(Received for publication, December 2, 1994; and in revised form, January 13, 1995)
From the
Cis-acting elements determining lung epithelial cell-selective transcription of the murine surfactant protein A (SP-A) gene were identified between nucleotide positions -255 and -57. This region of the murine SP-A gene contained nucleotide sequences consistent with thyroid transcription factor-1 (TTF-1) binding motifs. An SP-A-CAT plasmid containing the TTF-1 binding sites was transcriptionally active in mouse lung epithelial (MLE-15) cells but not in HeLa, 3T3, or H441 cells. However, transcription of the SP-A-CAT construct was activated after cotransfection of HeLa cells with a vector expressing recombinant TTF-1, pCMV-TTF-1. Recombinant TTF-1 homeodomain protein bound to four distinct binding sites located between nucleotides -231 to -168. Proteins in nuclear extracts of MLE-15 cells bound TTF-1 binding sites and were supershifted by TTF-1 antibody. Mutations of three of the TTF-1 binding sites in this region reduced expression of the SP-A-CAT construct in transfected MLE-15 cells and reduced transactivation in HeLa cells. TTF-1 interacts with complex protein/DNA binding sites located in the 5`-flanking region of the murine SP-A gene enhancing lung epithelial cell-specific expression in vitro.
Surfactant protein A (SP-A) ()is an abundant,
32-36-kDa glycoprotein secreted by respiratory epithelial cells
in the lung. SP-A plays an important role in the aggregation, function,
and regulation of pulmonary surfactant and enhances opsonization and
macrophage killing (see (1) and (2) for review).
SP-A gene expression is subject to cell-specific, developmental, and
humoral influences, being expressed by subsets of cells in the human
tracheal-bronchial glands and in the bronchiolar and alveolar
epithelium(3, 4) . SP-A mRNA increases with advancing
gestation and is up-regulated by cyclic AMP, glucocorticoids,
-interferon, and epidermal growth factor (1, 5, 6) and down-regulated by phorbol
esters (7) , tumor necrosis factor-
(8) , and
transforming growth factor-
(9) . Inhibitory and
stimulatory effects are mediated by changes in gene transcription and
mRNA stability(10) . Transcriptional activity of the murine
SP-A gene was previously demonstrated in pulmonary adenocarcinoma cells in vitro(11) . The 5`-flanking sequences of the rat
SP-A gene were transcriptionally active in nuclear extracts from
pulmonary tissues in vitro, supporting the concept that these
sequences contain cell-selective cis-active elements (12) .
DNA-protein interactions in the 5`-region of the rabbit SP-A gene were
demonstrated by DNase hypersensitivity and electrophoretic mobility
shift assays(13) . Two E-box-like motifs, binding unique lung
nuclear proteins, were identified in the rabbit SP-A gene promoter (13) . These motifs required a complex interaction with cAMP to
enhance lung cell-specific expression of SP-A in
vitro(13, 14) .
Recently, Bohinski et al.(15) demonstrated that thyroid transcription factor-1 (TTF-1) bound to and activated DNA binding sites located in the 5`-flanking region of the human surfactant protein B gene. Cotransfection of the TTF-1 cDNA and the SP-B gene promoter was sufficient to transactivate gene expression in non-pulmonary cells. In the present study, lung cell-selective transcription of the murine SP-A gene was mediated by a complex of TTF-1 binding sites located between nucleotide positions -231 and -168 from the start of transcription.
Figure 1: Transfection analysis of SP-A sequences. Culture and transfection of H441, MLE-15, HeLa, and 3T3 cells were described under ``Materials and Methods.'' To the left, the 5`-flanking region and portion of exon 1 of the mouse SP-A gene are depicted. Potential binding sites for TTF-1 or hepatocyte nuclear factor-5 are depicted above the line. Nucleotide positions are depicted below the line. cat indicates the position of the chloramphenicol acetyltransferase gene. To the right of each clone, CAT activity is plotted relative to the promoterless plasmid, pCPA-0. The transfection data are representative of at least five separate transfections for MLE-15 and 3T3 and two experiments for HeLa and H441. Presented data were calculated from two experiments with triplicate samples for each construct (n = 6). Values represent mean ± standard error. Values of pCPA 1.4 and pCPA 0.3 in HeLa or H441-4 cells were less than for pCPA-0 and therefore are not distinguished in the bar graph.
Figure 2: Transactivation of SP-A sequences by TTF-1 in HeLa cells. Cell culture and transfection were described under ``Materials and Methods.'' CAT activity is plotted relative to the activity of the promoterless plasmid. Activity was assessed with and without cotransfection with pCMV-TTF-1. CAT activity from pCPA-0.1 or pCPA-0 was not appreciably altered by cotransfection with pCMV-TTF-1. The transfection data are representative of four separate transfections. Presented data were calculated from two experiments with triplicate samples for each construct (n = 6). Value represents mean ± standard error. Absence of an errorbar means that the standard error was too small to be indicated on the graph. The standard error was not greater than ±20% in those lanes.
Figure 3: Sequences of oligomer probes. DNA probes from the SP-A 5`-flanking region were synthesized as described under ``Materials and Methods.'' Corresponding nucleotide positions of the SP-A 5`-flanking region are listed with the top sequence (probeA). Position of the TTF-1 binding motifs are underlined and numbered1, 2, 3, or 4.
Figure 4: EMSA of SP-A gene probes with TTF-1 homeodomain. Sequence of DNA probes are listed in Fig. 3. LettersA-D at the top of the figure indicate the probe used in each lane. Probe means the presence (+) of the labeled oligomer in each lane. TTF-1 is the presence (+) or absence(-) of TTF-1 homeodomain. With probeA, four bands were detected; two were detected with probeB, and one each was detected with probesC and D. The slowest migrating band for probeA is faint in this exposure, so its position is marked with an arrow. Freeprobe is marked with an arrowhead.
Figure 5:
EMSA of SP-A gene probes with MLE-15
nuclear extract proteins. Oligomer synthesis, preparation of MLE-15
nuclear extracts, and EMSA are described under ``Materials and
Methods.'' Sequences of DNA probes are listed in Fig. 3. LettersB-E at the top of the figure
indicate the probe used in each lane. Probe means the presence
(+) of labeled oligomer in each lane. MLE-15 means the
presence (+) of nuclear extracts; -TTF-1 means the presence
(+) or absence(-) of TTF-1 antibody. Position of major bands
are marked with arrowheads, and the supershifted band is
marked with an arrow. Exposures are 1 h at -80 °C
for B, 18 h at room temperature for C, 30 min at
-80 °C for D, and 24 h at room temperature for E.
Figure 6: Transfection analysis of TTF-1 binding site mutations. Cell culture, transfection, and plasmid construction were described under ``Materials and Methods.'' PanelA is a schematic representation of the TTF-1 sites with mutated sequences indicated with asterisks. PanelB is transfection analysis of MLE-15 cells, and relative CAT activity is presented relative to the activity of the promoterless pCPA-0 plasmid. The transfection data are representative of four separate transfections. Presented data were calculated from two experiments with triplicate samples for each construct (n = 6). Value represents mean ± standard error. PanelC is an autoradiogram of representative CAT assays of MLE-15 cells. Each construct is presented in duplicate. PanelD is transactivation with TTF-1 in HeLa cells. The transfection data are representative of two separate transfections. Relative CAT activity is presented relative to the activity of the promoterless pCPA-0 plasmid. Presented data were calculated from both experiments with triplicate samples for each construct (n = 6). Value represents mean ± standard error. PanelE is an autoradiogram of representative CAT assays of HeLa cells. Each construct is presented in duplicate. Absence of errorbars means that the standard error was too low to be represented in the graph. Standard error did not exceed ±20% in those lanes.
Transcriptional activity of flanking sequences of the murine SP-A gene was activated by recombinant TTF-1 through interactions with complex TTF-1 binding sites located within nucleotide positions -231 to -168. Mutations of TTF-1 motifs reduced cell-specific expression in transfected MLE-15 cells, an SV40 large TAg immortalized pulmonary adenocarcinoma cell that expresses murine SP-A, and reduced transactivation of SP-A sequences by TTF-1 in HeLa cells. TTF-1 enhances lung cell-specific expression of the SP-A gene by interactions with distinct TTF-1 binding sites.
TTF-1, a 38-kDa
nuclear transcription protein, was initially identified as an important
regulator of thyroid-specific gene expression and contains a highly
conserved homeobox domain capable of binding to regulatory regions of
target genes(19) . TTF-1 activated thyroglobulin and
thyroperoxidase gene transcription in thyroid adenocarcinoma cells (20, 21) and was detected in the developing and mature
thyroid epithelium(17) , consistent with its role in thyroid
epithelial cell gene expression. TTF-1 was also detected in the
embryonic forebrain and in the respiratory epithelium, where it appears
in the embryonic lung buds early in gestation. A potential role of
TTF-1 in lung epithelial cell gene expression was demonstrated by the
finding that TTF-1 activated transcription of the human surfactant
protein B gene(15) . TTF-1 bound to two closely apposed TTF-1
sites positioned within nucleotides -80 to -100 of the SP-B
gene in close proximity to a hepatic nuclear factor (HNF) binding site.
SP-A and SP-B are coexpressed in type II epithelia cells in the alveoli
and in overlapping subsets of respiratory epithelial cells in the
conducting airways(4, 22) . TTF-1 was detected in the
same subsets of respiratory epithelial cells, consistent with the
potential role of TTF-1 in the regulation of surfactant protein A
expression. TTF-1 was detected in the progenitor cells of the
developing bronchial tubules early in rat lung development. At the time
of birth, TTF-1 is expressed in both type II cells and in subsets of
respiratory nonciliated bronchiolar epithelial cells in a pattern
similar to that of SP-A and SP-B. ()Similarity of
distribution of SP-A, SP-B, and TTF-1 in the respiratory epithelial
cells provides further support for the role of TTF-1 in transcriptional
control of surfactant protein synthesis.
The proximal TTF-1 binding
sites in the SP-A gene are similar to but distinct from those in the
human SP-B gene, the former consisting of a complex site with at least
four distinct closely associated TTF-1 binding sites. These sites bound
the TTF-1 protein found in nuclear extracts as well as the recombinant
TTF-1 homeodomain protein as assessed by the EMSA analyses. The
consensus binding sites for TTF-1 in the SP-A gene include motifs of
CTCAAG, CTGAAG, TAAG, and GTTAAG. The human and murine SP-B genes
contain a TTF-1 binding site CCACTCTAAGT that was critical to lung cell
expression of SP-B-CAT gene constructs (15) . The surfactant
protein ATTF-1 sites only partially match the consensus TTF-1 binding
site compiled for the thyroid-specific gene CCACTCAAGT(23) . A
critical TTF-1 site in the SP-B gene contained two distinct TTF-1
binding sites, each of which contribute to activation of the SP-B-CAT
constructs. HNF-3 and HFH-8 bound to the proximal promoter region
of SP-B in close proximity to these TTF-1 binding sites and enhanced
SP-B promoter function(15, 24, 25) . However,
the HNF site was not critical to the lung epithelial cell gene
transcription, which was entirely dependent upon the TTF-1 binding
sites located -118 to -64 in the human SP-B gene. While
there were no discernible HNF binding sites within the SP-A gene region
from -255 to +45, a role of HNF in the regulation of SP-A
remains to be more fully explored.
The TTF-1 binding sites in the SP-A 5`-flanking region were sufficient to transactivate transcription of murine SP-A in transfected HeLa cells in vitro. Mutation of site 3 and sites 1 and 3 or 1 and 4 in combination reduced transcriptional activity in transfected MLE-15 cells about 4-6-fold. There was also a partial reduction in transactivation of mutated TTF-1 sequences in HeLa cells. These findings support the concept that the function of the TTF-1 complex binding region was dependent upon interactions between the three TTF-1 sites. While TTF-1 is thought to bind primarily as a monomer, TTF-1 can form protein oligomers that may be important to the function of binding sites in both thyroid- and lung-specific genes. Complex interactions among these closely approximated DNA binding sites and TTF-1 may be required for lung-specific gene transcription. The heterogeneity of the TTF-1 binding sites may also confer differences in TTF-1 binding affinity and further modulate the response of the promoter to varying concentrations of TTF-1.
Although recombinant TTF-1 transactivates the expression of SP-A, SP-B, SP-C, and CC10 gene promoters in HeLa cells(15) , expression of TTF-1 alone does not suffice to determine the observed heterogeneity of the expression of these four genes in the developing mammalian lung. In human lung, SP-C is expressed only in alveolar type II cells and excluded from the conducting airway(22) . In contrast, CC10 is expressed in the bronchiolar but not alveolar epithelium(26) . Thus, while TTF-1 appears to play an important role in lung cell-specific gene expression, other factors, such as combinatorial interactions with HNFs, may further modify gene expression, contributing to the distinct temporal and spatial pattern of gene expression.
The sequence of the rat SP-A gene differs from the mouse in containing a brain identifier sequence from position -316 to -211(12) . This sequence likely represents an insertion occurring after the divergence of the mouse and rat lineages and suggests that conserved sequences from position -210 to +1 of the rat gene may be critical to gene expression. Within this region of the rat gene, five potential TTF-1 binding sites are present. Allowing for degeneracy in TTF-1 binding motifs as identified in the present study, the following potential sites are present: CTGGAG at -194 to -189, CTCGAG at -162 to 157, TAAG at -151 to -148, GTTAAG at -137 to -132, and CTGAAG at -127 to -122. These sites are present in the regions active in cell- free transcription(12) . Four of these regions were encompassed in protected regions identified by DNase I footprint analyses of the rat gene(12) . Sites from -130 to -169 of the rat gene formed a single footprint on the coding strand called P5(12) . Similarly, we were unable to distinguish individual footprints (data not shown) because of the multiple DNA-protein interactions in a comparable region of the mouse SP-A gene. Footprints in this region of the rat SP-A gene were distinct in liver and lung, suggesting that these regions bind a combination of specific and ubiquitous factors (12) supporting the concept that combinatorial interactions of endodermally expressed factors control SP-A gene expression. The 5`-flanking region of the human SP-A gene diverges from the mouse and rat genes. However, four sites in the human gene with the consensus motif CTNNAG are present at positions -213 to -208, -180 to -175, -160 to -155, and -102 to -97(27) , consistent with a potential role for TTF-1 in control of the human SP-A gene.
Recently, two cis-active sites involved in cAMP-induced cell-specific expression of the rabbit SP-A gene were described(13, 14) . These sites correspond most closely to E-box motifs. TTF-1 is specific to endodermal derivatives of the foregut epithelium(17) , whereas E-box motifs are generally involved in regulation of cell-specific expression of mesodermal derivatives. A consensus sequence for an E-box binding site (CACTTG) was identified at position -8 to -3 of the murine SP-A gene. However, sequences from -57 to +45 of the murine gene were expressed at low levels in multiple cell types (Fig. 2). Thus, this E-box motif does not appear to play a regulatory role in lung cell-specific gene transcription of the murine SP-A gene assessed by transfection of MLE cells. Murine SP-A gene expression did not require cAMP and was induced only 1.5-2.0-fold by 1 mM dibutyryl cAMP (data not shown).
We previously demonstrated non-cell-specific expression of SP-A-CAT constructs (-1401 to +455) in transient transfections of H441-4 cells (11) . These studies included the sequences used in the present study, but the constructs also contained intron 1 and a portion of exon 2. Sequences from +46 to +455 of the mouse gene(11) , excluding the known transcription start site and 5`-flanking sequences, were tested in transient assays of MLE-15 and 3T3 cells. Expression from this construct was not lung cell-specific (data not shown), consistent with the presence of an alternate transcription site initiating downstream from the sequences tested in the present study. Since transcription initiation sites of the SP-A gene do not map to this region(11) , the function of this alternative transcription start site likely occurs only in the context of the chimeric gene constructs in transfection assays and is not active in the endogenous murine SP-A gene.
In summary, biochemical, site-specific mutagenesis, and functional assays demonstrate that 5`-flanking sequences of the murine SP-A gene from nucleotides -231 to -168 play an important role in determining its lung cell-specific gene expression. This region contains at least four distinct TTF-1 binding sites, supporting the important role of this homeodomain protein in control of SP-A gene expression in the lung.