Surfactant Protein D Gene Regulation
2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês
10.1074/jbc.m201126200
ISSN1083-351X
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoSurfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (−432, −340, −319, −140, and −90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (−432, −340, −319, and −90) efficiently competed for the binding of C/EBPα, C/EBPβ, or C/EBPδ to consensus oligomers. Cotransfection of C/EBPα, C/EBPβ, or C/EBPδ cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of −205. Independent site-directed mutagenesis of the sites at −432, −340, and −319 reduced C/EBP-mediated activation by ∼50%, and mutagenesis of the site at −432 in combination with either of the tandem sites at –340 and –319 blocked activation. The conserved AP-1 element at −109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D. Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (−432, −340, −319, −140, and −90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (−432, −340, −319, and −90) efficiently competed for the binding of C/EBPα, C/EBPβ, or C/EBPδ to consensus oligomers. Cotransfection of C/EBPα, C/EBPβ, or C/EBPδ cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of −205. Independent site-directed mutagenesis of the sites at −432, −340, and −319 reduced C/EBP-mediated activation by ∼50%, and mutagenesis of the site at −432 in combination with either of the tandem sites at –340 and –319 blocked activation. The conserved AP-1 element at −109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D. There is increasing evidence that surfactant protein D (SP-D) 1The abbreviations used are: SP-Dsurfactant protein DAPRacute-phase responseC/EBPCCAAT/enhancer-binding proteinSTAT3signal transducer and activator of transcription-3GAPDHglyceraldehyde-3-phosphate dehydrogenaseRbretinoblastoma proteinHNF-3hepatocyte nuclear factor-3 plays important roles in the lung's defense against inhaled microorganisms and organic particles and in the regulation of inflammatory and immune reactions within the lung (1Lawson P.R. Reid K.B. Immunol. Rev. 2000; 173: 66-78Crossref PubMed Scopus (126) Google Scholar, 2Crouch E. Wright J.R. Annu. Rev. Physiol. 2001; 63: 521-554Crossref PubMed Scopus (559) Google Scholar). SP-D, like pulmonary surfactant protein A and the serum mannose-binding protein (MBL), is a member of the collagenous lectin (collectin) subfamily of mammalian C-type lectins. SP-D is secreted into the distal airways and alveoli by non-ciliated bronchiolar epithelial cells and type II pneumocytes, respectively. Although the lung appears to be a major site of SP-D expression, there is evidence that SP-D is also synthesized by epithelial cells in a variety of extrapulmonary sites, consistent with more generalized roles in innate host defense (3Madsen J. Kliem A. Tornoe I. Skjodt K. Koch C. Holmskov U. J. Immunol. 2000; 164: 5866-5870Crossref PubMed Scopus (312) Google Scholar). Unlike other surfactant proteins, the regulation of SP-D promoter activity is dependent on the combinatorial interactions of relatively ubiquitous transcription factors, including members of the AP-1 family (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). surfactant protein D acute-phase response CCAAT/enhancer-binding protein signal transducer and activator of transcription-3 glyceraldehyde-3-phosphate dehydrogenase retinoblastoma protein hepatocyte nuclear factor-3 The expression of SP-D is increased following many forms of pulmonary injury (5Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (324) Google Scholar). For example, the levels of SP-D mRNA and immunoreactive protein in lung lavage increase within several hours to a few days following intratracheal instillation of bacterial endotoxin in rats (6McIntosh J.C. Swyers A.H. Fisher J.H. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1996; 15: 509-519Crossref PubMed Scopus (122) Google Scholar), following challenge of mice with Pseudomonas aeruginosa(7Jain-Vora S. LeVine A.M. Chroneos Z. Ross G.F. Hull W.M. Whitsett J.A. Infect. 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In this regard, a variety of hepatic acute-phase proteins are expressed in the lung, and at least some are elevated following lung injury (10Yang F. Friedrichs W.E. Navarijo-Ashbaugh A.L. deGraffenried L.A. Bowman B.H. Coalson J.J. Lab. Invest. 1995; 73: 433-440PubMed Google Scholar, 11D'Armiento J. Dalal S.S. Chada K. Gene (Amst.). 1997; 195: 19-27Crossref PubMed Scopus (68) Google Scholar, 12Heuertz R.M. Webster R.O. Mol. Med. Today. 1997; 3: 539-545Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 13Dentener M.A. Vreugdenhil A.C. Hoet P.H. Vernooy J.H. Nieman F.H. Heumann D. Janssen Y.M. Buurman W.A. Wouters E.F. Am. J. Respir. Cell Mol. Biol. 2000; 23: 146-153Crossref PubMed Scopus (135) Google Scholar, 14Lee P.T. Holt P.G. McWilliam A.S. Am. J. Respir. Cell Mol. Biol. 2000; 23: 652-661Crossref PubMed Scopus (30) Google Scholar). These include C-reactive protein and haptoglobin as well as the serum lipopolysaccharide-binding protein. Molecular regulation of the systemic APR, including the response to endotoxin, is complex and involves a variety of relatively ubiquitous transcription factors. However, members of the CCAAT/enhancer-binding protein (C/EBP) family of leucine zipper transcription factors figure prominently in the regulation of many APR genes, particularly members of the so-called "Class I" group of APR proteins (15Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 16Baumann H. Gauldie J. Immunol. Today. 1994; 15: 74-80Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 17Fey G.H. Fuller G.M. Mol. Biol. Med. 1987; 4: 323-338PubMed Google Scholar, 18Akira S. Kishimoto T. Immunol. Rev. 1992; 127: 25-50Crossref PubMed Scopus (471) Google Scholar). Like other leucine zipper transcription factors, C/EBPs bind to DNA as homo- or heterodimers and have a diversity of effects that in part reflect tissue and developmental stage-specific expression of various C/EBPs (19Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). The expression and activity of different C/EBP isoforms are differentially modulated in response to inflammatory stimuli, including pro-inflammatory cytokines and glucocorticoids. In addition, the activity of these proteins can be influenced by a variety of post-transcriptional mechanisms, including "leaky translation" with the production of truncated forms and protein phosphorylation. C/EBPα, C/EBPβ, and C/EBPδ are expressed by alveolar type II and non-ciliated bronchiolar epithelial cells, the known pulmonary sites of SP-D production (20Li F. Rosenberg E. Smith C.I. Notarfrancesco K. Reisher S.R. Shuman H. Feinstein S.I. Am. J. Physiol. 1995; 269: L241-L247PubMed Google Scholar, 21Breed D.R. Margraf L.R. Alcorn J.L. Mendelson C.R. Endocrinology. 1997; 138: 5527-5534Crossref PubMed Scopus (43) Google Scholar, 22Sugahara K. Sadohara T. Sugita M. Iyama K. Takiguchi M. Cell Tissue Res. 1999; 297: 261-270Crossref PubMed Scopus (39) Google Scholar, 23Lag M. Skarpen E. van Rozendaal B.A. Haagsman H.P. Huitfeldt H.S. Thrane E.V. Schwarze P.E. Exp. Lung Res. 2000; 26: 383-399Crossref PubMed Scopus (10) Google Scholar). C/EBPα and C/EBPδ are particularly abundant in the lung and increase in fetal rat lung in late gestation during a time when the production of surfactant-associated proteins, including SP-D, is increased. Mice deficient in C/EBPα show abnormalities in alveolar development and often die secondary to the respiratory abnormalities (24Flodby P. Barlow C. Kylefjord H. Ahrlund-Richter L. Xanthopoulos K.G. J. Biol. Chem. 1996; 271: 24753-24760Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Although mice deficient in C/EBPβ and C/EBPδ also expire perinatally, they have no obvious pulmonary phenotype (15Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Neonatal C/EBPα-null mice show no hepatic APR and fail to induce STAT3 binding in response to systemically administered endotoxin, despite marked increases in C/EBPβ or C/EBPδ (25Burgess-Beusse B.L. Darlington G.J. Mol. Cell. Biol. 1998; 18: 7269-7277Crossref PubMed Scopus (64) Google Scholar). However, the pulmonary APR to systemically administered endotoxin is not impaired in this model, and the levels of C/EBPβ and C/EBPδ are increased in the lung following systemic endotoxin administration (25Burgess-Beusse B.L. Darlington G.J. Mol. Cell. Biol. 1998; 18: 7269-7277Crossref PubMed Scopus (64) Google Scholar,26Alam T. An M.R. Papaconstantinou J. J. Biol. Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar). Inspection of the upstream sequence of the SP-D gene revealed five sites consistent with the consensus sequence for C/EBP binding. We have previously utilized H441 human lung adenocarcinoma cells as a model system for studying SP-D promoter activity (27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar). To characterize the regulatory role(s) of the putative C/EBP elements, we examined the interactions of oligomers containing these sequences with H441 nuclear proteins from cells cotransfected with cDNAs encoding the three major C/EBP isoforms. We also compared the activity of wild-type constructs and constructs containing mutated consensus sequences in transient transfection assays using luciferase reporter constructs and examined potential functional interactions with the conserved AP-1 element in the proximal promoter. An ∼7-kb EcoRI fragment of the previously described human genomic clone (H5), designated H5E7, containing human SP-D 5′-sequence was isolated and subcloned into pGEM-3Z (Promega) as previously described (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar). All experiments used restriction fragments containing 5′-regulatory sequence; each terminated at a SacI site within the untranslated first exon and were numbered from the transcription start site (27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar). Most studies employed a SpeI/SacI fragment (SS698) of the human SP-D gene containing 698 bp upstream of the transcription start site (see Fig. 1). For some experiments, we also used a StuI/SacI fragment containing 205 bp upstream of the start site (SS205) and a longer Hin dIII/SacI fragment containing 1674 bp of upstream sequence (HS1674). The restriction fragments were subcloned into a luciferase reporter plasmid (pGL3-Basic, Promega) between the KpnI and SacI sites. NCI H441 human lung adenocarcinoma cells were propagated as previously described (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar). In preliminary experiments performed in the late 1990s, we used cells provided by Dr. A. Gazdar (27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar). These experiments revealed detectable specific binding of H441 nuclear proteins to C/EBP consensus and NF-IL6 oligomers and to an oligomer containing one of the SP-D motifs, Oligo 340 (see Table I). However, all of the experiments presented here used H441 cells obtained more recently from the American Type Culture Collection. These cells, although similarly supporting SP-D promoter activity and demonstrating detectable levels of endogenous SP-D expression, showed only low levels of endogenous C/EBPα, C/EBPβ, and C/EBPδ as assessed in supershift assays. It is unclear whether these differences reflect substrain variation or differences in culture conditions. However, the reduced levels of endogenous C/EBPs facilitated the cotransfection experiments described below.Table IOligomers synthesized for electrophoretic mobility shift assays Open table in a new tab Several wild-type and mutant oligomers were synthesized (DNA International) for this study (see Table I). A commercial C/EBP consensus oligomer was obtained from Santa Cruz Biotechnology. Oligomers containing an authentic NF-IL6 site and a mutated NF-IL6 site were also synthesized (see Table I) based on published sequence (28Chen P.L. Riley D.J. Chen-Kiang S. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 465-469Crossref PubMed Scopus (198) Google Scholar). The oligomers and their reverse complements were annealed and used in electrophoretic mobility shift assays as described below. SS698 was subcloned into pGEM-3Z and used for thermal cycling-coupled mutagenesis. Forward- and reverse-directed oligomers were synthesized, each containing a mutated consensus sequence. Plasmid SS698 (SS698) was linearized outside the multiple cloning site by digestion with ScaI and used as template for thermal cycling reactions. Approximately 200 ng of template DNA, 200 ng of forward or reverse mutagenesis oligomer, and 200 ng of an oligomer directed to the appropriate SP6 or T7 RNA polymerase site in pGEM were combined with 200 μm dNTPs (Roche Molecular Biochemicals) and 1 unit of Taq polymerase (Fisher) in buffer supplied with the enzyme. Twenty to twenty-five cycles were performed, each consisting of 1 min at 95 °C (denaturing), 1 min at 45 °C (annealing), and 2 min at 70 °C (extension). Resultant DNA fragments were gel-purified using the QIAQuick gel extraction kit (QIAGEN Inc.). The 5′- and 3′-fragments of the mutated promoter DNA were joined together by extension thermal cycling using an overlapping internal oligomer sequence and oligomers to the flanking SP6 and T7 sites for amplification. The mutated fragments were subcloned into a luciferase reporter plasmid as described for the wild-type fragments. The orientation and sequence were verified by restriction mapping and DNA sequencing. Nuclear extracts were prepared from cultured cell lines using a rapid mini-extraction technique (29Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar) as previously described (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The protein content was analyzed by dye binding assay, and the extracts were frozen in liquid nitrogen and stored at −70 °C. Electrophoretic mobility shift (gel retardation) and supershift assays were performed by a modification of a method employed by Bingle and co-workers (30Toonen R.F.G. Gowan S. Bingle C.D. Biochem. J. 1996; 316: 467-473Crossref PubMed Scopus (60) Google Scholar) as previously described (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Antibodies to C/EBPα (14AA), C/EBPβ (C-19), and C/EBPδ (M-17) were from Santa Cruz Biotechnology. Specificity of each antibody was confirmed in supershift assays using nuclear extracts from cells transfected with C/EBPα, C/EBPβ, or C/EBPδ cDNA as described below. For experiments characterizing the promoter activity of mutant constructs, H441 target cells (5 × 105) were transferred to 35-mm plates in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Invitrogen), allowed to attach overnight, and washed twice with RPMI 1640 medium devoid of phenol red (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The cells were transfected with up to 1.5 μg of the luciferase reporter construct using Lipofectin (Invitrogen) and incubated for 5 h at 37 °C in the absence of serum. The medium was replaced with fresh growth medium, and the cells were incubated overnight. Cells were harvested at 48 h, with one media change at 24 h. C/EBPβ cDNA was obtained by thermal cycling gene amplification of a lung cDNA library (CLONTECH) using a pair of primers: one located at the 5′-end and the other at the 3′-end according to the known human C/EBPβ (NF-IL6) sequence. The full-length DNA sequence was confirmed by automated sequencing. The rat C/EBPα cDNA was a gift from Dr. Steven McKnight (University of Texas Southwestern Medical Center, Dallas, TX), and the rat C/EBPδ cDNA was a gift from Dr. Peter Rotwein (Oregon Health Sciences University, Portland, OR). All cDNAs were subcloned into the pcDNA3 vector (Invitrogen) at the Hin dIII and BamHI sites. This vector contains the cytomegalovirus immediate-early promoter, a polylinker, and the bovine growth hormone polyadenylation sequence. The plasmid concentration required for maximal transactivation of the wild-type reporter construct was determined in preliminary dose-response experiments. Although activation by C/EBPα was dose-dependent up to 1.5 μg, activation by C/EBPβ decreased at concentrations above 1.5 μg, whereas activation by C/EBPδ reached a plateau above 0.25 mg. Accordingly, most transfections were performed using 1.5 μg of pcDNA3 containing the desired cDNA or an equivalent weight of the pcDNA3 vector. Protein expression was confirmed by supershift assays. Cell layers were harvested, and transient transfection assays were performed using protein-equivalent amounts of cell extract containing the luciferase reporter constructs. Luciferase activity was measured using a Turner Designs Model TD20/20 luminometer. Transfection efficiency was internally controlled using the pRL-tk vector (Dual-Luciferase kit, Promega). All assays were performed on duplicate or triplicate plates. Except where indicated, at least three separate experiments were performed. In some initial experiments, chloramphenicol acetyltransferase assays were performed as previously described (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). SP-D mRNA was amplified from total RNA isolated from H441 cells. Primers for the full-length product contained untranslated sequence and a several bases of contiguous coding sequence: 5′-primer (+), CCT GCC ATG CTG CTC TTC CTC CTC TCT GC; and 3′-primer (−), CCA GTT GGC TCA GAA CTC GCA GAC CA. Five μg of RNA was reverse-transcribed for 30 min at 50 °C and then denatured for 2 min at 94 °C, followed by 10 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 68 °C. This was followed by 40 more cycles using the same denaturation and annealing conditions, but with the addition of 5 s/cycle of extension at 68 °C. The identity of the amplified fragment was initially confirmed by nested PCR using a different 5′-primer (+), AAT CCT GGA GAC AAA GGA GCA AAG GGA GAA. For this reaction, 1 μl of the full-length PCR product was amplified by conventional PCR. The full-length PCR product was subcloned into the pGEM-T vector (Promega), and the DNA was sequenced from both ends using T7 and SP6 primers. Increases in endogenous mRNA were also estimated by "comparative" PCR using a commercial protocol and reagents (Comparative PCR, Ambion Inc.). This technique is a variant of competitive PCR that competes the cDNAs derived from two preparations of RNA. Each cDNA was tagged with unique reverse transcriptase primers of different lengths. Known amounts of the two tagged cDNAs were mixed in various proportions and then subjected to PCR using an upstream tag-specific primer and a downstream SP-D-specific 3′-sense primer ∼300 nucleotides upstream from the stop codon (TGC TTT CCT GAG CAT GAC TGA T). Short and long tag primers for the amplification of control GAPDH message were provided with the kit. Purified RNA from the control and transfected cells gave essentially identical yields of cDNA/μg of RNA as assessed by incorporation of [α-32P]dATP during the reverse transcription. In most cases, members of the C/EBP family specifically interact with DNA sequences fulfilling the general consensus TT(G/T)NGNAA or TKDNGNAAK (K = G/T; D = A/G/T). Computer-assisted matrix analysis of the upstream sequence of the human SP-D gene using the TRANSFAC Database revealed five sequences consistent with this consensus within 698 bp of the transcription start site at −432, −340, −319, −140, and −90 (Figs. 1and 2A). Two motifs were found in an XbaI/SacI fragment that includes 285 bp upstream of the start site, which we have previously referred to as the "proximal" promoter (4He Y. Crouch E.C. Rust K. Spaite E. Brody S.L. J. Biol. Chem. 2000; 275: 31051-31060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The first three motifs are located in the region we have designated the "near-distal" promoter. The potential importance of the four most upstream motifs is suggested by the spatial conservation of homologous sequences in the rat and mouse SP-D promoters (27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar, 31Lawson P.R. Perkins V.C. Holmskov U. Reid K.B. Am. J. Respir. Cell Mol. Biol. 1999; 20: 953-963Crossref PubMed Scopus (18) Google Scholar) and/or in the promoter sequences of bovine conglutinin and CL-43, hepatic host defense collectins believed to have evolved from a primordial SP-D gene (32Kawasaki N. Itoh N. Kawasaki T. Biochem. Biophys. Res. Commun. 1994; 198: 597-604Crossref PubMed Scopus (20) Google Scholar, 33Kawasaki N. Satonaka M. Imagawa M. Naito H. Kawasaki T. J. Biochem. (Tokyo). 1998; 124: 1188-1197Crossref PubMed Scopus (6) Google Scholar, 34Hansen, S., Moeller, V., Holm, D., Vitved, L., Bendixen, C., Skjoedt, K., and Holmskov, U. (2002) Mol. Immunol., in pressGoogle Scholar). A complete C/EBP motif nearly identical to the human sequence at −432 is found in the mouse gene (TTGaGAAA, reverse) (Fig. 2B). The sequence for the corresponding region of the rat promoter is not available. Although the sequences at this position in the CL-43 and conglutinin promoters are quite highly conserved, key elements of the C/EBP motif are absent. Sequences in the region spanning −340 to −319 are highly conserved in the mouse and rat promoters, and the downstream sequences deviate from the C/EBP consensus sequence at only the first position (Fig. 2B) (27Rust K. Bingle L. Mariencheck W. Persson A. Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1996; 14: 121-130Crossref PubMed Scopus (45) Google Scholar, 31Lawson P.R. Perkins V.C. Holmskov U. Reid K.B. Am. J. Respir. Cell Mol. Biol. 1999; 20: 953-963Crossref PubMed Scopus (18) Google Scholar). In addition, these tandem motifs are conserved in the conglutinin and CL-43 promoters (Fig. 2) (32Kawasaki N. Itoh N. Kawasaki T. Biochem. Biophys. Res. Commun. 1994; 198: 597-604Crossref PubMed Scopus (20) Google Scholar, 34Hansen, S., Moeller, V., Holm, D., Vitved, L., Bendixen, C., Skjoedt, K., and Holmskov, U. (2002) Mol. Immunol., in pressGoogle Scholar). The C/EBP motif at −140 in the proximal promoter (TTcTGGAA) is nearly identical to the corresponding mouse (cTcTGGAA) and CL-43 or conglutinin (TTcTGGAc) sequences, but diverges significantly in the rat. Interestingly, this motif overlaps an H-APF-1 motif (CTGGRAA) that is completely conserved in the mouse and rat genes and conserved at all but one position in the bovine lectins. By contrast, the motif at –90 is unique to human SP-D. It is not conserved in the bovine collectins or mouse SP-D and is interrupted by a CA repeat in rat SP-D. Thus, the motifs appear to be most highly conserved in the near-distal promoter. Given the low levels of endogenous C/EBP-binding activity in our H441 nuclear extracts (see "Experimental Procedures"), we were able to utilize H441 cells that were transfected with cDNA for C/EBPα, C/EBPβ, or C/EBPδ as a model system for characterizing the interactions of C/EBPs with the motifs. We observed specific binding of the C/EBPs to oligomers containing the motifs at all five sites in electrophoretic mobility shift assays (Figs. Figure 3, Figure 4, Figure 5). In each case, binding was blocked by competition with the unlabeled oligomer, but not with a mutant oligomer (Table I). In supershift experiments, we also demonstrated binding of each of the three isoforms to each of the five C/EBP motifs.Figure 4C/EBPs bind to the motifs at –340 and −319. H441 cells were transfected with C/EBPδ cDNA, and binding of nuclear proteins was assessed using gel retardation assays. Comparable results were obtained when H441 cells were transfected with C/EBPα or C/EBPβ cDNA. A, binding of C/EBPδ to a radiolabeled oligomer containing the SP-D C/EBP sequence at −340 (Oligo 340; Table I). The presence of transfected C/EBPδ is indicated. Binding was competed by the unlabeled oligomer (Oligo 340; lane 3), but not by the corresponding mutant oligomer (Oligo 340m; lane 4). The supershifted complex generated with antibody to C/EBPδ is shown in lane 5. B, binding of C/EBPδ to a radiolabeled oligomer containing the SP-D C/EBP sequence at −319 (Oligo 319; Table I). As in A, binding was competed by the unlabeled oligomer (Oligo 319; lane 3) or by the consensus oligomer (lane 5), but not by the corresponding mutant oligomer (Oligo 319m;lane 4). The supershifted complex generated with antibody to C/EBPδ is shown in lane 6, and a representative negative supershift control using normal IgG (nl IgG) is shown in lane 7.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5C/EBPs bind to the motifs at −140 and −90. H441 cells were transfected with C/EBPδ cDNA, and binding of nuclear proteins was assessed using gel retardation assays. Comparable results were obtained when H441 cells were transfected with C/EBPα or C/EBPβ cDNA. A, binding of C/EBPδ to a radiolabeled oligomer containing the SP-D C/EBP sequence at −140 (Oligo 140; Table I). Binding was competed by the unlabeled oligomer (Oligo 140;lane 2), but not by the corresponding mutant oligomer (Oligo 140m; lane 3). The supershifted complex generated with antibody to C/EBPδ is shown in lane 4. B, binding of C/EBPδ to a radiolabeled oligomer containing the SP-D C/EBP sequence at −90 (Oligo 90; Table I). Binding was competed by the unlabeled oligomer (Oligo 90; lane 2), but not by the corresponding mutant oligomer (Oligo 90m; lane 3). The supershifted complex generated with antibody to C/EBPδ is shown in lane 4.View Large
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