Surfactant protein D gene regulation. Interactions among the conserved CCAAT/enhancer-binding protein elements.

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/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta 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 approximately 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) 1 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 (1,2). 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 nonciliated 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 (3). 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 (4).
The expression of SP-D is increased following many forms of pulmonary injury (5). 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 (6), following challenge of mice with Pseudomonas aeruginosa (7), and in rats exposed to hyperoxia (8). In addition, transgenic mice deficient in surfactant protein A or SP-D show abnormal microbial clearance or acute inflammatory responses to microbial challenge (9). Based on these and other data, it has been suggested that the lung collectins contribute to a pulmonary acute-phase response, somewhat analogous to the hepatic acute-phase response (APR) to systemic injury (6). In this regard, a variety of hepatic acute-phase proteins are expressed in the lung, and at least some are elevated following lung injury (10 -14). 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 (15)(16)(17)(18). 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 (19). 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/EBP␣, C/EBP␤, and C/EBP␦ are expressed by alveolar type II and non-ciliated bronchiolar epithelial cells, the known pulmonary sites of SP-D production (20 -23). C/EBP␣ and C/EBP␦ are particularly abundant in the lung and increase in fetal rat lung in late gestation during a time when the produc-tion of surfactant-associated proteins, including SP-D, is increased. Mice deficient in C/EBP␣ show abnormalities in alveolar development and often die secondary to the respiratory abnormalities (24). Although mice deficient in C/EBP␤ and C/EBP␦ also expire perinatally, they have no obvious pulmonary phenotype (15). Neonatal C/EBP␣-null mice show no hepatic APR and fail to induce STAT3 binding in response to systemically administered endotoxin, despite marked increases in C/EBP␤ or C/EBP␦ (25). However, the pulmonary APR to systemically administered endotoxin is not impaired in this model, and the levels of C/EBP␤ and C/EBP␦ are increased in the lung following systemic endotoxin administration (25,26).
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 (27). 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 wildtype 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.

EXPERIMENTAL PROCEDURES
Genomic Clones and Reporter Constructs-An ϳ7-kb EcoRI fragment of the previously described human genomic clone (H5), designated H5E7, containing human SP-D 5Ј-sequence was isolated and subcloned into pGEM-3Z (Promega) as previously described (4,27). All experiments used restriction fragments containing 5Ј-regulatory sequence; each terminated at a SacI site within the untranslated first exon and were numbered from the transcription start site (27). 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 HindIII/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.
Cells-NCI H441 human lung adenocarcinoma cells were propagated as previously described (4,27). In preliminary experiments performed in the late 1990s, we used cells provided by Dr. A. Gazdar (27). 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/EBP␣, C/EBP␤, and C/EBP␦ 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.
Oligomers-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 (28). The oligomers and their reverse complements were annealed and used in electrophoretic mobility shift assays as described below.
Mutagenesis by Overlap Extension-SS698 was subcloned into pGEM-3Z and used for thermal cycling-coupled mutagenesis. Forwardand 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 polymer-ase site in pGEM were combined with 200 M 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 gelpurified using the QIAQuick gel extraction kit (QIAGEN Inc.). The 5Јand 3Ј-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 and Electrophoretic Mobility Shift Assays-Nuclear extracts were prepared from cultured cell lines using a rapid miniextraction technique (29) as previously described (4). The protein content was analyzed by dye binding assay, and the extracts were frozen in liquid nitrogen and stored at Ϫ70°C. Electrophoretic mobility shift (gel retardation) and supershift assays were performed by a modification of a method employed by Bingle and co-workers (30) as previously described (4). Antibodies to C/EBP␣ (14AA), C/EBP␤ (C- 19), and C/EBP␦ (M-17) were from Santa Cruz Biotechnology. Specificity of each antibody was confirmed in supershift assays using nuclear extracts from cells transfected with C/EBP␣, C/EBP␤, or C/EBP␦ cDNA as described below.
Transient Transfection-For experiments characterizing the promoter activity of mutant constructs, H441 target cells (5 ϫ 10 5 ) 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 (4). The cells were transfected with up to 1.5 g 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.
Transfection with Nuclear Factor Expression Vectors-C/EBP␤ cDNA was obtained by thermal cycling gene amplification of a lung cDNA library (CLONTECH) using a pair of primers: one located at the 5Ј-end and the other at the 3Ј-end according to the known human C/EBP␤ (NF-IL6) sequence. The full-length DNA sequence was confirmed by automated sequencing. The rat C/EBP␣ cDNA was a gift from Dr. Steven McKnight (University of Texas Southwestern Medical Center, Dallas, TX), and the rat C/EBP␦ 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 HindIII 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/EBP␣ was dose-dependent up to 1.5 g, activation by C/EBP␤ decreased at concentrations above 1.5 g, whereas activation by C/EBP␦ reached a plateau above 0.25 mg. Accordingly, most transfections were performed using 1.5 g of pcDNA3 containing the desired cDNA or an equivalent weight of the pcDNA3 vector. Protein expression was confirmed by supershift assays.
Luciferase and Chloramphenicol Acetyltransferase 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 (4).
Thermal Cycling Assays of Endogenous SP-D mRNA-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: 5Ј-primer (ϩ), CCT GCC ATG CTG CTC TTC CTC CTC TCT GC; and 3Ј-primer (Ϫ), CCA GTT GGC TCA GAA CTC GCA GAC CA. Five g 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 5Ј-primer (ϩ), AAT CCT GGA GAC AAA GGA GCA AAG GGA GAA. For this reaction, 1 l 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 3Ј-sense primer ϳ300 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/g of RNA as assessed by incorporation of [␣-32 P]dATP during the reverse transcription.

RESULTS
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 ϭ G/T; D ϭ 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 Ϫ432, Ϫ340, Ϫ319, Ϫ140, and Ϫ90 (Figs. 1 and 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 (4). 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 (27,31) 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 (32)(33)(34). A complete C/EBP motif nearly identical to the human sequence at Ϫ432 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 Ϫ340 to Ϫ319 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) (27,31). In addition, these tandem motifs are conserved in the conglutinin and CL-43 promoters (Fig. 2) (32, 34). The C/EBP motif at Ϫ140 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.
C/EBP Isoforms Bind to the C/EBP Motifs-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/EBP␣, C/EBP␤, or C/EBP␦ 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. [3][4][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.
The sites at Ϫ340 and Ϫ319 also showed specific binding to all three C/EBP isoforms. For example, C/EBP␦ showed specific binding to Oligo 340 (Fig. 4A, lane 2) that was competed by the  Table I). The complex was specifically supershifted by antibody to C/EBP␦ (lane 5). The complex was also competed by the NF-IL6 oligomer, but not by the mutated NF-IL6 sequence (Table I; data not shown). Likewise, C/EBP␦ showed specific binding to Oligo 319 (Fig. 4B, lane 2) that was competed by the unlabeled oligomer (lane 3) or by the consensus oligomer (lane 5), but not by the mutant oligomer (lane 4) (Table I). In addition, the complex was specifically supershifted by antibody to C/EBP␦ (lane 6), but not by control IgG (lane 7). Comparable results were obtained in transfection assays using C/EBP␤ or C/EBP␣ cDNA (data not shown).
Similarly, the sites at Ϫ140 and Ϫ90 showed specific binding to C/EBP␣, C/EBP␤, and C/EBP␦. Representative binding data for Oligo 140 and Oligo 90 (Table I) and C/EBP␦ are shown in Fig. 5. Briefly, both oligomers specifically bound to C/EBP␦ (Fig. 5, A and B, lanes 1). Complex formation was blocked by the unlabeled oligomers (lanes 2), but not by the corresponding mutant oligomers (lanes 3). The complexes were also supershifted by antibody to C/EBP␦ (lanes 4), but not by control IgG (data not shown). Comparable results were obtained in transfection assays using C/EBP␤ or C/EBP␣ cDNA (data not shown). Thus, all five motifs are able to bind to the three C/EBP isoforms.
The relative affinities of the sites for transfected C/EBP were compared in parallel gel shift competition assays. Nuclear extracts from cells transfected with C/EBP␣, C/EBP␤, or C/EBP␦ were individually incubated with a radiolabeled commercial C/EBP consensus oligomer (Table I) in the presence of increasing concentrations of unlabeled SP-D oligomers encoding the five binding sites. As shown in Fig. 6, oligomers containing the sites at Ϫ432, Ϫ340, Ϫ319, and Ϫ90 efficiently inhibited the binding of all three C/EBPs to the consensus oligomer. The site at -432 showed the highest apparent affinity, followed by the sites at Ϫ319, Ϫ340, and Ϫ90. By contrast, the site at Ϫ140 was quite ineffective as a competitor. Oligomers encoding the near-distal motifs were particularly effective competitors of C/EBP␤ binding.
Cotransfection of C/EBPs Modulates SP-D Promoter Activity-To further examine the potential modulatory roles of specific C/EBPs, we performed cotransfection studies using luciferase reporter constructs and C/EBP expression constructs. For most of our initial experiments, we used an expression plasmid encoding a human lung C/EBP␤ (NF-IL6) cDNA, which we had isolated and characterized (see "Experimental Procedures"). As shown in Fig. 7, cotransfection of pGL3-SS698 with C/EBP␤ cDNA gave a 4-fold stimulation of promoter activity (n ϭ nine independent experiments). In a smaller number of experiments, cotransfection of pGL3-SS698 with plasmids encoding C/EBP␣ or C/EBP␦ cDNA gave a maximal 5.5fold (n ϭ 4) and 4.7-fold (n ϭ 3) stimulation of transactivation, respectively. Thus, there was no major difference in the level of activation when the three isoforms were compared using optimal amounts of plasmid as determined in dose-response assays. Comparable transactivation was obtained when a larger segment of DNA containing over 1.6 kilobase pairs of upstream sequence (HS1674) was used (Fig. 7). By contrast, cotransfection of the cDNA with the truncated construct (SS205), which lacks the three upstream motifs, showed no significant transactivation (Fig. 7). Accordingly, subsequent studies focused on the sequences in the near-distal promoter.
Additional experiments were performed to determine whether transfection of C/EBP cDNA can modulate endogenous SP-D gene expression. We have previously observed weak FIG. 3. C/EBPs bind to the motif at ؊432. H441 cells were transfected with C/EBP␣, C/EBP␤, or C/EBP␦ cDNA as indicated. Binding of nuclear proteins to a radiolabeled oligomer containing the SP-D C/EBP sequence at Ϫ432 (Oligo 432; Table I) was assessed in electrophoretic mobility shift assays as described under "Experimental Procedures." Binding was competed by the unlabeled oligomer (Oligo 432; lanes 3, 7, and 11) and by the consensus oligomer (lane 13), but not by the corresponding mutant oligomer (Oligo 432m; lanes 8 and 12). The complexes were supershifted by specific antibodies to C/EBP␣, C/EBP␤, and C/EBP␦ (lanes 4, 9, and 14, respectively). A representative negative control using normal IgG (nl IgG) is shown in lane 15.

FIG. 4. C/EBPs bind to the motifs at -340 and ؊319
. H441 cells were transfected with C/EBP␦ cDNA, and binding of nuclear proteins was assessed using gel retardation assays. Comparable results were obtained when H441 cells were transfected with C/EBP␣ or C/EBP␤ cDNA. A, binding of C/EBP␦ to a radiolabeled oligomer containing the SP-D C/EBP sequence at Ϫ340 (Oligo 340; Table I). The presence of transfected C/EBP␦ 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/EBP␦ is shown in lane 5. B, binding of C/EBP␦ to a radiolabeled oligomer containing the SP-D C/EBP sequence at Ϫ319 (Oligo 319; Table I) signals for human SP-D mRNA by Northern blotting of H441 RNA following stimulation of the cells with glucocorticoids (27). In the present study, we employed a more sensitive thermal cycling assay to assess possible changes in SP-D message in response to increased levels of C/EBP. The full-length H441 message was amplified and cloned, with verification of its identity by DNA sequencing as described under "Experimental Procedures." The SP-D PCR product was increased following transfection with C/EBP␤ cDNA (Fig. 8A). Virtually identical results were obtained in two different experiments using different cultures and plasmid preparations. Consistent with previous data, expression was also increased when cells were incubated in the presence of 50 nM dexamethasone (Fig. 8A,  Dex). In a semiquantitative assay, SP-D message levels were increased by at least 2.5-fold in response to C/EBP␤ as compared with the mock-transfected controls (data not shown). There was no detectable change in the level of GAPDH message (Fig. 8B).
The AP-1 Element at -109 Is Not Required for Transactivation-Our previous studies have shown that the conserved AP-1 element at Ϫ109 is important for basal or AP-1-stimulated expression (4). Site-directed mutagenesis of the AP-1 consensus sequence in the context of SS698 decreased C/EBPstimulated promoter activity by ϳ75% as compared with wildtype SS698 (0.27 Ϯ 0.06 (mean Ϯ S.D.), n ϭ four independent experiments). As a result, the ϳ4-fold stimulation of SS698 activity by C/EBP cDNA was largely abrogated. However, basal or unstimulated activity was decreased, and cotransfected C/EBP cDNA increased the low residual luciferase activity of the mutant reporter construct by ϳ4-fold (4.3 Ϯ 0.99 (mean Ϯ S.D.), n ϭ four independent experiments).

Mutagenesis of the Upstream C/EBP-binding Sites Decreases C/EBP-stimulated Promoter
Activity-To study the potential functional consequences of C/EBP binding, we employed a transient transfection assay utilizing H441 cells in conjunction with wild-type SP-D and mutant SP-D luciferase reporter constructs. Substitution mutations were identical to those shown for the mutant oligomers (Table I). Representative data using a cotransfected cDNA encoding C/EBP␤ are shown in Fig. 9. Mutagenesis of the C/EBP elements at Ϫ432, Ϫ340, and Ϫ319 significantly decreased the level of stimulation achieved following cotransfection of C/EBP␣, C/EBP␤, or C/EBP␦ cDNA. Individual mutations at Ϫ432, Ϫ340, and Ϫ319 decreased activity by ϳ50% as compared with the wild type (Fig. 7) based on at least six independent experiments.
Because mutagenesis of the individual sites gave similar but incomplete inhibition of C/EBP-stimulated promoter activity, we examined the effects of simultaneously mutating pairs of sites in the near-distal promoter (Fig. 9). Simultaneous mutation of the tandem sites at Ϫ340 and Ϫ319 did not significantly increase the extent of inhibition as compared with the singlesite mutations. Activity was decreased by ϳ50% based on four independent experiments. However, mutagenesis of the upstream site at Ϫ432 in combination with either one of the tandem sites reduced promoter activity to the level of the plasmid controls (Fig. 9).
C/EBP Binding to the Region Spanning -340 to -319 -To further characterize the tandem binding sites in the near-distal promoter, we generated an oligomer spanning both sites (Oligo 340/319) (Table I). This was in part motivated by the high degree of conservation of the sequence between the two binding sites (Fig. 2), which suggested that site-specific mutagenesis of the C/EBP sites might alter the binding of other transcription factors to this region. Gel shift experiments using radiolabeled wild-type Oligo 340/319 and nuclear extracts from cells transfected with C/EBP␣ showed a single specific band (Fig. 10, lane  10). In preliminary experiments, we observed that this complex was competed by unlabeled probe or oligomers containing the site at Ϫ340 or Ϫ319 and supershifted by antibody to C/EBP␣ (data not shown). In addition, no specific complexes were identified in the absence of cotransfected cDNA.
We then compared the wild-type complex with complexes formed using oligomers containing mutated sites at Ϫ340 or Ϫ319, designated Oligo 340m/319 and Oligo 340/319m, respectively ( Fig. 10 and Table I). Both mutant oligomers gave a single major complex that comigrated with the complex formed on the wild-type tandem oligomer (Fig. 10, compare lanes 1 and  7 with lane 10). These complexes, which were comparable in intensity to the wild-type complex, were competed by the un-  6 and 9). The complexes formed with the mutant oligomers were also competed by oligomers containing the individual sites or by tandem oligomers with single mutated sites. For example, the complex formed with Oligo 340m/319 (lane 1) was competed by Oligo 340, Oligo 319, and Oligo 340/319m (lanes 3-5). The findings suggest that the two sites are not simultaneously occupied within the context of the tandem oligomer and that other nuclear proteins do not interact with the intervening sequence. DISCUSSION We have previously shown that the proximal promoter of SP-D can mediate cell type-restricted, basal and glucocorticoidstimulated promoter activities (4,27). We further demonstrated that specific conserved sequences within this region interact with ubiquitous and lineage-dependent (but not lungspecific) transcription factors that are required for SP-D promoter activity in H441 cells. In particular, this region contains a functional AP-1 element at Ϫ109 and two interacting HNF-3-binding sites. In the present study, we identified five binding sites for three members of the C/EBP family that are expressed at varying levels by type II pneumocytes and Clara cells in normal or injured lung. C/EBP-mediated activation of AP-1-dependent promoter activity required interactions among the three C/EBP elements in the near-distal promoter.
Our current experiments demonstrate that the C/EBP motifs at Ϫ432 and the tandem motifs at Ϫ340 and Ϫ319 are functional C/EBP-binding sites capable of mediating C/EBP-dependent transactivation by C/EBP␣, C/EBP␤, or C/EBP␦ cDNA. Our data further suggest that the tandem sites in the human SP-D promoter, which are separated by 13 bp, constitute a compound response element that requires the integrity of both sites for function of the element. Mutagenesis of any one of the three sites (at Ϫ432, Ϫ340, and Ϫ319) inhibited (but did not block) activity, and mutagenesis of either tandem site or simultaneous mutation of both sites in the tandem sequence gave a comparable reduction in promoter activity (Fig. 9). By contrast, mutagenesis of the element at Ϫ432 in conjunction with either of the two downstream sites prevented transacti-vation of SS698 by cotransfected C/EBP cDNAs.
The truncated construct (SS205) showed no transactivation by cotransfected C/EBP cDNA, consistent with our ability to block C/EBP-dependent activation following mutagenesis of the site at Ϫ432 in combination with the site at Ϫ340 or Ϫ319. Thus, the binding sites at Ϫ140 and Ϫ90 are not sufficient to mediate significant C/EBP-dependent transactivation in H441 cells. In this regard, the site at Ϫ140 only very weakly competed the commercial consensus oligomer despite specific binding in gel shift assays. This appears to be consistent with the observed divergence of the motif from the consensus sequence (TTcTGGAA versus TKDNGNAA, where D ϭ A/G/T). Although oligomers containing the site at Ϫ90 showed high affinity binding, this site is not conserved in the rat or mouse.
Maximal stimulation of promoter activity by C/EBPs was dependent on the presence of a functional AP-1 element in the proximal promoter. Mutation of the conserved AP-1 element at Ϫ109 markedly decreased the stimulatory effect of C/EBPs on SS698 promoter activity. However, unstimulated promoter activity was decreased, and the low level of residual activity observed for the mutant AP-1 construct still showed stimulation by C/EBPs. Although some modulation of AP-1 activity by C/EBPs cannot yet be excluded, transactivation by C/EBPs does not require the AP-1 element.
Multiple C/EBP elements are present in many promoters, and tandem C/EBP-binding sites (referred to as contiguous, adjacent, or sequential) have been identified in the promoters of some APR proteins such as human C-reactive protein (35) and TSG-6 (tumor necrosis factor-inducible gene-6) (36), in C/EBP promoters (37), and in the promoter of the rat Clara cell-specific protein gene (38). The functional consequences of multiple C/EBP elements in such genes are quite varied, ranging from positive cooperativity to antagonism. In the case of C-reactive protein and TSG-6, it was shown that the two sequential sequences could be simultaneously occupied and that the inducibility of the intact promoter depends on a cooperative interaction between the two elements (35). This interaction was preserved when the elements were reduced from 13 to 8 base pairs. For Clara cell-specific protein, the two sites, which have 9 intervening base pairs, were required for transactivation by C/EBP␣ and/or C/EBP␦ cDNA. Although mutagenesis of both sites blocked transactivation, mutagenesis of the high affinity distal site markedly decreased activity, whereas mu-FIG. 5. C/EBPs bind to the motifs at ؊140 and ؊90. H441 cells were transfected with C/EBP␦ cDNA, and binding of nuclear proteins was assessed using gel retardation assays. Comparable results were obtained when H441 cells were transfected with C/EBP␣ or C/EBP␤ cDNA. A, binding of C/EBP␦ to a radiolabeled oligomer containing the SP-D C/EBP sequence at Ϫ140 (Oligo 140; Table I The resulting complexes were resolved in gel shift assays as described under "Experimental Procedures." The data for each C/EBP isoform were derived from two gels: one with the near-distal sites at Ϫ432, Ϫ340, and Ϫ319 and the other with the two proximal sites at Ϫ140 and Ϫ90. All the motifs, except the site at Ϫ140, were effective competitors of C/EBP binding. tagenesis of the low affinity proximal site gave a smaller decrease. Mutagenesis of the distal site also decreased binding to the proximal site. In the case of SP-D, mutagenesis of either site did not decrease binding to the other, and mutagenesis of one or both sites comparably decreased activation. In addition, the compound element interacts with an upstream site at -432 to effect transactivation. To our knowledge, this is the first demonstration of such a tandem element interacting with other, more distant C/EBP elements. Because only a single site was occupied on Oligo 340/319 under our assay conditions (Fig. 10), it is possible that simultaneous occupancy of both sites requires interactions with more remote cis-acting elements or that the two sites differentially interact with specific C/EBP homo-or heterodimers or modified forms of these proteins.
Our previous studies using the H441 model and chloramphenicol acetyltransferase reporter constructs demonstrated that SS698 is more active than shorter constructs (e.g. XS285) (27). We initially hypothesized that the multiple C/EBP elements contributed to the positive regulatory activity of this region. However, we observed significant levels of SP-D promoter activity in H441 cells, despite low levels of endogenous C/EBP. Furthermore, substitution mutagenesis of the C/EBP motifs in the near-distal region showed no more than a 20% decrease in promoter activity in the absence of exogenous C/EBP (data not shown). Thus, C/EBP-dependent transactivation does not play a major role in regulating basal expression in this system.
Based on our in vitro findings, we speculate that alterations in the levels or activity of C/EBP isoforms contribute to increased expression of SP-D by alveolar and bronchiolar epithelial cells in the setting of lung development or lung injury. As indicated in the Introduction, increases in SP-D message have been demonstrated in animal models of lung injury or infection, and such injuries are often associated with increases in the expression of C/EBP isoforms at known sites of SP-D expression. Although the three isoforms gave similar activation when expressed at levels sufficient to achieve their maximal stimulation, the near-distal sites appeared to preferentially compete for the binding of C/EBP␤ to the consensus oligomer, and  1 and 2). Parallel cultures were treated with 50 nM dexamethasone (Dex; lanes 5 and 6). The endogenous SP-D and GAPDH mRNAs in the H441 cells were then amplified by thermal cycling as described under "Experimental Procedures." Products were resolved on agarose gels and compared with DNA standards (Promega). A representative experiment is shown. A, RNAs from mock-transfected cells, C/EBP␤transfected cells, and cells treated with 50 nM dexamethasone were amplified using full-length human SP-D primers and resolved on a 0.8% agarose gel (lanes 1, 3, and 5). The major 1.3-kb products, which migrated at the expected positions, were reamplified using a nested human SP-D primer pair to further confirm the identity of the product. As predicted, an ϳ400-bp nested fragment was generated (lanes 2, 4, and 6). Cloning and DNA sequencing definitively established the identity of the full-length PCR product as SP-D. B, as a control for the comparative PCR assays, the levels of GAPDH mRNA were assessed using short or long primers as provided by the manufacturer. Reaction products using RNA from mock-transfected cells (lane 7) are compared with those using RNA from cells transfected with C/EBP␤ cDNA (lane 8).
FIG. 9. Mutagenesis of the near-distal C/EBP-binding sites decreases transactivation by C/EBP. H441 cells were cotransfected with mutant SS698 reporter constructs and 1.5 g of pcDNA3-C/EBP␤ or pcDNA3. Luciferase assays were performed as described in the legend to Fig. 7. The SS698 mutants contained mutated single sites (Ϫ432, Ϫ340, and Ϫ319) or combinations of the three sites (Ϫ340/Ϫ319, Ϫ432/Ϫ340, and Ϫ432/Ϫ319) as indicated. The mutations are the same as used for the mutant oligomers in Table I  Ϫ432 (n ϭ 6), Ϫ340 (n ϭ 7), Ϫ319 (n ϭ 6), Ϫ340/Ϫ319 (n ϭ 4), Ϫ432/Ϫ340 (n ϭ 3), and Ϫ432/Ϫ319 (n ϭ 5). The extent of activation can be directly compared with wild-type SS698, as shown in Fig. 7, which gave 4-fold activation relative to the control plasmid (n ϭ 9) (dashed line).  Table I) or tandem oligomers containing mutated sites (Oligo 340m/319 and Oligo 340/319m; Table I) was assessed using electrophoretic mobility shift (lanes 1-5, 7, 8, and 10) and supershift (lanes 6 and 9) assays. The labeled oligomers, which were identical in length and synthesized with equivalent specific activities, yielded major complexes of comparable size and intensity. The specificity of binding to Oligo 340/319 (lane 10) was confirmed in preliminary gel shift experiments as discussed under "Results." All complexes were efficiently competed by the C/EBP sites and supershifted by antibody to C/EBP␣. considerably lower amounts of C/EBP␦ were required for maximal transactivation. Thus, SP-D expression may be particularly sensitive to alterations in the levels of active C/EBP␤ and/or C/EBP␦, which may be preferentially increased with injury or inflammation. In this regard, hyperoxia in rats increases the expression of SP-D by alveolar and bronchiolar epithelial cells (8) and increases pulmonary C/EBP␤ and C/EBP␦ expression and binding activity (39). Likewise, intratracheal instillation of lipopolysaccharide in rats increases SP-D expression (6), and increases in both forms accompany systemic administration of lipopolysaccharide (26). Additional studies are needed to determine whether heterodimeric species, truncated forms, or post-translational modifications can differentially regulate promoter activity.
Transfection studies using cotransfected expression vectors have limitations. However, there is other evidence suggesting the physiological relevance of the current findings. In a recent study, Charles et al. (40) found that transfection of MLE-15 cells with pCMV-Rb increased the activity of the SS698 reporter construct by ϳ5-fold. Rb bound to the three endogenous C/EBP isoforms, with increased binding of Rb⅐C/EBP complexes to an oligomer containing the site at Ϫ340, which was the only site identified in our earlier studies. Deletion mutagenesis of the 9-bp C/EBP motif at Ϫ340 significantly decreased (but did not prevent) the activating effects of Rb while minimally decreasing unstimulated promoter activity. Consistent with our finding with C/EBP, a proximal promoter fragment containing the downstream sites (i.e. FS167) was insufficient to mediate activation by Rb. Thus, at least one of the near-distal sites can interact with endogenous C/EBP isoforms with activation of the promoter. Site-directed mutagenesis of the individual sites is needed to determine their specific roles in activation by Rb.
The observed 4 -5-fold maximal increase in SP-D promoter activity is consistent with the increase in endogenous SP-D mRNA in response to C/EBP. It is also consistent with the magnitude of alterations in lung message and protein levels observed following lung injury in vivo. For example, lipopolysaccharide instillation causes a 50% increase in SP-D message and a 2-fold increase in secreted protein at 24 h, with a 6-fold increase in lavage SP-D at 72 h (6). In addition, SP-D levels in bronchoalveolar lavage increase by a maximum of 3-fold within 3 days following challenge of mice with influenza A virus (41). Because the half-life of SP-D in the airspace is relatively long (42), a severalfold increase in epithelial gene expression might lead to a severalfold increase in secreted protein within this time frame. Notably, even a 2-fold alteration in SP-D concentration within the estimated physiological range can markedly alter the extent of inhibition of viral infectivity of epithelial cells in vitro (43). C/EBP activation and accumulation are often slower (but more prolonged) than the rapid transient activation of other APR modulators such as STAT3 and NFB (15). Thus, the time course for increased SP-D accumulation following lung injury is consistent with a potential role of newly synthesized C/EBPs in this response. Although the importance of C/EBPs in early development and the functional overlap of various isoforms limit transgenic models of C/EBP deficiency, transgenic studies using wild-type and mutant SP-D promoter reporter constructs could help define the contributions of C/EBP family members to increased SP-D expression in late gestation and in the setting of lung injury.