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• W2022124461 abstract "Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following lung injury. Because AP-1 proteins regulate cellular responses to diverse environmental stimuli, we hypothesized that the conserved AP-1 motif (at −109) and flanking sequences in the human SP-D promoter contribute to the regulation of SP-D expression. The AP-1 sequence specifically bound to fra-1, junD, andjunB in H441 lung adenocarcinoma nuclear extracts. Mutagenesis of the AP-1 motif in a chloramphenicol acetyltransferase reporter construct containing 285 base pairs of upstream sequence nearly abolished promoter activity, and co-transfection of junD significantly increased wild type but not mutant promoter activity. The sequence immediately downstream of the AP-1 element contained a binding site for HNF-3 (FOXA), and simultaneous mutation of this site (fox-d) and an upstream FoxA binding site (−277,fox-u) caused a 4-fold reduction in chloramphenicol acetyltransferase activity. Immediately upstream of the AP-1-binding site, we identified a GT box-containing positive regulatory element. Despite finding regions of limited homology to the thyroid transcription factor 1-binding site, SP-D promoter activity did not require thyroid transcription factor 1. Thus, transcriptional regulation of SP-D gene expression involves complex interactions with ubiquitous and lineage-dependent factors consistent with more generalized roles in innate immunity. Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following lung injury. Because AP-1 proteins regulate cellular responses to diverse environmental stimuli, we hypothesized that the conserved AP-1 motif (at −109) and flanking sequences in the human SP-D promoter contribute to the regulation of SP-D expression. The AP-1 sequence specifically bound to fra-1, junD, andjunB in H441 lung adenocarcinoma nuclear extracts. Mutagenesis of the AP-1 motif in a chloramphenicol acetyltransferase reporter construct containing 285 base pairs of upstream sequence nearly abolished promoter activity, and co-transfection of junD significantly increased wild type but not mutant promoter activity. The sequence immediately downstream of the AP-1 element contained a binding site for HNF-3 (FOXA), and simultaneous mutation of this site (fox-d) and an upstream FoxA binding site (−277,fox-u) caused a 4-fold reduction in chloramphenicol acetyltransferase activity. Immediately upstream of the AP-1-binding site, we identified a GT box-containing positive regulatory element. Despite finding regions of limited homology to the thyroid transcription factor 1-binding site, SP-D promoter activity did not require thyroid transcription factor 1. Thus, transcriptional regulation of SP-D gene expression involves complex interactions with ubiquitous and lineage-dependent factors consistent with more generalized roles in innate immunity. surfactant protein D surfactant protein A base pair(s) Clara cell-specific protein thyroid transcription factor 1 electrophoretic mobility shift assay chloramphenicol acetyltransferase dexamethasone 12-O-tetradecanoylphorbol-13-acetate Pulmonary surfactant protein D (SP-D)1 is a member of the collectin (collagenous lectin) subfamily of mammalian C-type lectins, which includes pulmonary surfactant protein A (SP-A), serum mannose-binding lectin, and at least two bovine serum lectins related to SP-D, conglutinin and CL-43 (1Eggleton P. Reid K.B. Curr. Opin. Immunol. 1999; 11: 28-33Crossref PubMed Scopus (72) Google Scholar, 2Crouch E.C. Am. J. Respir. Cell. Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (323) Google Scholar). The genes for both lung collectins and mannose-binding lectin are encoded in close proximity on human chromosome 10q. The pulmonary collectins, like the homologous serum proteins, are believed to contribute to innate (nonclonal) immunity and the host response to microorganisms (2Crouch E.C. Am. J. Respir. Cell. Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (323) Google Scholar). In addition, both SP-A and SP-D may contribute to the regulation of surfactant lipid homeostasis under certain circumstances in vivo (3Botas C. Poulain F. Akiyama J. Brown C. Allen L. Goerke J. Clements J. Carlson E. Gillespie A.M. Epstein C. Hawgood S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11869-11874Crossref PubMed Scopus (348) Google Scholar, 4Korfhagen T.R. Sheftelyevich V. Burhans M.S. Bruno M.D. Ross G.F. Wert S.E. Stahlmann M.T. Jobe A.H. Ikegami M. Whitsett J.A. Fisher J.H. J. Biol. Chem. 1998; 273: 28438-28443Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 5Korfhagen T.R. LeVine A.M. Whitsett J.A. Biochim. Biophys. Acta. 1998; 1408: 296-302Crossref PubMed Scopus (97) Google Scholar). SP-A and SP-D are secreted into the distal airways and pulmonary alveoli by Clara cells and type II pneumocytes. The expression of SP-A and SP-D by these cells is increased following many forms of pulmonary injury (2Crouch E.C. Am. J. Respir. Cell. Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (323) Google Scholar), and the rapid increase in SP-A and SP-D accumulation following intratracheal instillation of bacterial endotoxin suggests that they contribute to a pulmonary acute phase response (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). However, the regulation of these responses is not understood. We have previously shown that DNA sequences within 285 bp of the start site of transcription of the human SP-D promoter are able to confer glucocorticoid-responsive gene expression in H441 human lung adenocarcinoma cells but not liver HepG2 cells (7Rust 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), suggesting that the proximal sequence contains information sufficient to direct lung-restricted expression. Sequence analysis of the proximal promoter suggested potential regulatory roles for a variety of ubiquitous and lung-restricted transcription factors. In considering the potential contributions of these regulatory motifs to SP-D gene regulation, we initially focused our attention on the AP-1 consensus (5′-TGAGTCA-3′) at −109 bp relative to the start site of transcription (7Rust 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). Preliminary footprinting assays showed preferential protection of this motif and its contiguous flanking sequences by nuclear extracts from H441 cells. In addition, an identical AP-1 motif is spatially conserved in the rat and mouse promoters (7Rust 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, 8Lawson 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 in the promoter of the homologous bovine conglutinin gene (9Kawasaki N. Itoh N. Kawasaki T. Biochem. Biophys. Res. Commun. 1994; 198: 597-604Crossref PubMed Scopus (20) Google Scholar, 10Liou L.S. Sastry R. Hartshorn K.L. Lee Y.M. Okarma T.B. Tauber A.I. Sastry K.N. J. Immunol. 1994; 153: 173-180PubMed Google Scholar) (Fig.1). Because alterations in the composition of the AP-1 complex regulate a wide variety of transcriptional events associated with cellular differentiation and the response to environmental stimuli including cell injury, inflammation, and the systemic acute phase response, we hypothesized that the AP-1 motif plays important roles in the regulation of SP-D expression. The flanking regions of the conserved AP-1 motif also contain sequences resembling binding sites for factors known to regulate respiratory epithelial cell differentiation and the expression of various secreted lung proteins including Clara cell-specific protein (CCSP) and SP-A, -B, and -C (11Perl A.K. Whitsett J.A. Clin. Genet. 1999; 56: 14-27Crossref PubMed Scopus (125) Google Scholar). These include potential binding sites for the homeodomain-containing protein, thyroid transcription factor 1 (TTF-1), and the forkhead/winged helix transcription factors, such as HNF-3 (FOXA). Previous studies have shown that TTF-1 and HNF-3 can act in combinatorial fashion with other ubiquitous and cell-specific transcription factors to regulate lung-specific genes (12Hackett B.P. Bingle C.D. Gitlin J.D. Annu. Rev. Physiol. 1996; 58: 51-71Crossref PubMed Scopus (49) Google Scholar, 13Whitsett J.A. Glasser S.W. Biochim. Biophys. Acta. 1998; 1408: 303-311Crossref PubMed Scopus (89) Google Scholar). Because pulmonary cells known to express TTF-1 and forkhead box proteins secrete SP-D in vivo, we hypothesized that these nuclear factors similarly contribute to the regulation of SP-D expression. To characterize the regulatory role(s) of the putative AP-1 element and other potential regulatory motifs in its conserved flanking sequences, we examined the interactions of oligomers containing these sequences with H441 nuclear proteins using electrophoretic mobility shift and antibody supershift assays. We also selectively mutated these sequences and compared the activity of wild type and mutant constructs in transient transfection assays. These studies are the first to demonstrate specific cis-acting elements in the human SP-D gene. The regulatory profile is consistent with more generalized roles in pulmonary and nonpulmonary host defense. An approximately 7-kilobase EcoRI fragment of the previously described human genomic clone (H5), designated H5E7, containing 5′ hSP-D sequence was isolated and subcloned into pGEM3Z as described previously (7Rust 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 XbaI/SacI fragment of the hSP-D gene containing 285 bp immediately upstream of the start site of transcription (XS285 or XS) (Fig. 1). NCI-H441 human lung adenocarcinoma cells were obtained as a gift from Dr. A. Gazdar (University of Texas Medical Center, Dallas, TX) and propagated as described previously (7Rust 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). HeLa (CCL-2), A549 (CCL-185), and HepG2 (HB-8065) cell lines were obtained from the American Type Culture Collection and maintained under recommended conditions of cell culture. Several wild type and mutant oligomers were synthesized (DNA International) for this study (see Table I). In addition, oligomers corresponding to the CCSP upstream and/or downstream HNF-3-binding sites (14Bingle C.D. Gitlin J.D. Biochem. J. 1993; 295: 227-232Crossref PubMed Scopus (59) Google Scholar), FREAC2 (15Pierrou S. Hellqvist M. Samuelsson L. Enerback S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (373) Google Scholar), TTR-S (16Qian X. Samadani U. Porcella A. Costa R.H. Mol. Cell. Biol. 1995; 15: 1364-1376Crossref PubMed Google Scholar), and wild type and mutant TTF-1 (17Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biochem. 1994; 94: 5671-5681Crossref Scopus (484) Google Scholar) were synthesized based on published sequences. The GT box oligomer corresponds to the GT box-binding site in human SP-A (18Young P.P. Mendelson C.R. Mol. Endocrinol. 1997; 11: 1082-1093PubMed Google Scholar). Commercial consensus oligomers to AP-1 and AP-2 were purchased from Promega, and commercial Sp1, E box, and GATA (1Eggleton P. Reid K.B. Curr. Opin. Immunol. 1999; 11: 28-33Crossref PubMed Scopus (72) Google Scholar, 2Crouch E.C. Am. J. Respir. Cell. Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (323) Google Scholar, 3Botas C. Poulain F. Akiyama J. Brown C. Allen L. Goerke J. Clements J. Carlson E. Gillespie A.M. Epstein C. Hawgood S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11869-11874Crossref PubMed Scopus (348) Google Scholar, 4Korfhagen T.R. Sheftelyevich V. Burhans M.S. Bruno M.D. Ross G.F. Wert S.E. Stahlmann M.T. Jobe A.H. Ikegami M. Whitsett J.A. Fisher J.H. J. Biol. Chem. 1998; 273: 28438-28443Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 5Korfhagen T.R. LeVine A.M. Whitsett J.A. Biochim. Biophys. Acta. 1998; 1408: 296-302Crossref PubMed Scopus (97) Google Scholar, 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) oligomers were obtained from Santa Cruz. The oligomers and their reverse complements were annealed and used in electrophoretic mobility shift assays (EMSAs) as described below.Table IOligomers synthesized for electrophoretic mobility shift assaysAP-1 oligomers and mutants Oligo15′-GAGTGAGTGAGTCAATAAAGSP-D AP-1 motif (at −109) Oligo1m5′-GAGTGAGgGgtgacCTAAAGMutated SP-D AP-1 Oligo2 5′-GAGTGAGTCAATTruncated SP-D AP-1 oligomer AP1/fox-d 5′-GAGTGAGTCAATAAAGAAGAAAATTGSP-D AP-1 and forkhead boxDownstream of AP-1 and forkhead fox-d5′-ATAAAGAAGAAAATTGDownstream forkhead site (at −104) fox-dm5′-ATAACtggatccATTGMutated downstream forkhead site fox-u5′-TTCTAGACATCTATAAATACAGACACTGATGAUpstream forkhead site fox-um5′-TTCTAGACATCTggatccACAGACACTGATGAMutated upstream forkhead siteUpstream of AP-1 Oligo35′-AGAACGCAGGTGGGGATAAGAGTGAGSP-D sequence upstream of AP-1 Oligo3m15′-AGAACGCAGGatccGATAAGAGTGAGMutated SP-D GT-box in Oligo3 Oligo3m25′-AGAACGCAGGTGGGGgatccAGTGAGMutated SP-D GATAA in Oligo3 Oligo4 5′-GGTGGGGATAAGAGTGAGSequence between E-box and AP-1 Oligo4m 5′-GGTGGGGgatccAGTGAGOligo4 with mutated GATAA Open table in a new tab XS285 (Fig. 1) was subcloned into pGEM-3Z (Promega) and used for thermal cycling-coupled mutagenesis. Forward and reverse directed oligomers were synthesized, each containing a mutated consensus sequence. pXS 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. 20–25 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 QIA Quick Gel extraction kit (Qiagen). 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 wild type or final mutated fragment was subcloned into pSK-CAT as described previously (7Rust 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) or pBLCAT3 (plasmid backbone of pTK-CAT), and the orientation was verified by restriction digestion and sequencing. All SP-D sequences terminated at aSacI site within the untranslated first exon and were numbered from the start site of transcription (7Rust 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). Nuclear extracts were prepared from cultured cell lines using a rapid mini-extraction technique (19Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3916) Google Scholar). Briefly, cells were scraped into 7 ml of Tris-buffered saline, pH 7.5, and centrifuged. The pellet was resuspended in 1 ml of Tris-buffered saline, transferred to a 1.5-ml microcentrifuge tube, and recentrifuged. The resultant pellet was thoroughly resuspended in 400 μl of ice-cold Buffer A (10 mm HEPES, pH 7.8, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, and 2 μg/ml aprotinin) and incubated on ice for 15 min. Nonidet P-40 (Sigma) was then added to a final concentration of 0.625%, and the sample was vortexed for 10 s prior to microcentrifugation at 1350 rpm in a Hermle Z233M centrifuge for 30 s at 4 °C. The resultant nuclear pellet was resuspended in 50 μl of cold Buffer C (20% glycerol, 400 mm NaCl, 10 mm HEPES, pH 7.8, 1 mm EDTA, 1 mmEGTA, 1 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, and 2 μg/ml aprotinin). The pellet was vigorously agitated on a rocker platform for 15 min at 4 °C and then clarified by centrifugation for 15 min at 4 °C in a microcentrifuge (12,000 rpm). The supernatant was transferred to a chilled fresh tube, and the protein content was analyzed by dye binding assay. Yields were in the range of 300–500 μg/75-cm2 flask. Once quantified, the extract was frozen in liquid nitrogen and stored at −70 °C. Gel retardation assays were performed by a modification of a method employed by Bingle and co-workers (20Toonen R.F.G. Gowan S. Bingle C.D. Biochem. J. 1996; 316: 467-473Crossref PubMed Scopus (60) Google Scholar). Briefly, 5–10 μg of nuclear extract in the absence or presence of 50–100-fold excess of competitive inhibitor was incubated for 10 min at 18 °C in a buffer consisting of 1 mmMgCl2, 20 mm HEPES, pH 7.8, 40 mmKCl, 1 mm dithiothreitol, 100 mm EGTA, 4% (w/v) Ficoll (Sigma), and 50 μg/ml poly(dI-dC) (Sigma). Approximately 1.75 pmol of kinase-labeled double-stranded oligomer were then added, and the incubation was continued for 10 min. For supershift experiments purified antibodies were then added to samples at the optimal concentration and incubated for an additional 10 min. Antibodies tojun or fos proteins, GATA-6, Sp1, and Sp3 were from Santa Cruz Biotechnology; antibodies to HNF-3α and HNF-3β were from Dr. Robert Costa (University of Illinois, Chicago, IL), and the antibody to TTF-1 was provided by Dr. Roberto Di Lauro (Stazione Zoologica “Anton Dohrn,” Naples, Italy). Prior to loading on polyacrylamide gel electrophoresis, 110 volume of loading buffer (250 mm Tris-HCl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene cyanol, and 40% glycerol) was added to each sample. Complexes were resolved by polyacrylamide gel electrophoresis (2.5–3 h, 250 V, 4 °C) using nondenaturing 4% bisacrylamide, 2.5% glycerol gels, and 0.5× TBE running buffer. Gels were dried to 3MM paper (Whatman) and exposed to X-Omat AR film (Eastman Kodak) for a period of 1 h to 1 week with an intensifying screen. In brief, for experiments characterizing the promoter activity of 5′ deletion or mutant constructs, H441 target cells (5 × 105) were plated on 35-mm plates in RPMI medium (Life Technologies, Inc.) supplemented with 10% (v/v) newborn calf serum (Life Technologies, Inc.), allowed to attach overnight, and washed twice with RPMI devoid of phenol red (7Rust 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). The cells were transfected with 2 μg of CAT reporter construct and 1 μg of pCMV-β-gal (Promega) using LipofectAMINE (Life Technologies, Inc.) and incubated for 2 h at 37 °C in the absence of serum. Parallel transfections were performed using pSK-CAT vector controls and/or a glucocorticoid-responsive control promoter, pMSG-CAT (Amersham Pharmacia Biotech). Concentrated serum-containing medium was then added, and the cells were incubated overnight. Cells were harvested at various times up to 48 h with one medium change at 24 h when needed. Where indicated, a previously optimized concentration of dexamethasone (Dex; final concentration, 50 nm) was added to the concentrated medium and with the succeeding medium change. All assays were performed on duplicate or triplicate plates. For some experiments, similar studies were performed using HeLa or HepG2 cells. Cell layers were harvested, and transient transfection assays were performed using protein equivalent amounts of cell extract using and CAT promoter constructs as described previously (7Rust 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). Prior to each transfection the quality of the plasmids was reassessed by gel electrophoresis. CAT activity was measured by phase partitioning and thin layer chromatography, followed by autoradiography. To quantify relative acetylation, gels were exposed to a phosphorimaging screen (Storage phosphor Screen GP; Eastman Kodak Co.) and scanned using a STORM image reader (Molecular Dynamics). To prepare figures the data files were imported to ImageQuant (Molecular Dynamics) and transferred to Illustrator (Adobe) as unmanipulated TIFF files. Each assay was performed in duplicate, and each figure is representative of at least three experiments. When indicated, conversion data were normalized to β-galactosidase activity as described previously (7Rust 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). ThejunD expression plasmid (HA1-junD) was a gift of Dr. Lester Lau (University of Illinois). The fra-1 cDNA was kindly provided by Dr. Thomas Curran (St. Jude's Hospital, Memphis, TN). Expression plasmids for c-fos and c-jun were a gift of Dr. G. Doyle (University of Wisconsin, Madison, WI). These were constructed by inserting the nuclear factor cDNA in the pcDNA3 vector (Invitrogen, Carlsbad, CA; contains the cytomegalovirus immediate-early promoter, a polylinker, and the bovine growth hormone polyadenylation sequence). The pCMV-human HNF-3α cDNA (21Bingle C.D. Gowan S. Biochim. Biophys. Acta. 1996; 1307: 17-20Crossref PubMed Scopus (12) Google Scholar) and TTF-1 cDNA (20Toonen R.F.G. Gowan S. Bingle C.D. Biochem. J. 1996; 316: 467-473Crossref PubMed Scopus (60) Google Scholar) were gifts from Dr. Colin Bingle (University of Sheffield Medical School, Sheffield, UK). Dr. James Darnell (Rockefeller University, New York, NY) kindly provided the rat HNF-3α and HNF-3β cDNAs. Transfections were performed using up to 1 μg of pcDNA3 containing the desired cDNA or an equivalent weight of the pcDNA3 vector. For some experiments, expression of mRNA or protein encoded by the cotransfected plasmid was confirmed by Northern hybridization or gel supershift assays, respectively. A labeled SP-D oligomer (20-mer) containing the AP-1 sequence (TableI, Oligo1) showed binding to proteins in H441 nuclear extracts (Fig.2 A, lane 2). The major complex was specific as demonstrated by competition with unlabeled Oligo1 (Fig. 2 A, lane 3) or a commercial AP-1 oligomer (Promega pAP-1; Fig. 2 A, lane 4). There was no competition by a mutant oligomer with base substitutions at six of seven positions (Fig. 2 A, lane 5; Oligo1m, Table I) or an unrelated oligomer (AP-2; Fig.2 A, lane 6) with a similar base composition. Because surrounding sequences can influence the binding of specific AP-1 proteins (22Ryseck R.P. Bravo R. Oncogene. 1991; 6: 533-542PubMed Google Scholar), we also examined the interactions of H441 nuclear proteins with a commercial AP-1 consensus oligomer (pAP-1) (Fig.2 B). Binding was competed by Oligo1 or pAP-1 (Fig.2 B, lanes 3 and 4), but not by Oligo1m or AP-2 (Fig. 2 B, lanes 5 and 6). Binding was also competed with a smaller SP-D AP-1 oligomer (Oligo 2, Table I) (data not shown). Thus, the data demonstrate that the SP-D AP-1 motif can specifically bind to nuclear proteins expressed by H441 cells. Complexes of comparable mobility were also identified using nuclear extracts of an SP-D producing lung adenocarcinoma cell line (NCI-H969) (23Persson A. Moeller P. Am. J. Respir. Cell. Mol. Biol. 1996; 153 (abstr.): 109Google Scholar) and freshly isolated rat type II cells (data not shown). AP-1 complexes consist of homodimers of two jun family members (c-jun, junB, and junD), or heterodimers of one jun family member with c-fos or one of the fos-related proteins (fosB, fra-1, and fra-2) (24Ransone L.J. Verma I.M. Annu. Rev. Cell Biol. 1990; 6: 539-557Crossref PubMed Scopus (344) Google Scholar). Supershift assays using pan-fosand pan-jun antibodies and radiolabeled Oligo1 incubated with H441 nuclear extracts demonstrated binding of components immunologically related to jun and fos family members (Fig. 3, lanes 2 and6). In other experiments, stronger supershift bands were obtained with the pan-jun antibody (data not shown). Supershift assays with antibodies for specific fos andjun proteins demonstrated relatively strong and specific supershift bands with antibodies to junB, junD, and fra-1 (lanes 3, 5, and 9) and much weaker bands for c-jun, c-fos, orfra-2 (lanes 4, 7, and 10), and no detectable signal for fosB (lane 8). Thefra-1, junD, and junB supershift bands comigrated with the major bands detected using the pan-fosand pan-jun antibodies but had a reproducibly lower mobility than the faint complexes formed with antibodies to c-fos and c-jun. Consistent with the finding shown in Fig. 2, binding was blocked by competition with the commercial AP-1 oligomer or Oligo1 but not by Oligo1m (data not shown). Thus, the data indicate that selected AP-1 family members are binding to the SP-D sequence and suggest that the binding of fra-1,junD, and junB reflects the predominating species of AP-1 proteins in H441 cells rather than an intrinsic property of the AP-1-binding sequence. Because prior studies have describedfra-1 and junB but not junD in H441 cells (25Sawaya P.L. Stripp B.R. Whitsett J.A. Luse D.S. Mol. Cell. Biol. 1993; 13: 3860-3871Crossref PubMed Scopus (113) Google Scholar), we performed Northern hybridization assays to further confirm the presence of junD. Although there was a predominance of junB message, junD mRNA was also identified (data not shown). To study the potential functional consequences of AP-1 binding, we employed a transient transfection assay utilizing H441 lung adenocarcinoma cells in conjunction with wild type and mutant CAT reporter constructs. Mutagenesis of the conserved AP-1 motif in pXS (pXSm) resulted in a marked decrease in both basal and promoter activity (Fig.4 A). The reduction in normalized basal activity was 3.9 ± 0.9 in five separate experiments. Decreases in promoter activity were also observed when the mutation was examined within the context of two shorter restriction fragments, pPS and pFS (Fig. 1; data not shown). Dex increases the production of SP-D in lung tissue in vivoand in vitro, and these effects are mediated at the level of transcription (7Rust 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). However, the stimulation of pXS activity achieved with Dex is indirect (e.g. requires 24–48 h for maximal stimulation) and does not involve direct interactions of glucocorticoid receptor with classical response elements in the proximal promoter (7Rust 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). We therefore sought to determine whether mutation of the AP-1 could inhibit the 2–3-fold higher levels of promoter activity observed in the presence of glucocorticoids. As shown in Fig. 4 A, mutation of the AP-1 element (pXSm) markedly decreased promoter activity in the absence or presence of Dex. Although the residual activity of pXSm showed a detectable increase in the presence of Dex, the pSK vector control often showed a similar increase (Fig.4 A, compare pSK with pXSm). We occasionally observed a slight decrease in mobility and intensity of the complexes formed on Oligo1 in the presence of Dex; however, there were no major or reproducible effects on AP-1 binding (data not shown). Thus, the effects of Dex do not appear to be secondary to increased occupancy of the AP-1 site. To further examine the potential modulatory roles of specific AP-1 proteins we performed cotransfection studies using the selected CAT reporter constructs and jun and fosexpression constructs. The activities of pXS were increased 3–4-fold by cotransfection with junD cDNA in the absence or presence of Dex, and mutation of the AP-1 site markedly decreased basal and glucocorticoid stimulated expression (Fig. 4 B). The effects of junD and Dex appeared additive rather than synergistic. Cotransfection with junD did not alter the activity of a pMSG-CAT control plasmid, which lacks a known AP-1 site, consistent with specific stimulation. Slightly lower specific stimulation was observed with fra-1 cotransfection, but inhibition was reproducibly observed with c-fos or c-jun (data not shown). Plasmids containing the mutated AP-1 showed some residual modulation by co-transfected AP-1 proteins under conditions where there was no significant alteration in the activity of control plasmids (Fig.4 B and data not shown). However, the only other sequence in the proximal promoter with significant homology to an AP-1 consensus (−215, TGAGTTCA) (7Rust 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) did not bind AP-1 proteins in supershift assays using either pan-jun or pan-fos antibodies (data not shown), suggesting that the residual modulatory effects are secondary to interactions of AP-1 proteins with components of the transcriptional initiation complex (26Martin M.L. Lieberman P.M. Curran T. Mol. Cell. Biol. 1996; 16: 2110-2118Crossref PubMed Google Scholar) or other potential regulatory factors such as C/EBPβ (27Klampfer L. Lee T.H. Hsu W. Vilcek J. Chen-Kiang S. Mol. Cell. Biol. 1994; 14: 6561-6569Crossref PubMed Scopu" @default.
• W2022124461 created "2016-06-24" @default.
• W2022124461 creator A5036335582 @default.
• W2022124461 creator A5039056574 @default.
• W2022124461 creator A5041204313 @default.
• W2022124461 creator A5077664115 @default.
• W2022124461 creator A5088462075 @default.
• W2022124461 date "2000-10-01" @default.
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• W2022124461 title "Proximal Promoter of the Surfactant Protein D Gene" @default.
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