homologous Promoter in Monocytic THP-1 Cells
1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês
10.1074/jbc.274.31.21714
ISSN1083-351X
AutoresAnne Seiler-Tuyns, Nathalie Dufour, François Spertini,
Tópico(s)T-cell and B-cell Immunology
ResumoTo better define the role of 3′ untranslated region (3′UTR) on transcriptional regulation of the human tumor necrosis factor (TNF)-α gene, monocytic human THP-1 cells were transfected with two TNF-α promoter constructs spanning base pairs −1897/−1 and −1214/−1, respectively, and linked to the rabbit β-globin gene. Quantitative globin gene expression of chimerae was measured by reverse transcription-polymerase chain reaction. A construct linking the chicken β-actin promoter and a deleted portion of the β-globin gene was cotransfected and used as internal standard. Unexpectedly, when THP-1 cells were stimulated with lipopolysaccharide or toxic shock syndrome toxin-1, gene regulation was hardly detected. In contrast, endogenous TNF-α gene regulation measured by the same reverse transcription-polymerase chain reaction procedure was vigorous. Remarkably, ligation of 3′UTR to chimeric constructs led to a drastic drop in the basal level of chimeric gene expression, resulting in a 15- to 40-fold induction of the reporter gene. Consistently, when the TNF-α promoter was replaced by the cytomegalovirus early immediate promoter, gene expression was also uniformly reduced but was no longer up-regulated upon stimulation with lipopolysaccharide and toxic shock syndrome toxin-1. These data provide the first line of evidence that, in addition to its role in TNF-α transcript stability and translation, human TNF-α 3′UTR also participates in modulating gene expression at the transcriptional level. To better define the role of 3′ untranslated region (3′UTR) on transcriptional regulation of the human tumor necrosis factor (TNF)-α gene, monocytic human THP-1 cells were transfected with two TNF-α promoter constructs spanning base pairs −1897/−1 and −1214/−1, respectively, and linked to the rabbit β-globin gene. Quantitative globin gene expression of chimerae was measured by reverse transcription-polymerase chain reaction. A construct linking the chicken β-actin promoter and a deleted portion of the β-globin gene was cotransfected and used as internal standard. Unexpectedly, when THP-1 cells were stimulated with lipopolysaccharide or toxic shock syndrome toxin-1, gene regulation was hardly detected. In contrast, endogenous TNF-α gene regulation measured by the same reverse transcription-polymerase chain reaction procedure was vigorous. Remarkably, ligation of 3′UTR to chimeric constructs led to a drastic drop in the basal level of chimeric gene expression, resulting in a 15- to 40-fold induction of the reporter gene. Consistently, when the TNF-α promoter was replaced by the cytomegalovirus early immediate promoter, gene expression was also uniformly reduced but was no longer up-regulated upon stimulation with lipopolysaccharide and toxic shock syndrome toxin-1. These data provide the first line of evidence that, in addition to its role in TNF-α transcript stability and translation, human TNF-α 3′UTR also participates in modulating gene expression at the transcriptional level. TNF-α, 1The abbreviations used are: TNF, tumor necrosis factor; huTNF, human tumor necrosis factor; 3′UTR, 3′ untranslated region; LPS, lipopolysaccharide; TSST-1, toxic shock syndrome toxin-1; SEB, staphylococcal enterotoxin B; PCR, polymerase chain reaction; CMV, cytomegalovirus; bp, base pair(s); CAT, chloramphenicol acetyltransferase.1The abbreviations used are: TNF, tumor necrosis factor; huTNF, human tumor necrosis factor; 3′UTR, 3′ untranslated region; LPS, lipopolysaccharide; TSST-1, toxic shock syndrome toxin-1; SEB, staphylococcal enterotoxin B; PCR, polymerase chain reaction; CMV, cytomegalovirus; bp, base pair(s); CAT, chloramphenicol acetyltransferase. a pleiotropic cytokine produced mainly by macrophages, plays a central role in cell immune responses (1Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1290) Google Scholar, 2Le J. Vilcek J. Lab. Invest. 1987; 56: 234-247PubMed Google Scholar, 3Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar), host defense (4Beutler B. Cerami A. Biochemistry. 1988; 27: 7575-7582Crossref PubMed Scopus (114) Google Scholar), and inflammation. TNF-α gene expression mediated by lipopolysaccharide (LPS) or by MHC class II ligands such as bacterial superantigen toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB) appears to be regulated at both the transcriptional and post-transcriptional levels (5Beutler B. Cerami A. Nature. 1986; 320: 584-588Crossref PubMed Scopus (1293) Google Scholar, 6Trede N.S. Geha R.S. Chatila T. J. Immunol. 1991; 146: 2310-2315PubMed Google Scholar, 7Espel E. Garcia-Sanz J.A. Aubert V. Menoud V. Sperisen P. Fernandez N. Spertini F. Eur. J. Immunol. 1996; 26: 2417-2424Crossref PubMed Scopus (48) Google Scholar). Remarkably, TNF-α promoter responses were only weakly induced by LPS, and even in non-stimulated cells, a significant constitutive gene expression was detected (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar, 9Takashiba S. Van Dyke T.E. Shapira L. Amar S. Infect. Immun. 1995; 63: 1529-1534Crossref PubMed Google Scholar). The role of human TNF-α 3′ untranslated region (3′UTR) in post-transcriptional control of TNF-α mRNA has been well documented (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar). A conserved sequence element in the 3′UTR of several cytokines in several species, the TTATTTAT element, normally confers translational repression (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar, 11Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). Whereas this UA-rich motif also confers instability to many cytokine mRNAs, TNF-α transcript stability appears to be unchanged after cell stimulation by LPS (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar, 12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 13Han J. Beutler B. Huez G. Biochim. Biophys. Acta. 1991; 1090: 22-28Crossref PubMed Scopus (56) Google Scholar). Interestingly, it was first demonstrated in the mouse that TNF-α promoter and 3′UTR synergized to regulate murine TNF-α gene expression (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar). Moreover, constitutive expression of mouse TNF-α promoter in non-macrophage cell line L929 could be suppressed by ligating mouse 3′UTR to chimeric CAT constructs, indicating that mouse 3′UTR also played a role in silencing TNF-α gene in cells in which it was not expressed (14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar). In this study, we extend these observations to the human cell line THP-1 and demonstrate, using constructs derived from the human TNF-α promoter, that despite a vigorous induction of endogenous TNF-α, no significant regulation of a reporter gene could be detected upon cell stimulation by LPS or TSST-1. Regulation of two TNF-α chimeric constructs could only be obtained after TNF-α 3′UTR ligation to promoter constructs. These data indicate that the TNF-α gene promoter and its 3′UTR cooperate in regulating gene expression at the transcriptional level in humans as well.DISCUSSIONWe have shown here that the TNF-α 3′UTR plays a crucial role in human TNF-α gene transcriptional regulation, an observation that further extends its role in RNA transcript stability and translation as described previously (23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar). In our approach, we took advantage of a reporter gene system that allows direct analysis of RNA expression, in contrast to CAT or luciferase gene assays, which are less appropriate to tackle this issue. These latter systems involve protein enzymatic assays that not only reflect transcriptional regulation but, depending on the transfected chimera, also reflect the post-transcriptional and/or translational regulations that can affect the final enzymatic activity. Because TNF-α 3′UTR contains sequences that can modulate translation (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar), we overcame this pitfall by directly measuring RNA transcripts. Thus, it appeared that chimeric constructs driven by large fragments of the human TNF-α promoter were strongly expressed in the monocytic cell line THP-1 but only weakly regulated by stimuli such as TSST-1 and LPS. These data are consistent with results from Goldfeldet al. (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar), who transfected human TNF-α promoter-CAT chimeric constructs in the murine monocytic cell line P3888D1 and found a significant level of expression of the chimeras in uninduced cells. In agreement with our data, only weak (1.5- to 2-fold) induction by LPS could be detected. Takashiba et al. (9Takashiba S. Van Dyke T.E. Shapira L. Amar S. Infect. Immun. 1995; 63: 1529-1534Crossref PubMed Google Scholar), who also used CAT assays, found similar induction ratios. Very different results were obtained for the mouse gene because a strong induction by LPS was detectable in chimeric constructs containing mouse TNF promoter only (24Drouet C. Shakhov A.N. Jongeneel C.V. J. Immunol. 1991; 147: 1694-1700PubMed Google Scholar). Furthermore, kB-type enhancers were involved in the transcription of the murine gene (25Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (729) Google Scholar) but not the human gene (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar). Although the regulation of human and mouse TNF genes differs in many respects, we took advantage of previous observations on the murine gene to design the chimeric constructs studied here. The role of mouse 3′UTR on chimeric gene expression has been well documented (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 13Han J. Beutler B. Huez G. Biochim. Biophys. Acta. 1991; 1090: 22-28Crossref PubMed Scopus (56) Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar, 23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar, 26Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). In particular, mouse TNF-α 3′UTR is able to suppress TNF-α promoter constitutive activity in non-macrophage cell lines (14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar). Furthermore, it interacts with the mouse TNF-α 5′ end to modulate chimeric CAT construct expression (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar). We have shown in this study that human TNF-α 3′UTR suppressed the strong basal expression of TNF-α promoter constructs observed in its absence in unstimulated THP-1 cells and partially restored gene regulation in induced cells. This regulation could be due to transcriptional and/or post-transcriptional phenomena. Evidence for transcriptional regulation was provided by the analysis of constructs in which TNF-α promoter fragments were replaced with the CMV immediate early promoter. CMV chimeric constructs linked to the TNF-α 3′UTR were expressed at a level comparable to TNF-α promoter constructs. However, the expression of CMV chimeric construct CMV/3′s was poorly regulated or was not regulated, in contrast to chimeric constructs 1/3′s and 2/3′s. These results are in agreement with previous studies on the murine TNF-α gene (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar) and indicate that these observations hold true for human TNF-α gene regulation. Taken together, our data suggest that 5′ TNF-α promoter and 3′UTR regions interact with one another to regulate TNF-α gene expression at transcriptional level as well. The interaction between the 3′ and 5′ regions appears to be independent of the type of stimulation. DNA fragments used in these experiments are large, and the identification of potential cis- and trans- regulatory elements in both the 5′ or 3′ regions will require the study of smaller gene fragments. TNF-α transcription may be enhanced by stimulatory mechanisms and/or the release of a pre-existing block. The precise characterization of transcription factors binding to DNA regions essential for regulated expression of the TNF-α gene may open new perspectives in the understanding of human diseases related to abnormal TNF-α gene expression. Interestingly, mutated genomic sequences in regions flanking the 3′ TTATTTAT signal element of TNF-α gene have been described in murine models (27Jacob C.O. Lee S.K. Strassmann G. J. Immunol. 1996; 156: 3043-3050PubMed Google Scholar), but not in young patients with autoimmune diseases (28Becker L. Brown T. Fink C. Marks J. Lvandosky G. Giroir B.P. Pediatr. Res. 1995; 37: 165-168Crossref PubMed Scopus (7) Google Scholar). Because our results demonstrate the presence of other crucial regulatory domains interacting with promoter regions, the precise identification of discrete regulatory elements may help us to better understand the regulation of the TNF-α gene in inflammatory diseases. TNF-α, 1The abbreviations used are: TNF, tumor necrosis factor; huTNF, human tumor necrosis factor; 3′UTR, 3′ untranslated region; LPS, lipopolysaccharide; TSST-1, toxic shock syndrome toxin-1; SEB, staphylococcal enterotoxin B; PCR, polymerase chain reaction; CMV, cytomegalovirus; bp, base pair(s); CAT, chloramphenicol acetyltransferase.1The abbreviations used are: TNF, tumor necrosis factor; huTNF, human tumor necrosis factor; 3′UTR, 3′ untranslated region; LPS, lipopolysaccharide; TSST-1, toxic shock syndrome toxin-1; SEB, staphylococcal enterotoxin B; PCR, polymerase chain reaction; CMV, cytomegalovirus; bp, base pair(s); CAT, chloramphenicol acetyltransferase. a pleiotropic cytokine produced mainly by macrophages, plays a central role in cell immune responses (1Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1290) Google Scholar, 2Le J. Vilcek J. Lab. Invest. 1987; 56: 234-247PubMed Google Scholar, 3Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar), host defense (4Beutler B. Cerami A. Biochemistry. 1988; 27: 7575-7582Crossref PubMed Scopus (114) Google Scholar), and inflammation. TNF-α gene expression mediated by lipopolysaccharide (LPS) or by MHC class II ligands such as bacterial superantigen toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB) appears to be regulated at both the transcriptional and post-transcriptional levels (5Beutler B. Cerami A. Nature. 1986; 320: 584-588Crossref PubMed Scopus (1293) Google Scholar, 6Trede N.S. Geha R.S. Chatila T. J. Immunol. 1991; 146: 2310-2315PubMed Google Scholar, 7Espel E. Garcia-Sanz J.A. Aubert V. Menoud V. Sperisen P. Fernandez N. Spertini F. Eur. J. Immunol. 1996; 26: 2417-2424Crossref PubMed Scopus (48) Google Scholar). Remarkably, TNF-α promoter responses were only weakly induced by LPS, and even in non-stimulated cells, a significant constitutive gene expression was detected (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar, 9Takashiba S. Van Dyke T.E. Shapira L. Amar S. Infect. Immun. 1995; 63: 1529-1534Crossref PubMed Google Scholar). The role of human TNF-α 3′ untranslated region (3′UTR) in post-transcriptional control of TNF-α mRNA has been well documented (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar). A conserved sequence element in the 3′UTR of several cytokines in several species, the TTATTTAT element, normally confers translational repression (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar, 11Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). Whereas this UA-rich motif also confers instability to many cytokine mRNAs, TNF-α transcript stability appears to be unchanged after cell stimulation by LPS (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar, 12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 13Han J. Beutler B. Huez G. Biochim. Biophys. Acta. 1991; 1090: 22-28Crossref PubMed Scopus (56) Google Scholar). Interestingly, it was first demonstrated in the mouse that TNF-α promoter and 3′UTR synergized to regulate murine TNF-α gene expression (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar). Moreover, constitutive expression of mouse TNF-α promoter in non-macrophage cell line L929 could be suppressed by ligating mouse 3′UTR to chimeric CAT constructs, indicating that mouse 3′UTR also played a role in silencing TNF-α gene in cells in which it was not expressed (14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar). In this study, we extend these observations to the human cell line THP-1 and demonstrate, using constructs derived from the human TNF-α promoter, that despite a vigorous induction of endogenous TNF-α, no significant regulation of a reporter gene could be detected upon cell stimulation by LPS or TSST-1. Regulation of two TNF-α chimeric constructs could only be obtained after TNF-α 3′UTR ligation to promoter constructs. These data indicate that the TNF-α gene promoter and its 3′UTR cooperate in regulating gene expression at the transcriptional level in humans as well. DISCUSSIONWe have shown here that the TNF-α 3′UTR plays a crucial role in human TNF-α gene transcriptional regulation, an observation that further extends its role in RNA transcript stability and translation as described previously (23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar). In our approach, we took advantage of a reporter gene system that allows direct analysis of RNA expression, in contrast to CAT or luciferase gene assays, which are less appropriate to tackle this issue. These latter systems involve protein enzymatic assays that not only reflect transcriptional regulation but, depending on the transfected chimera, also reflect the post-transcriptional and/or translational regulations that can affect the final enzymatic activity. Because TNF-α 3′UTR contains sequences that can modulate translation (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar), we overcame this pitfall by directly measuring RNA transcripts. Thus, it appeared that chimeric constructs driven by large fragments of the human TNF-α promoter were strongly expressed in the monocytic cell line THP-1 but only weakly regulated by stimuli such as TSST-1 and LPS. These data are consistent with results from Goldfeldet al. (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar), who transfected human TNF-α promoter-CAT chimeric constructs in the murine monocytic cell line P3888D1 and found a significant level of expression of the chimeras in uninduced cells. In agreement with our data, only weak (1.5- to 2-fold) induction by LPS could be detected. Takashiba et al. (9Takashiba S. Van Dyke T.E. Shapira L. Amar S. Infect. Immun. 1995; 63: 1529-1534Crossref PubMed Google Scholar), who also used CAT assays, found similar induction ratios. Very different results were obtained for the mouse gene because a strong induction by LPS was detectable in chimeric constructs containing mouse TNF promoter only (24Drouet C. Shakhov A.N. Jongeneel C.V. J. Immunol. 1991; 147: 1694-1700PubMed Google Scholar). Furthermore, kB-type enhancers were involved in the transcription of the murine gene (25Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (729) Google Scholar) but not the human gene (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar). Although the regulation of human and mouse TNF genes differs in many respects, we took advantage of previous observations on the murine gene to design the chimeric constructs studied here. The role of mouse 3′UTR on chimeric gene expression has been well documented (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 13Han J. Beutler B. Huez G. Biochim. Biophys. Acta. 1991; 1090: 22-28Crossref PubMed Scopus (56) Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar, 23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar, 26Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). In particular, mouse TNF-α 3′UTR is able to suppress TNF-α promoter constitutive activity in non-macrophage cell lines (14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar). Furthermore, it interacts with the mouse TNF-α 5′ end to modulate chimeric CAT construct expression (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar). We have shown in this study that human TNF-α 3′UTR suppressed the strong basal expression of TNF-α promoter constructs observed in its absence in unstimulated THP-1 cells and partially restored gene regulation in induced cells. This regulation could be due to transcriptional and/or post-transcriptional phenomena. Evidence for transcriptional regulation was provided by the analysis of constructs in which TNF-α promoter fragments were replaced with the CMV immediate early promoter. CMV chimeric constructs linked to the TNF-α 3′UTR were expressed at a level comparable to TNF-α promoter constructs. However, the expression of CMV chimeric construct CMV/3′s was poorly regulated or was not regulated, in contrast to chimeric constructs 1/3′s and 2/3′s. These results are in agreement with previous studies on the murine TNF-α gene (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar) and indicate that these observations hold true for human TNF-α gene regulation. Taken together, our data suggest that 5′ TNF-α promoter and 3′UTR regions interact with one another to regulate TNF-α gene expression at transcriptional level as well. The interaction between the 3′ and 5′ regions appears to be independent of the type of stimulation. DNA fragments used in these experiments are large, and the identification of potential cis- and trans- regulatory elements in both the 5′ or 3′ regions will require the study of smaller gene fragments. TNF-α transcription may be enhanced by stimulatory mechanisms and/or the release of a pre-existing block. The precise characterization of transcription factors binding to DNA regions essential for regulated expression of the TNF-α gene may open new perspectives in the understanding of human diseases related to abnormal TNF-α gene expression. Interestingly, mutated genomic sequences in regions flanking the 3′ TTATTTAT signal element of TNF-α gene have been described in murine models (27Jacob C.O. Lee S.K. Strassmann G. J. Immunol. 1996; 156: 3043-3050PubMed Google Scholar), but not in young patients with autoimmune diseases (28Becker L. Brown T. Fink C. Marks J. Lvandosky G. Giroir B.P. Pediatr. Res. 1995; 37: 165-168Crossref PubMed Scopus (7) Google Scholar). Because our results demonstrate the presence of other crucial regulatory domains interacting with promoter regions, the precise identification of discrete regulatory elements may help us to better understand the regulation of the TNF-α gene in inflammatory diseases. We have shown here that the TNF-α 3′UTR plays a crucial role in human TNF-α gene transcriptional regulation, an observation that further extends its role in RNA transcript stability and translation as described previously (23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar). In our approach, we took advantage of a reporter gene system that allows direct analysis of RNA expression, in contrast to CAT or luciferase gene assays, which are less appropriate to tackle this issue. These latter systems involve protein enzymatic assays that not only reflect transcriptional regulation but, depending on the transfected chimera, also reflect the post-transcriptional and/or translational regulations that can affect the final enzymatic activity. Because TNF-α 3′UTR contains sequences that can modulate translation (10Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (430) Google Scholar), we overcame this pitfall by directly measuring RNA transcripts. Thus, it appeared that chimeric constructs driven by large fragments of the human TNF-α promoter were strongly expressed in the monocytic cell line THP-1 but only weakly regulated by stimuli such as TSST-1 and LPS. These data are consistent with results from Goldfeldet al. (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar), who transfected human TNF-α promoter-CAT chimeric constructs in the murine monocytic cell line P3888D1 and found a significant level of expression of the chimeras in uninduced cells. In agreement with our data, only weak (1.5- to 2-fold) induction by LPS could be detected. Takashiba et al. (9Takashiba S. Van Dyke T.E. Shapira L. Amar S. Infect. Immun. 1995; 63: 1529-1534Crossref PubMed Google Scholar), who also used CAT assays, found similar induction ratios. Very different results were obtained for the mouse gene because a strong induction by LPS was detectable in chimeric constructs containing mouse TNF promoter only (24Drouet C. Shakhov A.N. Jongeneel C.V. J. Immunol. 1991; 147: 1694-1700PubMed Google Scholar). Furthermore, kB-type enhancers were involved in the transcription of the murine gene (25Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (729) Google Scholar) but not the human gene (8Goldfeld A.E. Doyle C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9769-9773Crossref PubMed Scopus (214) Google Scholar). Although the regulation of human and mouse TNF genes differs in many respects, we took advantage of previous observations on the murine gene to design the chimeric constructs studied here. The role of mouse 3′UTR on chimeric gene expression has been well documented (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 13Han J. Beutler B. Huez G. Biochim. Biophys. Acta. 1991; 1090: 22-28Crossref PubMed Scopus (56) Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar, 23Beutler B. Han J. Kruys V. Giroir B.P. Beutler B. Tumor Necrosis Factors. Raven Press, New York1992: 561-574Google Scholar, 26Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). In particular, mouse TNF-α 3′UTR is able to suppress TNF-α promoter constitutive activity in non-macrophage cell lines (14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar). Furthermore, it interacts with the mouse TNF-α 5′ end to modulate chimeric CAT construct expression (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar). We have shown in this study that human TNF-α 3′UTR suppressed the strong basal expression of TNF-α promoter constructs observed in its absence in unstimulated THP-1 cells and partially restored gene regulation in induced cells. This regulation could be due to transcriptional and/or post-transcriptional phenomena. Evidence for transcriptional regulation was provided by the analysis of constructs in which TNF-α promoter fragments were replaced with the CMV immediate early promoter. CMV chimeric constructs linked to the TNF-α 3′UTR were expressed at a level comparable to TNF-α promoter constructs. However, the expression of CMV chimeric construct CMV/3′s was poorly regulated or was not regulated, in contrast to chimeric constructs 1/3′s and 2/3′s. These results are in agreement with previous studies on the murine TNF-α gene (12Han J. Huez G. Beutler B. J. Immunol. 1991; 146: 1843-1848PubMed Google Scholar, 14Kruys V. Kemmer K. Shakhov A. Jongeneel V. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 673-677Crossref PubMed Scopus (63) Google Scholar) and indicate that these observations hold true for human TNF-α gene regulation. Taken together, our data suggest that 5′ TNF-α promoter and 3′UTR regions interact with one another to regulate TNF-α gene expression at transcriptional level as well. The interaction between the 3′ and 5′ regions appears to be independent of the type of stimulation. DNA fragments used in these experiments are large, and the identification of potential cis- and trans- regulatory elements in both the 5′ or 3′ regions will require the study of smaller gene fragments. TNF-α transcription may be enhanced by stimulatory mechanisms and/or the release of a pre-existing block. The precise characterization of transcription factors binding to DNA regions essential for regulated expression of the TNF-α gene may open new perspectives in the understanding of human diseases related to abnormal TNF-α gene expression. Interestingly, mutated genomic sequences in regions flanking the 3′ TTATTTAT signal element of TNF-α gene have been described in murine models (27Jacob C.O. Lee S.K. Strassmann G. J. Immunol. 1996; 156: 3043-3050PubMed Google Scholar), but not in young patients with autoimmune diseases (28Becker L. Brown T. Fink C. Marks J. Lvandosky G. Giroir B.P. Pediatr. Res. 1995; 37: 165-168Crossref PubMed Scopus (7) Google Scholar). Because our results demonstrate the presence of other crucial regulatory domains interacting with promoter regions, the precise identification of discrete regulatory elements may help us to better understand the regulation of the TNF-α gene in inflammatory diseases. We thank Dr. B. Corthésy (Division of Immunology and Allergy) and Dr. F. Feihl (Division of Pathophysiology) for critical review of the manuscript.
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