Cell Type-specific Transactivation of the VCAM-1 Promoter through an NF-κB Enhancer Motif
1995; Elsevier BV; Volume: 270; Issue: 15 Linguagem: Inglês
10.1074/jbc.270.15.8976
ISSN1083-351X
AutoresMushtaq Ahmad, Nobuyuki Marui, R. Wayne Alexander, Russell M. Medford,
Tópico(s)Natural product bioactivities and synthesis
ResumoCytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-κB enhancer motifs, κL-κR (positions −77 and −63). However, cytokine activation of the dimeric NF-κB transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor-α markedly transactivated a transiently transfected minimal κL-κR motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the κL-κR motif. Both cell types exhibited similar inductions of NF-κB DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-κB(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-κB may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression. Cytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-κB enhancer motifs, κL-κR (positions −77 and −63). However, cytokine activation of the dimeric NF-κB transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor-α markedly transactivated a transiently transfected minimal κL-κR motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the κL-κR motif. Both cell types exhibited similar inductions of NF-κB DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-κB(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-κB may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression. Vascular cell adhesion molecule1-1 (VCAM-1) 1The abbreviations used are:VCAMvascular cell adhesion moleculeCATchloramphenicol acetyltransferaseCMVcytomegalovirusHUVEhuman umbilical vein endothelialTNFtumor necrosis factor. 1The abbreviations used are:VCAMvascular cell adhesion moleculeCATchloramphenicol acetyltransferaseCMVcytomegalovirusHUVEhuman umbilical vein endothelialTNFtumor necrosis factor. is an inducible cell surface protein of vascular endothelial cells that mediates the adhesion of mononuclear leukocytes to endothelial cells in response to a wide variety of inflammatory signals (1Osborn L. Hession C. Tizard R. Vassallo C. Luhowskyj S. Chi-Rossó G. Lobb R. Cell. 1989; 59: 1203-1211Abstract Full Text PDF PubMed Scopus (1403) Google Scholar, 2Rice G.E. Munro J.M. Bevilacqua M.P. J. Exp. Med. 1990; 171: 1369-1374Crossref PubMed Scopus (310) Google Scholar). Endothelial expression of VCAM-1 is observed in early atherosclerotic lesions of the vessel wall, as well as other inflammatory processes, suggesting the importance of VCAM-1 in these disease states (3Cybulsky M.I. Gimbrone Jr., M.A. Science. 1991; 251: 788-791Crossref PubMed Scopus (1384) Google Scholar). However, the molecular mechanisms regulating VCAM-1 gene expression are not well understood. The human VCAM-1 promoter contains two closely linked κB-like elements, κL -κR, separated by five nucleotides, at positions −77 and −63 relative to the transcription start site, respectively (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). κR but not κL completely conforms to the κB consensus sequence for the binding of the inducible, transcriptional regulatory factor NF-κB (5Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar). Yet, both of these sequences are necessary for the induction of VCAM-1 promoter by tumor necrosis factor α (TNF-α) in endothelial cells (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar, 6Neish A. Williams A. Palmer H. Whitley M. Collins T. J. Exp. Med. 1992; 176: 1583-1593Crossref PubMed Scopus (384) Google Scholar, 7Shu H.B. Agranoff A.B. Nabel E.G. Leung K. Duckett C.S. Neish A.S. Collins T. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6283-6289Crossref PubMed Google Scholar).The role of the NF-κB transcriptional factor in transactivating VCAM-1 in response to cytokine stimulation of endothelial cells is not fully understood. NF-κB is a member of the Rel family of transcriptional regulatory proteins and consists of two distinct polypeptides of 50 and 65 kDa, termed p50 and p65. In most unstimulated cell types, NF-κB is complexed with an inhibitory protein, IκB, in a non-DNA-binding form that is localized to the cytosol (8Baeuerle P.A. Lenardo M. Pierce J.W. Baltimore D. Cold Spring Harbor Symp. Quant. Biol. 1988; 2: 789-798Crossref Google Scholar). Stimulation of cells with cytokines (9Osborn L. Kunkle S. Nabel G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1353) Google Scholar) and several other activators (5Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar, 10Grilli M. Chiu J.J.-S. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (880) Google Scholar) results in the release of NF-κB from IκB and its translocation into the nucleus where it transcriptionally regulates the expression of a wide variety of genes through specific κB enhancer motifs (5Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar, 11Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1253) Google Scholar). An important feature of the Rel family is its ability to form a wide range of homodimers (e.g. p65 and p50) as well as heterodimers not only within the Rel family, but also with other classes of transcriptional factors (12Stein B. Baldwin Jr., A.S. Ballard D.W. Greene W.C. Angel P. Herrlich P. EMBO J. 1993; 12: 3879-3891Crossref PubMed Scopus (567) Google Scholar, 13Stein B. Cogswell P.C. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Crossref PubMed Google Scholar, 14Gutsch D.E. Holley-Guthrie E.A. Zhang Q. Stein B. Blanar M.A. Baldwin A.S. Kenney S.C. Mol. Cell. Biol. 1994; 14: 1939-1948Crossref PubMed Google Scholar). These complexes exhibit significant differences in their binding affinity for, and transactivation through, several κB DNA motifs. Recently, it has been shown that the p65 subunit of NF-κB can form homodimers that can also function as transactivators (15Nolan G.P. Ghosh S. Liou H.C. Tempst P. Baltimore D. Cell. 1991; 64: 961-969Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 16Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 17Ruben S. Narayanan R. Klement J. Chen C.-H. Rosen C. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar, 18Schmid R.M. Perkins N.D. Duckett C.S. Andrews P.C. Nabel G.J. Nature. 1991; 352: 733-736Crossref PubMed Scopus (274) Google Scholar, 19Ballard D.W. Walker W.H. Doerre S. Sista P. Molitor J.A. Dixon E.P. Peffer N.J. Hannink M. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1875-1879Crossref PubMed Scopus (217) Google Scholar, 20Fujita T. Nolan G. Ghosh S. Baltimore D. Genes & Dev. 1992; 6: 775-787Crossref PubMed Scopus (301) Google Scholar). Studies with the p50 subunit have shown that it can also act as a transactivator in vitro (20Fujita T. Nolan G. Ghosh S. Baltimore D. Genes & Dev. 1992; 6: 775-787Crossref PubMed Scopus (301) Google Scholar, 21Kretzschmar M. Meisterernst M. Scheidereit C. Li G. Roeder G.R. Genes & Dev. 1992; 6: 761-774Crossref PubMed Scopus (97) Google Scholar) as well as in yeast (22Moore P.A. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1993; 13: 1666-1674Crossref PubMed Google Scholar), although this has not yet been observed in mammalian cells (16Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 17Ruben S. Narayanan R. Klement J. Chen C.-H. Rosen C. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar, 19Ballard D.W. Walker W.H. Doerre S. Sista P. Molitor J.A. Dixon E.P. Peffer N.J. Hannink M. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1875-1879Crossref PubMed Scopus (217) Google Scholar, 23Richardson P.M. Gilmore T.D. J. Virol. 1991; 65: 3122-3130Crossref PubMed Google Scholar). Thus, cell type-specific differences in the activation of this family of NF-κB-like DNA-binding proteins may play an important role in regulating VCAM-1 gene expression through its κL-κR motif.Two observations suggest that transcriptional regulatory factors other than the NF-κB heterodimer itself may mediate transactivation through the VCAM-1 κL-κR elements. First, the κL-κR elements bind at least two different NF-κB-like binding proteins (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). Second, TNF-α transactivates the VCAM-1 promoter through the κL-κR elements in a cell type-specific manner. Non-endothelial cell lines such as Jurkat and HeLa activate NF-κB when stimulated with cytokine TNF-α as well as other activators (5Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar, 10Grilli M. Chiu J.J.-S. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (880) Google Scholar). However, although active in TNF-α-stimulated human vascular endothelial (HUVE) cells, constructs of the VCAM-1 promoter containing κL-κR are not transactivated in the TNF-α-stimulated T-cell line Jurkat (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). This raises the possibility that instead of NF-κB, other homo- or heterodimers of members of NF-κB/Rel family may be involved in activating the VCAM-1 promoter in TNF-α-stimulated HUVE cells.To explore the relative roles of NF-κB and NF-κB-like factors in regulating VCAM-1 gene transcription through the κL-κR motif, we have characterized the ability of these factors to mediate transcription of the VCAM-1 promoter through cytokine activation of endogenous NF-κB as well as to reconstituted homo- and heterodimers of p50 and p65 using expression vectors in HeLa cells. Our studies suggest that while Rel family members, such as the p65 homodimer, are potent transactivators, the NF-κB (p50+p65 heterodimer) does not effectively transactivate the VCAM-1 promoter through the κL-κR motif. DNA binding studies suggest that TNF-α induces NF-κB (p50+p65 heterodimer) and p65 homodimer or homodimer-like protein both in HUVE and HeLa cells. However, functional p65 homodimer was mainly present in HUVE cells. Thus, VCAM-1 transactivation through κL-κR may be a function of differential activation of NF-κB (p50+p65 heterodimer) relative to other members of the Rel family, such as p65 homodimers.MATERIALS AND METHODSCell CultureHeLa cells (CCL2) were obtained from American Type Culture Collection (Rockville, MD) and maintained in minimal essential medium supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA). HUVE cells were purchased from Clonetics (San Diego, CA) and were cultured in M199 medium supplemented with 20% fetal bovine serum, 16 units/ml heparin (ESI Pharmaceuticals, Cherry Hill, NJ), 50 μg/ml endothelial cell growth supplement (Collaborative Research Incorporated, Bedford, MA), and 25 m M HEPES buffer. The medium for both cells contains 2 m ML-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. HUVE cells were grown on tissue culture plates coated with 0.1% gelatin and were used within the first 6 passages. Human recombinant TNF-α was obtained from Boehringer Mannheim. All other reagents were of reagent grade.CAT AssayOne day prior to transfection, HeLa and HUVE cells were split at the ratio that would give 60-70% confluence. The transfection was done by the calcium phosphate co-precipitation technique. For HeLa cells, 5-10 μg of reporter plasmids were used. For HUVE cells, 30 μg of reporter plasmids were transfected as described previously (24Marui N. Offermann M.K. Swerlick R. Kunsch C. Rosen C.A. Ahmad M. Alexander R.W. Medford R.M. J. Clin. Invest. 1993; 92: 1866-1874Crossref PubMed Scopus (973) Google Scholar). The promoterless plasmid, poLUC (25Brasier A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar), was used for adjusting the amount of transfected DNA. The cells were harvested and cell extracts were prepared by three cycles of rapid freeze-thaw in 0.25 M Tris, pH 8.0. Protein content was determined using the Bradford (26Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213254) Google Scholar) technique. The same amounts of proteins were assayed for CAT activity according to standard protocols (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The CAT activity was expressed as percent of chloramphenicol converted to acetyl chloramphenicol. Acetylated and unacetylated forms of chloramphenicol were separated on thin layer chromatography and their amounts were determined after scraping by counting the radioactivity of the respective bands in scintillation vials. Each assay was performed in duplicate or triplicate and the results reported are the average of at least two separate experiments.Expression Vectors and CAT Reporter GenesThe eukaryotic expression vectors CMV-p50, CMV-p65, CMV-p50/65, and CMV-rel/p65 contain the respective cDNAs cloned between a cytomegalovirus (CMV) promoter, β-globin intron and simian virus 40 poly(A) signal (17Ruben S. Narayanan R. Klement J. Chen C.-H. Rosen C. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar). CMV-p50/65 encodes a chimeric protein consisting of the DNA binding domain of p50 (amino acids 1-370) and the transactivation domain of p65 (amino acids 309-550) (28Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). The reporter plasmid, p(HIVκB)4CAT, contains four tandem copies of the κB DNA sequences cloned upstream of the human immunodeficiency virus type-1 (HIV-1) long terminal repeat and fused to the coding region of the bacterial CAT gene (28Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). The sequence of the κB motif of p(HIVκB)4CAT is 5′-GGGGACTTTCC-3′, and is identical to the κB sequence found in the mouse immunoglobulin gene promoter (IgκB). These vectors were generous gifts of Dr. C. Rosen (Human Genome Sciences, Rockville, MD). The reporter gene, p85VCAMCAT, contains coordinates −85 to +12 of the human VCAM-1 promoter (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). The κL-κR driven heterologous promoter pTA(−77/−63)CAT has been described previously (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). These reporter genes were generous gifts of Dr. D. Dean (Washington University, St. Louis, MO).Nuclear Extracts PreparationConfluent HUVE and HeLa cells were exposed to TNF-α (100 units/ml) for 1-3 h. Nuclear extracts were prepared by a modification of the method of Dignam et al. (29Dignam J. Lebovitz R. Roeder R. Nucleic Acids Res. 1983; 11: 1476-1489Crossref Scopus (9141) Google Scholar). Briefly, after washing with phosphate buffered saline, cells were centrifuged and the cell pellet suspended in 500 μl of buffer A (10 m M HEPES, pH 7.9, 1.5 m M MgCl2, 10 m M KCl, and 1.0 m M dithiothreitol). After recentrifugation, the cells were resuspended in 80 μl buffer A containing 0.1% Triton X-100 by gentle pipetting up and down. After incubating for 10 min at 4°C, the homogenate was centrifuged and the nuclear pellet was washed once with buffer A and resuspended in 70 μl of buffer C (20 m M HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 m M MgCl2, 0.2 m M EDTA, 1 m M dithiothreitol). This suspension was incubated for 30 min at 4°C followed by centrifugation at 20,000 × g for 10 min. The resulting supernatant (nuclear extract) was stored at −70°C. Protein concentrations were determined by the Bradford (26Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213254) Google Scholar) method. To minimize proteolysis, all buffers contained 1.0 m M phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), leupeptin (10 μg/ml), and antipain (10 μg/ml).Gel Shift AssaysThe oligonucleotide containing κL-κR of the VCAM-1 promoter (VCAM-1 wild type oligo) was synthesized. Its sequence is as follows: 5′CTGCCCTGGGTTTCCCCTTGAAGGGATT-TCCCTCCGCCTCTGCAACAAGCTCGAGATCCTATG-3′. The sequences of κL and κR are underlined with a single line. The double underlined sequences represent an unrelated tail sequence added to serve as a template for synthesis of the double-stranded DNA. To prepare double-stranded DNA, first an oligonucleotide 5′-CATAGGATCTCGAGC-3′ (complementary to the 3′-unrelated tail, double underlined sequence) was annealed to VCAM-1 wild type oligo. The second strand was extended with DNA polymerase (Klenow fragment) in a reaction mixture containing 50 μCi of [32P]dCTP and 0.5 m M of cold dATP, dGTP, and dTTP. The reaction was followed by the addition of 0.5 m M cold dCTP to insure completion of the second strand. Unincorporated nucleotides were removed by column chromatography over a Sephadex G-50 column. The DNA binding reaction was performed at 30°C for 15 min in a volume of 20 μl, which contained 225 μg/ml bovine serum albumin, 1.0 × 105cpm of 32P-labeled probe, 0.1 μg/ml poly(dI-dC), and 15 μl of binding buffer (12 m M HEPES pH 7.9, 4 m M Tris, 60 m M KCl, 1 m M EDTA, 12% glycerol, 1 m M dithiothreitol, and 1 m M phenylmethylsulfonyl fluoride). After the binding reaction, the samples were subjected to electrophoresis in 1 × Tris-glycine buffer using 4% native polyacrylamide gels.Antibody p50 was a generous gift of Dr. C. Rosen (Human Genome Sciences Inc., Rockville, MD) and was characterized to completely shift both the p50 homodimer and the NF-κB(p50+p65 heterodimer) in gel shift assays (data not shown). Antibodies against p65, c-Rel (raised against a peptide in the carboxyl terminus region), and RelB were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Bacterially expressed proteins, p50 and truncated p65 (containing amino acids 1-309), were prepared and purified as described previously (17Ruben S. Narayanan R. Klement J. Chen C.-H. Rosen C. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar). Due to the low amounts of available purified proteins, the concentration of the purified proteins was roughly estimated from the concentration of proteins before dialysis, the final step of purification. The homo- and heterodimers of p50 and truncated p65 were prepared by incubating them alone or in combination with each other for one hour at 37°C as described earlier (20Fujita T. Nolan G. Ghosh S. Baltimore D. Genes & Dev. 1992; 6: 775-787Crossref PubMed Scopus (301) Google Scholar) except that DNA binding buffer was used for dimerization. These dimers were used for DNA binding studies. The purified proteins were generous gifts of Dr. C. Rosen (Human Genome Sciences Inc., Rockville, MD).The sequences of phosphorothioate antisense p50, p65 (30Narayanan R. Higgins K.A. Perez J.R. Coleman T.A. Rosen C.A. Mol. Cell. Biol. 1993; 13: 3802-3810Crossref PubMed Google Scholar), and the unrelated antisense (intercellular adhesion molecule-1) (31Chiang M.Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Abstract Full Text PDF PubMed Google Scholar) were 5′-TGGATCATCTTCTGCCATTCT-3′, 5′-GGGGAACAGTTCGTCCATGGC-3′ and 5′-CCCCCACCACTTCCCCTCTC-3′, respectively. These sequences have been used previously to inhibit the translation of the respective mRNAs (30Narayanan R. Higgins K.A. Perez J.R. Coleman T.A. Rosen C.A. Mol. Cell. Biol. 1993; 13: 3802-3810Crossref PubMed Google Scholar, 31Chiang M.Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Abstract Full Text PDF PubMed Google Scholar).RESULTSTNF-α Activates NF-κB-mediated Transcription in Both HUVE and HeLa Cells but Induces the VCAM-1 Promoter Only in HUVE CellsTo explore the mechanisms underlying cell type-specific expression of VCAM-1, we chose to compare the activation of the VCAM-1 promoter in passaged HUVE cells that markedly induce VCAM-1 expression in response to cytokines (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar) with the epithelial cell line, HeLa, that does not express VCAM-1 at either the mRNA or protein levels (data not shown). This lack of VCAM-1 expression in HeLa cells occurs despite its well described NF-κB activation in response to several cytokines and to the phorbol ester phorbol 12-myristate 13-acetate (32van Huijsduijnen R.H. Whelan J. Pescini R. Becker-Andre M. Schenk A. DeLamarter J.F. J. Biol. Chem. 1992; 267: 22385-22391Abstract Full Text PDF PubMed Google Scholar, 33Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-23234Crossref PubMed Google Scholar, 34McDonnell P.C. Kumar S. Rabson A.B. Gelinas C. Oncogene. 1992; 7: 163-170PubMed Google Scholar, 35Salvetti A. Lilienbaum A. Li Z. Paulin D. Gazzolo L. Mol. Cell. Biol. 1993; 13: 89-97Crossref PubMed Scopus (37) Google Scholar). HeLa and HUVE cells were transfected with p85VCAMCAT, a deletion construct of the human VCAM-1 promoter (coordinates −85 to +12) that contains the κL-κR elements and has been previously characterized as the minimal TNF-α-inducible VCAM-1 promoter (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). To assess for functional NF-κB, both cell types were also transfected in parallel with p(HIVκB)4CAT, an NF-κB-responsive promoter construct containing a tetramer of canonical κB elements, p(HIVκB)4CAT, well characterized to be activated by NF-κB(p50+p65 heterodimers) (28Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar).As expected, TNF-α markedly induced p(HIVκB)4CAT in both HUVE (Fig. 1 B, lanes 1 and 2) and HeLa cells (Fig. 1 A, lanes 1 and 2), demonstrating functional activation of NF-κB. TNF-α also markedly induced p85VCAMCAT activity in HUVE cells (Fig. 1 B, lanes 3 and 4). In contrast, TNF-α caused little or no activation of p85VCAMCAT (Fig. 1 A, lanes 3 and 4) in HeLa cells. Similar results were obtained when the TNF-α concentration was increased up to 1000 units/ml (data not shown). Similar to studies in Jurkat T cells (4Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar), these results suggest that activated NF-κB in TNF-α-stimulated HeLa cells is not sufficient to effectively transactivate the minimal inducible VCAM-1 promoter, containing the κL-κR elements.Co-expression of the p65 Subunit of NF-κB Potently Transactivates the VCAM-1 Promoter in HeLa CellsAs an endogenous activation of NF-κB in HeLa cells appeared not to be effective, we undertook to determine which members of the NF-κB/Rel family could transactivate the VCAM-1 promoter through the κL-κR elements. In functional reconstitution experiments Rel-like subunits were co-transfected into HeLa cells with p85VCAMCAT or p(HIVκB)4CAT (a p65 homodimer and NF-κB (p50+p65 heterodimer) driven reporter gene) as a control. These included expression vectors for the p50 and p65 subunits of NF-κB as well as for the chimeric protein, p50/65, that contains the DNA binding domain of p50 and transactivation domain of p65 and is a functional mimic of NF-κB (p50+p65 heterodimer) (28Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). As shown in Fig. 2 B, co-expression of the p65 subunit markedly transactivated p85VCAMCAT (7Shu H.B. Agranoff A.B. Nabel E.G. Leung K. Duckett C.S. Neish A.S. Collins T. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6283-6289Crossref PubMed Google Scholar). In contrast, co-expression of the chimeric protein (p50/65) failed to significantly induce p85VCAMCAT. Similarly, p50 did not induce p85VCAMCAT. However, both p65 and the chimeric protein p50/65 potently induced p(HIVκB)4CAT (Fig. 2 A) (28Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). p50 did not induce p(HIVκB)4CAT. These data suggest that expression of p65 either as a homodimer or in combination with a component distinct from p50 already present in HeLa cells is sufficient to transactivate the minimal inducible VCAM-1 promoter containing the κL-κR elements.FIG. 2The p65 subunit of NF-κB is a potent activator of the VCAM-1 promoter. HeLa cells were transfected with 10 μg of p85VCAMCAT (Panel B) or p(HIVκB)4CAT (Panel A) along with 5 μg of the expression vectors encoding p65 and p50, and the chimeric protein p50/65 as described under "Materials and Methods." The CAT activity was determined using equal amount of protein. The CAT activity of p85VCAMCAT (Panel B) and p(HIVκB)4CAT (Panel A) under different conditions is expressed as percentage of acetylated chloramphenicol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reconstitution of a Functional NF-κB (p50+p65 Heterodimer) in Vivo Enhances p(HIVκB)4CAT but Blocks p85VCAMCATTo further elucidate the relative roles of p65 and the NF-κB (p50+p65 heterodimer) on transactivation of the VCAM-1 promoter, functional NF-κB (p50+p65 heterodimer) was reconstituted in HeLa cells by co-transfection of the expression vectors encoding p50 and p65. Recent studies have shown that the NF-κB(p50+p65 heterodimer) can be efficiently formed when expression vectors of both p50 and p65 are co-transfected in different cell lines. This has been shown by gel mobility shift assays and by increases in the transcriptional activation of p(HIVκB)4CAT reporter genes (16Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 17Ruben S. Narayanan R. Klement J. Chen C.-H. Rosen C. Mol. Cell. Biol. 1992; 12: 444-454Crossref PubMed Google Scholar, 18Schmid R.M. Perkins N.D. Duckett C.S. Andrews P.C. Nabel G.J. Nature. 1991; 352: 733-736Crossref PubMed Scopus (274) Google Scholar, 19Ballard D.W. Walker W.H. Doerre S. Sista P. Molitor J.A. Dixon E.P. Peffer N.J. Hannink M. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1875-1879Crossref PubMed Scopus (217) Google Scholar). Studies in vitro have also suggested that the p50+p65 heterodimer is preferentially formed when purified proteins of p50 and p65 are incubated together (20Fujita T. Nolan G. Ghosh S. Baltimore D. Genes & Dev. 1992; 6: 775-787Crossref PubMed Scopus (301) Google Scholar).We first determined the concentration of p65 that was required for the maximal activation of p85VCAMCAT. p85VCAMCAT was transfected into HeLa cells along with different concentrations (0.1-10 μg) of the p65 expression vector. Increasing amounts of p65 activated p85VCAMCAT in a dose-dependent manner (7Shu H.B. Agranoff A.B. Nabel E.G. Leung K. Duckett C.S. Neish A.S. Collins T. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6283-6289Crossref PubMed Google Scholar) (data not shown). The amount of p65 required for the maximal CAT activity was approximately 5 μg. Further increase in the amount of expression vector did not significantly increase or decrease CAT activity. Based on these studies, the concentration of p65 expression vector used for the activation of p85VCAMCAT was 5 μg or lower. To generate NF-κB (p50+p65 heterodimer), HeLa cells were transfected with a constant amount of p65 and increasing amounts of p50 expression vectors. The effect of the co-expressed p50+p65 heterodimers on promoter function was assessed in p85VCAMCAT. The functional formation of the p50+p65 heterodimer and its ability to drive a
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