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Identification of multiple regulatory regions in the TNF-α gene
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ISOLATION OF A RECEPTOR FOR TUMOR NECROSIS FACTOR FROM HUMAN LUNG TISSUE. VdAhepherd and R. Abdolrasulnia. Vanderbilt Universitv and the VAMC. Nashville. TN 37212 Tumor hecrosis fact&o (TNF) ii an important mediator of inflammation that is secreted predominantly by macrophages in response to bacterial lipopolysaccharide. TNF has been show" to have a variety of effects on both transformed and nontransformed cells that appear to be mediated by specific We have initiated studies on the cell surface receptors. characterization of TNF receptors on macrophages and endothelial cells. As a part of this work, we have purified a TNF receptor from human lung tissue using affinity chromatography on TNF-Sepharose. TNF was covalently attached to Sepharose. The immobilized TNF retained the ability to kill L-929 cells, and anti-TNF antibodies could block this killing. A detergent extract of human lung tissue was incubated with TNF-Sepharose, and TNF binding proteins were eluted with a low pH buffer. The specific TNF binding activity of each fraction was determined by incubation with "'I-TNF in the presence and absence of excess unlabeled TNF. The receptor-TNF complex was specifically precipitated with ammonium sulfate, and the complex collected on filters. The active fractions contained a major protein band at 75 kD and a minor band at 50 kD by SDS-PAGE analysis. We have preliminary data from ligand blotting studies that TNF binds specifically to both bands. We are currently raising antiTNF receptor antibodies for use in biosynthesis and turnover studies in q acrophages and endothelial cells.
IDENTIFICATION OF MULTIPLE REGULATORY REGIONS IN THE TNF-Q GENE. S_M. S.U.N.Y. Health Science Center, Syracuse, NY 13210. Tumor necrosis factor (TNF-(r) is induced in macrophages in response to activating stimuli. In order to study the regulation of TNF-a expression we constructed a plasmid containing part of the 5' flanking region of the TNF-a gene coupled to a marker gene, chloramphenicol acetyl transferase (CAT). This construct was transfected into the RAW264 macrophage-like ccl line using a calcium phosphate co-precipitation procedure. After transfection the cells were stimulated with 300 "e/ml LPS and allowed to incubate at 37-C overnight before the cells were lysed and CAT activity was assayed. There was little detectable promotor activity using this construct. When a downstream enhancer element, present in the third intro", was cloned 3' to the CAT gene it was possible to obtain LPSinducible CAT activity. Regulation of TNF-Q gene expression by LPS, CAMP or dexamethasone was determined to be a function of the 5' flanking region and not the enhancer element, which was always active. Initial analysis of a series of Bal 31 deletion of the 5' flanking region indicated that sequences located between 43 and 109 bases upstream of the transcriptional start site were required for TNF-(r transcription. These results indicate that at least two elements regulate the expression of TNF-0, a LPS sensitive promoter in the 5' flanking region and a downstream enhancer element. Supported by NIH grant A124236.
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HUMAN B CELL GROWTH FACTOR ENHANCES HIV EXPRESSION IN PERIPHERAL BLOOD LYMPHOCYTES. P. Thampatty, R. Balachandran, C.R. Rinaldo, and P. Gupta. Univ. Pittsburgh, Pittsburgh, PA 15261 Role 0f human B cell growth factor (BCGF) i" HIV e~pt33si0n is studied in human peripheral blood lymphocytes (PBL). PBL were preincubated with BCGF for 72 hrs, infected with HIV and then cultured In presence of BCGF. HIV expression was monitored by the antigen capture test and by measurement of HIVspecific RNA. BCGF increased the production of HIV lo-20 fold in PBL in a dose dependent manner. The increased virus production was associated with a" increase in HIV RNA production. BCGF hd no stimulatory effect on HIV production in T cell enriched cultures. This indicates that BCGF-induced stimulation of HIV is a B cell mediated phenomenon. Anti CD4 antibody completely blocked BCGF induced HIV production indicating BCGF enhanced expression of HIV was the role of CD4 receptor. not associated with induction of GM-CSF, IL-1 6 or IL-Z. Anti-IgM antibody was found to inhibit the stimulatory effect of BCGF. Such suppressive effect was found to be associated with an increased production of interferon. Data suggest that HIV replication in B cells may be a significance in the immunopathogenesis of the retrovirus.
5’ FLANKING REGIONS AND TRANSCRIPTIONAL REGULATION OF THE TNF-a AND TNF-8 GENES. A.N. Shakhov. C.V. Jonaeneel. R.L. Turetskava, S.A. Nedosoasov Ludwig Inst. Cant. Res., 1066 Epalinges, Switzerland, and Engelhardt Inst. Mol. Biol., Moscow 117984, USSR We have compared the sequences of the tandemly arranged genes for TNF-8 (lymphotoxin) and TNF-a (tumor necrosis factor) in the human, mouse, and rabbit genomes, and searched for the presence of conserved regions. In addition to the expected conservation of coding regions, we found significant homologies among presumed regulatory elements. In the promoter of the TNF-8 gene, there is a 300 bp conserved region containing in all three species consensus binding sites for the transcription factors Spl, NF-~6, and AP-2. Homology in the TNF-a promoter is more dispersed, with conserved patches stretching over more than 700 bp. Conserved elements include promoterproximal Sp-1 and MHC class II “Y box” elements, and a &-like motif (also known as “cytokine-1”) located about 800 bp upstream of the mRNA initiation site. The mouse TNF-a promoter additionally contains a strong consensus KB enhancer at nt -510. which is important in its inducibility by LPS. We are mapping regulatory elements in the TNF-8 promoter by constructing recombinants containing the chloramphenicol acetyl transferase gene under the control of portions of the promoter, and transfecting these into the human B-cell line RPMI64lOt.
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AND EXPRESSION OF HUMAN IL-l RECEPTOR. -Young, Jay Lillquist, Peter Xmetz, Philip Simon, Philip K!Cas"ey and Ganesh Sathe. Depts. of Molecular Genetics and Immunoloqy, King of Prussia, PA 19406-0939. Smith Kline & French Labs., We have isolated and sequenced a 3.3kb cDNA from a human liver cDNA library which encodes the human IL-1 receptor. The predicted protein is 569 amino acids lonq, and shares a 67.5% amino acid identity with the published mouse IL-1 Science 241: 585, 1988). receptor sequence (SlrnS. et al., The putative cytoplasmic domain is the mOst conserved with the mouse sequence (78% identity), followed by the extracellular domain (64% identity1 and transmembrane region The three extracellular immunoglobulin V(48% identity). like domains are approximately equally conserved between mouse and human (66-70% identity). The rest of the CDNA encodes more poorly conserved 5' (394 nt) and 3' untransTransient expression of the human IL-1 lated sequence. receptor cDNA in COSlqf$lls resulted in a" increase in specific binding of I-labelled human IL-1B to these cells. Northern blots hybridized with the cDNA indicate a" mRNA of ca. 5.2kb. which is expressed in LPS activated human peripheral blood monocytes and the human aetrocytoma cell line T24. The mla?A is not easily detected in unstimuindicating that, like IL-1 itself, it is lated monocytes, induced by LPS in these cells.
A 23-bp MULTIPLE CYTOKINE- AND SECOND MESSENGER- RESPONSIVE ELEMENT (MRE) IN THE HUMAN INTERLEUKIN-6 ENHANCER: MODULATION OF FUNCTION BY c-fos DNA. Anuradha Rav and Pravinkumar B, M. The Rockefeller University, New York, N.Y. 10021. Interleukin-6 (IL-6) represents amajor systemic alarm signal which denotes the occurrence of tissue damage. A transcriptional enhancer present within the 115-bp region from -225 to -111 in the IL-6 5'-flanking region is the major contributor to the inducibility of this gene by serum, several different inflammation-associated cytokines, viruses and second messenger agonists. Serum as well as activators of the protein kinase A and protein kinase C signal transduction pathways converge on to a 23-bp IL-6 "multi-response element" (MRE) (-173 to -151) which shares nucleotide sequence similarities with the SRE and the adjacent AP-l-like site in c-fos (-326 to -277). Mutations in the AP-1 like site within the 23-bp IL-6 MRE decreases inducibility of a chimeric IL-6/CAT gene by phorbol ester and by forskolin. These mutations do not affect induction by serum, IL-lo or TNF. Appropriate IL-6 and c-fos DNA fragments cros.scompete in gel-shift assays using nuclear extracts from induced HeLa cells and in cotransfection assays in intact cells suggesting that the transcription factors that act collectively through the IL-6 MRE are essentially the same as those that act on the dispersed individual regulatory elements in the c-fos promoter. Furthermore, Fos, a key negative transcription factor that downregulates c-fos expression, also transrepresses the IL-6 promoter in serum-stimulated HeLa cells.
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ISOLATION OF A RECEPTOR FOR TUMOR NECROSIS FACTOR FROM HUMAN LUNG TISSUE. VdAhepherd and R. Abdolrasulnia. Vanderbilt Universitv and the VAMC. Nashville. TN 37212 Tumor hecrosis fact&o (TNF) ii an important mediator of inflammation that is secreted predominantly by macrophages in response to bacterial lipopolysaccharide. TNF has been show" to have a variety of effects on both transformed and nontransformed cells that appear to be mediated by specific We have initiated studies on the cell surface receptors. characterization of TNF receptors on macrophages and endothelial cells. As a part of this work, we have purified a TNF receptor from human lung tissue using affinity chromatography on TNF-Sepharose. TNF was covalently attached to Sepharose. The immobilized TNF retained the ability to kill L-929 cells, and anti-TNF antibodies could block this killing. A detergent extract of human lung tissue was incubated with TNF-Sepharose, and TNF binding proteins were eluted with a low pH buffer. The specific TNF binding activity of each fraction was determined by incubation with "'I-TNF in the presence and absence of excess unlabeled TNF. The receptor-TNF complex was specifically precipitated with ammonium sulfate, and the complex collected on filters. The active fractions contained a major protein band at 75 kD and a minor band at 50 kD by SDS-PAGE analysis. We have preliminary data from ligand blotting studies that TNF binds specifically to both bands. We are currently raising antiTNF receptor antibodies for use in biosynthesis and turnover studies in q acrophages and endothelial cells.
IDENTIFICATION OF MULTIPLE REGULATORY REGIONS IN THE TNF-Q GENE. S_M. S.U.N.Y. Health Science Center, Syracuse, NY 13210. Tumor necrosis factor (TNF-(r) is induced in macrophages in response to activating stimuli. In order to study the regulation of TNF-a expression we constructed a plasmid containing part of the 5' flanking region of the TNF-a gene coupled to a marker gene, chloramphenicol acetyl transferase (CAT). This construct was transfected into the RAW264 macrophage-like ccl line using a calcium phosphate co-precipitation procedure. After transfection the cells were stimulated with 300 "e/ml LPS and allowed to incubate at 37-C overnight before the cells were lysed and CAT activity was assayed. There was little detectable promotor activity using this construct. When a downstream enhancer element, present in the third intro", was cloned 3' to the CAT gene it was possible to obtain LPSinducible CAT activity. Regulation of TNF-Q gene expression by LPS, CAMP or dexamethasone was determined to be a function of the 5' flanking region and not the enhancer element, which was always active. Initial analysis of a series of Bal 31 deletion of the 5' flanking region indicated that sequences located between 43 and 109 bases upstream of the transcriptional start site were required for TNF-(r transcription. These results indicate that at least two elements regulate the expression of TNF-0, a LPS sensitive promoter in the 5' flanking region and a downstream enhancer element. Supported by NIH grant A124236.
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HUMAN B CELL GROWTH FACTOR ENHANCES HIV EXPRESSION IN PERIPHERAL BLOOD LYMPHOCYTES. P. Thampatty, R. Balachandran, C.R. Rinaldo, and P. Gupta. Univ. Pittsburgh, Pittsburgh, PA 15261 Role 0f human B cell growth factor (BCGF) i" HIV e~pt33si0n is studied in human peripheral blood lymphocytes (PBL). PBL were preincubated with BCGF for 72 hrs, infected with HIV and then cultured In presence of BCGF. HIV expression was monitored by the antigen capture test and by measurement of HIVspecific RNA. BCGF increased the production of HIV lo-20 fold in PBL in a dose dependent manner. The increased virus production was associated with a" increase in HIV RNA production. BCGF hd no stimulatory effect on HIV production in T cell enriched cultures. This indicates that BCGF-induced stimulation of HIV is a B cell mediated phenomenon. Anti CD4 antibody completely blocked BCGF induced HIV production indicating BCGF enhanced expression of HIV was the role of CD4 receptor. not associated with induction of GM-CSF, IL-1 6 or IL-Z. Anti-IgM antibody was found to inhibit the stimulatory effect of BCGF. Such suppressive effect was found to be associated with an increased production of interferon. Data suggest that HIV replication in B cells may be a significance in the immunopathogenesis of the retrovirus.
5’ FLANKING REGIONS AND TRANSCRIPTIONAL REGULATION OF THE TNF-a AND TNF-8 GENES. A.N. Shakhov. C.V. Jonaeneel. R.L. Turetskava, S.A. Nedosoasov Ludwig Inst. Cant. Res., 1066 Epalinges, Switzerland, and Engelhardt Inst. Mol. Biol., Moscow 117984, USSR We have compared the sequences of the tandemly arranged genes for TNF-8 (lymphotoxin) and TNF-a (tumor necrosis factor) in the human, mouse, and rabbit genomes, and searched for the presence of conserved regions. In addition to the expected conservation of coding regions, we found significant homologies among presumed regulatory elements. In the promoter of the TNF-8 gene, there is a 300 bp conserved region containing in all three species consensus binding sites for the transcription factors Spl, NF-~6, and AP-2. Homology in the TNF-a promoter is more dispersed, with conserved patches stretching over more than 700 bp. Conserved elements include promoterproximal Sp-1 and MHC class II “Y box” elements, and a &-like motif (also known as “cytokine-1”) located about 800 bp upstream of the mRNA initiation site. The mouse TNF-a promoter additionally contains a strong consensus KB enhancer at nt -510. which is important in its inducibility by LPS. We are mapping regulatory elements in the TNF-8 promoter by constructing recombinants containing the chloramphenicol acetyl transferase gene under the control of portions of the promoter, and transfecting these into the human B-cell line RPMI64lOt.
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AND EXPRESSION OF HUMAN IL-l RECEPTOR. -Young, Jay Lillquist, Peter Xmetz, Philip Simon, Philip K!Cas"ey and Ganesh Sathe. Depts. of Molecular Genetics and Immunoloqy, King of Prussia, PA 19406-0939. Smith Kline & French Labs., We have isolated and sequenced a 3.3kb cDNA from a human liver cDNA library which encodes the human IL-1 receptor. The predicted protein is 569 amino acids lonq, and shares a 67.5% amino acid identity with the published mouse IL-1 Science 241: 585, 1988). receptor sequence (SlrnS. et al., The putative cytoplasmic domain is the mOst conserved with the mouse sequence (78% identity), followed by the extracellular domain (64% identity1 and transmembrane region The three extracellular immunoglobulin V(48% identity). like domains are approximately equally conserved between mouse and human (66-70% identity). The rest of the CDNA encodes more poorly conserved 5' (394 nt) and 3' untransTransient expression of the human IL-1 lated sequence. receptor cDNA in COSlqf$lls resulted in a" increase in specific binding of I-labelled human IL-1B to these cells. Northern blots hybridized with the cDNA indicate a" mRNA of ca. 5.2kb. which is expressed in LPS activated human peripheral blood monocytes and the human aetrocytoma cell line T24. The mla?A is not easily detected in unstimuindicating that, like IL-1 itself, it is lated monocytes, induced by LPS in these cells.
A 23-bp MULTIPLE CYTOKINE- AND SECOND MESSENGER- RESPONSIVE ELEMENT (MRE) IN THE HUMAN INTERLEUKIN-6 ENHANCER: MODULATION OF FUNCTION BY c-fos DNA. Anuradha Rav and Pravinkumar B, M. The Rockefeller University, New York, N.Y. 10021. Interleukin-6 (IL-6) represents amajor systemic alarm signal which denotes the occurrence of tissue damage. A transcriptional enhancer present within the 115-bp region from -225 to -111 in the IL-6 5'-flanking region is the major contributor to the inducibility of this gene by serum, several different inflammation-associated cytokines, viruses and second messenger agonists. Serum as well as activators of the protein kinase A and protein kinase C signal transduction pathways converge on to a 23-bp IL-6 "multi-response element" (MRE) (-173 to -151) which shares nucleotide sequence similarities with the SRE and the adjacent AP-l-like site in c-fos (-326 to -277). Mutations in the AP-1 like site within the 23-bp IL-6 MRE decreases inducibility of a chimeric IL-6/CAT gene by phorbol ester and by forskolin. These mutations do not affect induction by serum, IL-lo or TNF. Appropriate IL-6 and c-fos DNA fragments cros.scompete in gel-shift assays using nuclear extracts from induced HeLa cells and in cotransfection assays in intact cells suggesting that the transcription factors that act collectively through the IL-6 MRE are essentially the same as those that act on the dispersed individual regulatory elements in the c-fos promoter. Furthermore, Fos, a key negative transcription factor that downregulates c-fos expression, also transrepresses the IL-6 promoter in serum-stimulated HeLa cells.
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