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• W2019937507 abstract "Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family play important roles in myeloid cell differentiation. CD14 is a monocyte/macrophage differentiation marker and is strongly up-regulated during monocytic cell differentiation. Here, we report the direct binding of C/EBP to the monocyte-specific promoter of CD14. Transactivation analyses demonstrate that C/EBP family members significantly activate the CD14 promoter. These data indicate that C/EBP is directly involved in the regulation of CD14 gene expression. When myelomonoblastic U937 cells are treated with vitamin D3 and TGF-β, they differentiate toward monocytic cells. Using specific antibodies against different C/EBP family members in electrophoretic mobility shift assays and Western blot assays, we have identified a specific increase in the DNA binding and the expression of C/EBPα and C/EBPβ during U937 monocytic cell differentiation, and we found C/EBPα and C/EBPβ bind to the promoter in heterodimer. Furthermore, with stably transfected cell lines, we demonstrate that the C/EBP binding site in the CD14 promoter plays a critical role for mediating TGF-β signaling in the synergistic activation of CD14 expression by vitamin D3 and TGF-β during U937 differentiation. This may indicate that C/EBPs have important functions in the process of TGF-β signal transduction during monocyte differentiation. Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family play important roles in myeloid cell differentiation. CD14 is a monocyte/macrophage differentiation marker and is strongly up-regulated during monocytic cell differentiation. Here, we report the direct binding of C/EBP to the monocyte-specific promoter of CD14. Transactivation analyses demonstrate that C/EBP family members significantly activate the CD14 promoter. These data indicate that C/EBP is directly involved in the regulation of CD14 gene expression. When myelomonoblastic U937 cells are treated with vitamin D3 and TGF-β, they differentiate toward monocytic cells. Using specific antibodies against different C/EBP family members in electrophoretic mobility shift assays and Western blot assays, we have identified a specific increase in the DNA binding and the expression of C/EBPα and C/EBPβ during U937 monocytic cell differentiation, and we found C/EBPα and C/EBPβ bind to the promoter in heterodimer. Furthermore, with stably transfected cell lines, we demonstrate that the C/EBP binding site in the CD14 promoter plays a critical role for mediating TGF-β signaling in the synergistic activation of CD14 expression by vitamin D3 and TGF-β during U937 differentiation. This may indicate that C/EBPs have important functions in the process of TGF-β signal transduction during monocyte differentiation. CCAAT/enhancer-binding protein transforming growth factor colony-stimulating factor base pair(s) polymerase chain reaction electrophoretic mobility shift assay Rous sarcoma virus promoter directing human growth hormone gene expression Hematopoiesis is the process by which distinct blood cell lineages, including monocytes, granulocytes, erythrocytes, megakaryocytes, and lymphocytes, are generated from pluripotential stem cells. The combined action of various transcription factors, which mediate the differentiation signals, determines the lineage-specific gene expression and subsequent cell differentiation (1Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 2Shivdasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar). A differentiation block is a major determinant in the appearance of leukemia (3Nichols J. Nimer S.D. Blood. 1992; 80: 2953-2963Crossref PubMed Google Scholar, 4Look A.T. Science. 1997; 278: 1059-1064Crossref PubMed Scopus (991) Google Scholar). Numerous studies have investigated monocyte/macrophage development. Cell-specific transcription factors, such as PU.1 and C/EBP, and ubiquitously expressed transcription factors, such as AML1 and Sp1, are involved in monocyte development and specific gene expression (5Scott E.W. Simon M.C. Anastasi J. Singh H. Science. 1994; 265: 1573-1577Crossref PubMed Scopus (1282) Google Scholar, 6McKercher S.R. Torbett B.E. Anderson K.L. Henkel G.W. Vestal D.J. Baribault H. Klemsz M. Feeney A.J. Wu G.E. Paige C.J. Maki R.A. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (934) Google Scholar, 7Zhang D.E. Fujioka K. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar, 8Zhang D.E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar, 9Zhang D.E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 10Zhang D.E. Hetherington C.J. Tan S. Dziennis S.E. Gonzalez D.A. Chen H.M. Tenen D.G. J. Biol. Chem. 1994; 269: 11425-11434Abstract Full Text PDF PubMed Google Scholar, 11Shapiro L.H. Look A.T. Curr. Opin. Hematol. 1995; 2: 3-11Crossref PubMed Scopus (14) Google Scholar, 12Klemsz M.J. McKercher S.R. Celada A. Van B.C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (759) Google Scholar, 13Ross I.L. Yue X. Ostrowski M.C. Hume D.A. J. Biol. Chem. 1998; 273: 6662-6669Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the mechanism associated with the differentiation of monocytes from hematopoietic progenitor cells has not yet been fully elucidated. CD14 is highly expressed on the surface of monocytes/macrophages and is strongly up-regulated during the differentiation of monocytic precursors into monocytes (14Griffin J.D. Ritz J. Nadler L.M. Schlossman S.F. J. Clin. Invest. 1981; 68: 932-941Crossref PubMed Scopus (358) Google Scholar, 15Goyert S.M. Ferrero E. Rettig W.J. Yenamandra A.K. Obata F. Le B.M. Science. 1988; 239: 497-500Crossref PubMed Scopus (297) Google Scholar, 16Simmons D.L. Tan S. Tenen D.G. Nicholson-Weller A. Seed B. Blood. 1989; 73: 284-289Crossref PubMed Google Scholar). Therefore, the regulation of CD14 expression has been investigated as a model to provide critical insight into the mechanisms of monocyte differentiation. During monocyte differentiation, the expression of CD14 is specifically and strongly up-regulated at the transcription level (17Zhang D.E. Hetherington C.J. Gonzalez D.A. Chen H.M. Tenen D.G. J. Immunol. 1994; 153: 3276-3284Crossref PubMed Google Scholar). The upstream sequence of the CD14 gene shows a strong tissue-specific promoter activity (10Zhang D.E. Hetherington C.J. Tan S. Dziennis S.E. Gonzalez D.A. Chen H.M. Tenen D.G. J. Biol. Chem. 1994; 269: 11425-11434Abstract Full Text PDF PubMed Google Scholar). Functionally, CD14 is a receptor for the complex of lipopolysaccharide and its binding protein (18Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3420) Google Scholar); the recognition between CD14 and the complex is a crucial step in trigging macrophage function during bacterial infection (19Ziegler-Heitbrock H.W. Ulevitch R.J. Immunol. Today. 1993; 14: 121-125Abstract Full Text PDF PubMed Scopus (503) Google Scholar). Recently, CD14 has also been reported to play an important role in apoptosis (20Heidenreich S. Schmidt M. August C. Cullen P. Rademaekers A. Pauels H.G. J. Immunol. 1997; 159: 3178-3188PubMed Google Scholar, 21Devitt A. Moffatt O.D. Raykundalia C. Capra J.D. Simmons D.L. Gregory C.D. Nature. 1998; 392: 505-509Crossref PubMed Scopus (566) Google Scholar). C/EBPs1 are a family of transcription factors containing an activation domain, a DNA-binding basic region, and a leucine-rich dimerization domain (22Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). C/EBPα was initially identified in the liver (23Johnson P.F. Landschulz W.H. Graves B.J. McKnight S.L. Genes Dev. 1987; 1: 133-146Crossref PubMed Scopus (341) Google Scholar), where it was found to be important for cell-specific gene expression and differentiation (24Friedman A.D. Landschulz W.H. McKnight S.L. Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (363) Google Scholar,25Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (838) Google Scholar). There are currently six known members of the C/EBP transcription factor family. C/EBP regulates the expression of three critical growth factor receptors for myeloid cell development, namely, macrophage-CSF, granulocyte-CSF, and granulocyte/macrophage-CSF (8Zhang D.E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar, 26Smith L.T. Hohaus S. Gonzalez D.A. Dziennis S.E. Tenen D.G. Blood. 1996; 88: 1234-1247Crossref PubMed Google Scholar, 27Hohaus S. Petrovick M.S. Voso M.T. Sun Z. Zhang D.E. Tenen D.G. Mol. Cell. Biol. 1995; 15: 5830-5845Crossref PubMed Google Scholar). Knockout experiments in mice provide direct evidence to demonstrate that C/EBPs play critical roles in hematopoietic cell development (28Zhang D.E. Zhang P. Wang N.D. Hetherington C.J. Darlington G.J. Tenen D.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 569-574Crossref PubMed Scopus (760) Google Scholar, 29Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (376) Google Scholar, 30Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 31Yamanaka R. Kim G.D. Radomska H.S. Lekstrom-Himes J. Smith L.T. Antonson P. Tenen D.G. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6462-6467Crossref PubMed Scopus (153) Google Scholar, 32Chumakov A.M. Grillier I. Chumakova E. Chih D. Slater J. Koeffler H.P. Mol. Cell. Biol. 1997; 17: 1375-1386Crossref PubMed Google Scholar, 33Yamanaka R. Barlow C. Lekstrom-Himes J. Castilla L.H. Liu P.P. Eckhaus M. Decker T. Wynshaw-Boris A. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13187-13192Crossref PubMed Scopus (310) Google Scholar). In the present study, we have identified a C/EBP site in the critical region of the CD14 promoter and investigated the expression of the CD14 gene under the regulation of C/EBP. Furthermore, we have analyzed the regulation of CD14 expression during vitamin D3- and TGF-β-induced monocyte differentiation with U937 cells. Our results demonstrate that C/EBP is an important transcription factor for CD14 promoter activity and that the C/EBP binding site is crucial for the synergistic signaling from vitamin D3 and TGF-β to induce monocyte differentiation, and we are the first to demonstrate that C/EBPs mediate TGF-β signaling in a model of monocyte development. Human promonocytic THP-1 cells (American Type Culture Collection (ATCC) TIP202, Manassas, VA) were grown in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Sigma), 2 mm l-glutamine (Life Technologies, Inc.), and 2 × 10−5m2-mercaptoethanol (Sigma). Human myelomonoblastic U937 cells (ATCC, CRL 1593) were maintained in RPMI 1640 medium with 10% fetal bovine serum and 2 mm l-glutamine. Human cervical carcinoma HeLa cells (ATCC, CCL2) and African green monkey kidney fibroblast-like CV-1 cells (ATCC, CCL70) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) with 10% calf serum (Sigma) and 2 mm l-glutamine. Human Mono Mac 6 cells were propagated as described (34Ziegler-Heitbrock H.W. Thiel E. Futterer A. Herzog V. Wirtz A. Riethmuller G. Int. J. Cancer. 1988; 41: 456-461Crossref PubMed Scopus (492) Google Scholar). Vitamin D3 (kindly provided by Dr. Milan R. Uskokovic of Hoffman-La Roche, Nutley, NJ) was dissolved in 100% ethanol at 10−3m and diluted in cell culture medium to 1 × 10−7m or the concentrations described in the figure legends. TGF-β was purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer's protocol at 1 ng/ml. The characterization of monocyte differentiation of U937 cells induced by vitamin D3 and/or TGF-β was based on a previous report (35Testa U. Masciulli R. Tritarelli E. Pustorino R. Mariani G. Martucci R. Barberi T. Camagna A. Valtieri M. Peschle C. J. Immunol. 1993; 150: 2418-2430PubMed Google Scholar). According to the analysis with fluorescent-activated cell sorter, 99% of U937 cells were positive for CD14 expression after 48 h induction by vitamin D3 and TGF-β. pXP2 is a promoterless luciferase construct (36Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar). The construction of p-227CD14-luc was described previously (10Zhang D.E. Hetherington C.J. Tan S. Dziennis S.E. Gonzalez D.A. Chen H.M. Tenen D.G. J. Biol. Chem. 1994; 269: 11425-11434Abstract Full Text PDF PubMed Google Scholar). p-227CD14(m135)-luc, which contains a mutation in the C/EBP binding site (bp −129 to −135) of p-227CD14-luc, was generated by polymerase chain reaction. Plasmid p-227CD14-luc was used as template with a pair of primers, 5′-GGGGTACCCCGATAAGTCTTCCGAACCTC-3′ and 5′-CGGGATCCCTGAGTCATCAGGACAC-3′, to generate PCR fragment A, or with another pair of primers, 5′-GGGGTACCCCGATAAGTCTTCCGAACCTC-3′ and 5′-GGGGTACCATTTCCAGGAAAGG-3′, to generate PCR fragment B. PCR fragment A was digested with Kpn I and Bam HI, and PCR fragment B was digested with Kpn I and Sac I. The digested fragments were then ligated into the Bam HI andSac I sites of pXP2 to create p-227CD14(m135)-luc. pCMV-hC/EBPα was kindly provided by G. Darlington (37Timchenko N. Wilson D.R. Taylor L.R. Abdelsayed S. Wilde M. Sawadogo M. Darlington G.J. Mol. Cell. Biol. 1995; 15: 1192-1202Crossref PubMed Google Scholar). pCMV-hC/EBPβ was constructed by inserting the human C/EBPβ cDNA (provided by Dr. P. Auron) into the Eco RI/Xho I sites of pcDNA1. pCMV-hC/EBPδ was generated by inserting a 2.6-kilobase Pst I fragment of human C/EBPδ (kindly provided by Dr. K. G. Xanthopoulos) (38Cleutjens C.B. van E.C. van D.H. Smit E.M. Hagemeijer A. Wagner M.J. Wells D.E. Trapman J. Genomics. 1993; 16: 520-523Crossref PubMed Scopus (28) Google Scholar) into the Pst I site of pCMV5. THP-1 and U937 cells were cultured to a concentration of 4 × 105 cells/ml, and HeLa cells were cultured to 50–70% confluence. The cells were subsequently harvested for nuclear protein preparation. Nuclear extracts were prepared as described previously (39Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3918) Google Scholar). Double-stranded oligonucleotides (oligo A: 5′-ACATCCTTCATTGCAATATTTCCATTAAAGG-3′ containing bp −144 to −115 of the CD14 promoter; the underlined sequence is a putative C/EBP binding site) for EMSA were labeled with [γ-32P]ATP using T4 kinase and purified by polyacrylamide gel electrophoresis. EMSAs were performed by incubating 5 μg of nuclear protein extracts with labeled double-stranded oligonucleotide in a 20-μl reaction mixture containing 10 mm HEPES-KOH buffer (pH 7.9), 50 mm KCl, 2.5 mm MgCl2, 1 mm dithiothreitol, 10% glycerol, 1 μg of bovine serum albumin, and 1 μg of poly(dI-dC) at room temperature for 20 min. For competition analysis, a 50-fold molar excess (except as otherwise noted in the figure legends) of unlabeled double-stranded oligonucleotide was added to the nuclear extracts prior to the addition of the labeled probe. For the supershift assays, polyclonal antibodies against C/EBPα, -β, -δ, Sp1, normal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA), or C/EBPε (kindly provided by Dr. K. G. Xanthopoulos), were incubated with the nuclear extracts for 15 min followed by the addition of radiolabeled probe. The binding reactions were resolved on 6% polyacrylamide gels in 1× TBE. The transfection procedure is described elsewhere (40Pahl H.L. Burn T.C. Tenen D.G. Exp. Hematol. 1991; 19: 1038-1041PubMed Google Scholar). The cells were harvested 5 h after transfection in 0.5 ml of lysis buffer, and luciferase assays were performed as described previously (40Pahl H.L. Burn T.C. Tenen D.G. Exp. Hematol. 1991; 19: 1038-1041PubMed Google Scholar). The transfection efficiency was normalized by the levels of growth hormone expressed from 2 μg of co-transfected plasmid containing the Rous sarcoma virus promoter directing human growth hormone gene expression (RSV-hGH). Growth hormone concentrations were measured by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA). 2 × 105 CV-1 cells were plated in 4 ml of Dulbecco's modified Eagle's medium with 10% calf serum in 60 mm plates 1 day prior to the transfection. Cells were transfected by the calcium phosphate method as described elsewhere (41Chen C.A. Okayama H. BioTechniques. 1988; 6: 632-638Crossref PubMed Scopus (28) Google Scholar) with 10 μg of reporter plasmid (p-227CD14-luc or p-227CD14(m135)-luc), 1 μg of expression plasmid (pCMV-hC/EBPα, pCMV-hC/EBPβ or pCMV-hC/EBPδ), 0.5 μg of RSV-hGH (as an internal transfection efficiency control), and sheared salmon sperm DNA (Sigma) to a total of 20 μg of DNA per transfection. Control plates received 1 μg of empty expression vector to allow a precise comparison. The medium was changed 12–16 h after transfection. The luciferase activities were measured 48 h after transfection. The transfection efficiency was normalized as described above by human growth hormone expression. Total RNA was isolated using the Tri-Reagent method according to the manufacturer's protocol (Molecular Research Center, Inc., Cincinnati, OH). Northern blot analysis was performed as described previously (17Zhang D.E. Hetherington C.J. Gonzalez D.A. Chen H.M. Tenen D.G. J. Immunol. 1994; 153: 3276-3284Crossref PubMed Google Scholar, 42Radomska H.S. Huettner C.S. Zhang P. Cheng T. Scadden D.T. Tenen D.G. Mol. Cell. Biol. 1998; 18: 4301-4314Crossref PubMed Scopus (411) Google Scholar). CD14 cDNA was labeled by the random priming method. The blot was washed twice with 0.2× SSC, 0.1% SDS for 20 min at 65 °C. Autoradiography was performed using Kodak BioMax MR film at −80 °C with Kodak BioMax MS intensifying screens. Quantitation of related mRNA levels was performed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Nuclear extracts were prepared as described above. Proteins (80 μg) were diluted 1:1 with Laemmli 2× sample buffer, boiled for 5 min, and electrophoresed on 10% SDS-polyacrylamide gels. Proteins were transferred by electroblotting (Bio-Rad, Hercules, CA) to nitrocellulose membranes. Human C/EBPα and C/EBPβ were detected after 1 h of incubation with a 1:500 dilution of rabbit polyclonal anti-rat C/EBPα and C/EBPβ antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. They were visualized by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech) according to the manufacturer's protocol using a horseradish peroxidase-conjugated secondary antibody (diluted 1:1000). Quantitation of relative protein levels was performed with ImageQuant software from Molecular Dynamics. U937 cells were transfected by electroporation in RPMI 1640 medium at 960 μF and 250 V. Linearized pXP2, p-227CD14-luc or p-227CD14(m135)-luc (10 μg) were co-transfected with PGK-neo (1 μg) into 1 × 107cells. Geneticin (G418) (1 mg/ml, Life Technologies, Inc.) was added to the cell medium 48 h after transfection. Positive clones were assayed for luciferase activity using a luciferase assay kit from Promega (Madison, WI). Four individual transfected stable lines for each luciferase construct were assayed after induction with 1 × 10−7m vitamin D3 and/or 1 ng/ml TGF-β for 24 h in three independent experiments. In a previous study (10Zhang D.E. Hetherington C.J. Tan S. Dziennis S.E. Gonzalez D.A. Chen H.M. Tenen D.G. J. Biol. Chem. 1994; 269: 11425-11434Abstract Full Text PDF PubMed Google Scholar), we defined the proximal promoter of the CD14 gene, in which four regions of the CD14 proximal promoter were protected by nuclear proteins from monocytic cells in DNase I footprinting analysis. Three Sp1 sites were located in this region. The Sp1 binding site at bp −110 is critical for the tissue-specific activity of the CD14 promoter. However, the function of a protected site further upstream (bp −121 to −154) and the identity of the protein interacting with this site remained unknown. In order to elucidate this protein/DNA interaction, the sequence of this region was analyzed. We found that the sequence between bp −128 and −136 (TATTGCAAT) of the noncoding strand was almost identical to the C/EBP consensus sequence TTNNGNAAT (43Natsuka S. Akira S. Nishio Y. Hashimoto S. Sugita T. Isshiki H. Kishimoto T. Blood. 1992; 79: 460-466Crossref PubMed Google Scholar). To verify the functional importance of the putative C/EBP site for CD14 promoter activity, six base pairs (bp −129 to −135) in the core C/EBP binding site were mutated by PCR-mediated site-specific mutagenesis from TGCAAT in the CD14 promoter-luciferase reporter gene construct, p-227CD14-luc, to GGTACC in p-227CD14(m135)-luc. This mutation destroyed the ability of the region from bp −115 to −144 to interact with nuclear protein(s) in EMSA (data not shown). Luciferase constructs with and without this mutation in the CD14 promoter were used in transient transfection assays in the myelomonoblast cell line U937 and the monocytic cell line Mono Mac 6 (Fig. 1). The mutation reduced CD14 promoter activity to 40% of the wild type promoter in U937 cells and to 30% of the wild type in Mono Mac 6 cells. These results demonstrate that the putative C/EBP site is important for CD14 promoter activity. To demonstrate that the nuclear protein(s) interacting with the putative C/EBP site of the CD14 promoter are C/EBP family members, EMSAs were performed. Oligo A was radiolabeled and incubated with nuclear extracts from THP-1 cells. The nuclear protein(s) showed a strong interaction with oligo A (Fig.2 A, lane 2 ). The major shifted complex (labeled with C/EBP) can be efficiently competed with unlabeled self oligonucleotide and a C/EBP binding oligonucleotide from the M-CSF receptor promoter (Fig. 2 A, lanes 3 and 4 ) (8Zhang D.E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar). In contrast, non-C/EBP binding oligonucleotides with the consensus sequences of Sp1 or PU.1 failed to compete with labeled oligo A (Fig.2 A, lanes 5 and 6 ). In addition, nuclear extracts from nonmonocytic HeLa cells do not form the same complex with oligo A (Fig. 2 A, lane 7 ). C/EBPs are not normally expressed in HeLa cells (7Zhang D.E. Fujioka K. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar, 8Zhang D.E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar). These results demonstrate that the protein(s) binding to bp −115 to −144 of the CD14 promoter belongs to the C/EBP family. C/EBPs are a family of transcription factors that have high homologies at their C-terminal dimerization domains and DNA binding domains. In order to verify which C/EBP member interacts with the CD14 promoter, specific antibodies against different members of the C/EBP family were used in an EMSA. As shown in Fig. 2 B, a strong supershifted band was observed with the C/EBPα antibody, and the original shifted band was significantly reduced in the presence of the C/EBPα antibody (Fig. 2 B, lanes 2 and 3 ). The antibodies against C/EBPβ and C/EBPδ also reacted with the DNA-C/EBP complex (Fig. 2 B, lanes 4 and5 ). However, in comparison to the C/EBPα antibody, the reduction of the complex by C/EBPβ and C/EBPδ antibodies was less significant. As negative controls, the antiserum against Sp1 and normal rabbit serum were used and do not have any effect on the DNA-C/EBP complex (Fig. 2 B, lanes 6 and 7 ). The results indicate that C/EBPα, -β, and -δ are able to bind to the CD14 promoter. Furthermore, C/EBPα is the major C/EBP family member interacting with the CD14 promoter in promonocytic THP-1 cells. To further demonstrate the importance of C/EBPs for CD14 promoter activity, C/EBP transactivation experiments were conducted in the C/EBP negative cell line CV-1 (Fig.3). Compared with CD14 promoter activity in the absence of any C/EBP expression, C/EBPα activated the CD14 promoter 11-fold, whereas C/EBPβ and C/EBPδ activated the promoter 4.8- and 8.5-fold, respectively. When the construct with the mutated C/EBP site was used in parallel experiments, C/EBPα, -β, and -δ significantly lost their ability to transactivate the CD14 promoter. These data indicate that C/EBPα, -β, and -δ can activate the CD14 promoter and that this transactivation by C/EBPs is through the identified C/EBP site at bp −129 to −136 of the CD14 promoter. The above results demonstrate that C/EBPs are important transcription factors for CD14 expression. To further study the role of C/EBP in CD14 expression during monocyte differentiation, U937 cells were treated with vitamin D3; these myelomonoblastic cells undergo differentiation toward monocytes (44Olsson I. Gullberg U. Ivhed I. Nilsson K. Cancer Res. 1983; 43: 5862-5867PubMed Google Scholar, 45Munker R. Norman A. Koeffler H.P. J. Clin. Invest. 1986; 78: 424-430Crossref PubMed Scopus (173) Google Scholar, 46Liu M. Lee M.H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (843) Google Scholar). TGF-β can synergize with vitamin D3 to strongly induce monocyte differentiation (35Testa U. Masciulli R. Tritarelli E. Pustorino R. Mariani G. Martucci R. Barberi T. Camagna A. Valtieri M. Peschle C. J. Immunol. 1993; 150: 2418-2430PubMed Google Scholar). As shown in Fig. 4, CD14 expression was elevated upon such induction. CD14 mRNA was detectable by Northern blot analysis when cells were treated with vitamin D3 (Fig. 4, lanes 2 and 3 ). CD14 expression was highly up-regulated when cells were treated with both vitamin D3 and TGF-β (Fig. 4, lanes 5 and6 ) but not by TGF-β (lane 4 ). Densitometry analysis revealed a 30-fold increase relative to treatment by vitamin D3 alone. To investigate whether C/EBP is involved in the induction of CD14 expression, an EMSA was performed with nuclear extracts from untreated and variously treated U937 cells, as shown in Fig. 5. Under both treatments, an increase of C/EBP binding to the CD14 promoter was detected. Treatment with a combination of vitamin D3 and TGF-β showed a much higher level of the increase than is seen in treatment with vitamin D3 alone (Fig. 5, lanes 2, 7, and12 ). To identify which C/EBP family members were responsible for the increase of C/EBP binding during monocyte differentiation, antibodies against C/EBPα, -β, -δ, and -ε were independently added to the reaction mixtures. The results showed that the majority of the binding before the inductions was C/EBPα (Fig. 5, lanes 2–6 ). C/EBPα and C/EBPβ were the major contributors in the increase of the DNA-C/EBP complex after the cells were treated with vitamin D3 or vitamin D3 plus TGF-β (Fig. 5,lanes 7–16 ). Because the addition of either C/EBPα or C/EBPβ antibody removed almost the entire complex (Fig. 5,lanes 8, 9, 13, and 14 ), it indicates not only that C/EBPα and C/EBPβ are the major binding factors but also that they form heterodimers.Figure 5The increase of C/EBP binding during monocyte differentiation. Promonocytic U937 cells were treated with 1 × 10−7m vitamin D3 or with 1 × 10−7m vitamin D3 and 1 ng/ml TGF-β to induce differentiation. A double-stranded oligonucleotide containing bp −144 to −115 of the CD14 promoter was radiolabeled with 32P and incubated in the absence (lane 1 ) or the presence of 5 μg of nuclear extracts from wild type U937 cells (lanes 2–6 ), U937 cells treated with vitamin D3 (lanes 7–11 ), or U937 cells treated with vitamin D3 and TGF-β (lanes 12–16 ). Various C/EBP antisera were added to the binding reaction mixtures. Theasterisk marks a relatively nonspecific complex. Thedots mark the complexes possibly formed between DNA and the short forms of C/EBPβ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the reason for the elevation of C/EBP binding during monocyte differentiation, Western blot analyses were performed with nuclear proteins from U937 cells after various inductions. The expression of C/EBPα and C/EBPβ was increased when U937 cells were treated with either vitamin D3 or TGF-β (Fig. 6). However, the strongest increases of both C/EBP factors were seen upon treatment with both vitamin D3 and TGF-β. Densitometry analysis revealed a 13-fold increase for both C/EBPα and C/EBPβ. It has been reported that phosphorylation regulates the ability of C/EBPs to interact with DNA (47Mahoney C.W. Shuman J. McKnight S.L. Chen H.C. Huang K.P. J. Biol. Chem. 1992; 267: 19396-19403Abstract Full Text PDF PubMed Google Scholar, 48Trautwein C. van de Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Crossref PubMed Scopus (129) Google Scholar, 49Ray A. Ray B.K. Mol. Cell. Biol. 1994; 14: 4324-4332Crossref PubMed Google Scholar, 50Ford A.M. Bennett C.A. Healy L.E. Towatari M. Greaves M.F. Enver T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10838-10843Crossref PubMed Scopus (121) Google Scholar). Therefore, we also treated these nuclear extracts with a phosphatase. The dephosphorylation did not alter the increasing interaction bet" @default.
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• W2019937507 title "CCAAT/Enhancer-binding Protein Activates the CD14 Promoter and Mediates Transforming Growth Factor β Signaling in Monocyte Development" @default.
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