(Received for publication, October 6, 1995; and in revised form, January 10, 1996)
From the
Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing -13 kilobase pairs to -320 base pairs (bp) of the 5` flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between -320 and -180 bp from the start of transcription by transfection analysis. Five DNase-protected regions, indicating multiple protein-DNA interactions within the -320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from -197 to -158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The -197 to -158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription.
Pulmonary surfactant is a mixture of phospholipids and proteins,
which functions to reduce surface tension at the air/liquid interface
preventing alveolar collapse during respiration(1) . A
surfactant deficiency is the basis of lethal respiratory distress
syndrome in infants born prematurely. Surfactant protein C (SP-C) ()is a 3.7-kDa hydrophobic protein that associates with
surfactant lipids and is a component of replacement surfactants used in
the treatment of neonatal respiratory distress
syndrome(2, 3) . The alveolus is lined by two
morphologically distinct epithelial cell types: the alveolar Type I
cells responsible for gas exchange and the cuboidal Type II cells,
which contain numerous lamellar inclusion bodies, the intracellular
form of surfactant(4) . In the adult lung, SP-C is exclusively
synthesized and secreted by Type II
cells(5, 6, 7) . In contrast, the other
surfactant-associated proteins (SP-A, SP-B, and SP-D) are expressed in
both Type II and subsets of airway epithelial cells in the conducting
airway(5, 8, 9) . SP-C transcriptional
activity is detected in primordial respiratory epithelial cells at the
earliest stages of lung development (fetal day 11 in the mouse), is
restricted to the distal most portions of the developing fetal lung,
and is maintained in alveolar Type II epithelial cells in the postnatal
lung(6, 7) .
As a first step in identifying the
cis-active regulatory elements that confer the Type II cell-specific,
developmental, and humoral regulation of SP-C gene expression, we have
cloned and sequenced both the human and the murine SP-C genes and their
flanking sequences(10, 11) . In transgenic mice, 3.7
kb of the 5` flanking sequences of the human SP-C gene is sufficient to
produce lung-specific expression of several reporter
constructs(11, 12, 13, 14) . The
developmental expression of the 3.7huSP-C-CAT transgene mimics the
endogenous SP-C gene, indicating that the cis-active regulatory
elements essential for both cell-specific and developmental expression
are located within 3.7 kb of the transcriptional start site of the
human gene(9, 15) . Parallel experiments with the
murine SP-C promoter (which shows extensive sequence homology to the
human promoter) support these same conclusions for 4.8 kb of the murine
promoter. ()
In the current study we demonstrate the critical role of TTF-1 in the transcriptional regulation of the murine SP-C gene. TTF-1 is a homeodomain containing transcription factor expressed in the developing thyroid, brain, and lung(16) . The spatial and temporal pattern of TTF-1 expression in the lung parallels that of SP-C. TTF-1 is detected in the lung rudiments at the earliest stages of epithelial migration into the pulmonary mesenchyme and is maintained in epithelial cells of the distal conducting airways and alveoli(16, 17) . SP-C expression appears only slightly later on murine fetal day 11, but is restricted only to alveolar expression with further development. Two recent studies demonstrate that TTF-1 activates the transcription of surfactant proteins A and B(18, 19) . Here we extend those observations to SP-C and identify specific cis-acting sequences involved in transactivation of the SP-C promoter by TTF-1.
To produce herpes virus-thymidine kinase (HSV-TK) promoter constructs, fragments of the murine SP-C 5` flanking regions were blunt end-cloned into the filled-in BamHI site distal to the HSV-TK promoter of pBLCAT5(20) . The orientation and oligomerization of the inserts were determined by sequencing.
For site-directed mutagenesis of potential TTF-1 sites T4 and T5, the SP-C promoter region was liberated with PstI and XhoI from p0.32muCAT. This 320-bp fragment was subcloned into the pGL2Basic (Promega) producing pSP-C wt. Site-specific mutations in the SP-C promoter were created by two separate PCR reactions using either the sense or antisense mT4 or mT5 oligonucleotide and the corresponding vector oligonucleotides (GL1 or GL2, Promega) to generate PCR products that overlap at the site of mutation. A second PCR reaction using the vector oligonucleotide primers and the products of the first PCR reaction generated the mutant 320-bp SP-C promoters. The presence of the expected mutations was verified by sequencing.
-Galactosidase assays were run as described
previously(19) , and optical density at 410 nm was determined
on a Dynatech MR600 plate reader. Reporter assays were normalized for
transfection efficiency based on the
-galactosidase activity. CAT
activity was determined on 10-40 µl of extract. Acetylated
reaction products were separated from the substrate by thin layer
chromatography as described(19) . The CAT activity was
quantitated on an Molecular Dynamics PhosphorImager, relative to the
activity of the promoterless plasmid, pBLCAT6. Luciferase activity was
assayed on 20 µl of extract at room temperature in 100 µl of
luciferase assay reagent (Promega) for 10 s after a 2-s delay in a
Monolight 2010 luminometer.
Figure 1:
Deletional analysis of the SP-C
promoter. A, a representative autoradiogram shows the TLC
separation of acetylated chloramphenicol products (dark
arrowheads) from unreacted substrate (light arrowhead)
catalyzed by extracts from MLE-15 cells transiently cotransfected
(duplicate plates) with the p4.8 muCAT test construct and pCMV-TTF-1,
pCMV-HNF-3, or pCMV-HFH-8. The transactivators are expressed under
the control of the CMV promoter, and the control assays are
transfections with pRcCMV, the empty vector. The extracts were
normalized for transfection efficiency as determined by
-galactosidase activity. B, a representative
autoradiogram shows the TLC separation of acetylated chloramphenicol
products from unreacted substrate as catalyzed by extracts from MLE-15
cells transiently transfected with the deletion constructs. The length
of the murine SP-C promoter is indicated (in kilobases) beneath the
autoradiogram. The plus signs indicate cotransfection with
recombinant rat TTF-1 (from plasmid pCMV-TTF-1), and the minus
signs indicate cotransfection with the empty vector pRcCMV.
pBLCAT6 is the promoterless reporter vector; pBLCAT5 has the reporter
gene under the control of the HSV-TK promoter. The extracts were
normalized to transfection efficiency as determined by
-galactosidase activity. C, the relative CAT activity of
MLE-15 cells transiently transfected with the indicated deletion test
constructs. The CAT activity is expressed relative to pBLCAT6, the
promoterless plasmid, with black bars representing CAT
activity in the presence of coexpressed TTF-1 (from plasmid pCMV-TTF-1)
and white bars in its absence (cotransfected with pRcCMV). The
plasmid pBLCAT5 has the constitutive HSV-TK promoter, which is
unresponsive to TTF-1. The bars each represent a mean relative
CAT activity from at least three independent transfections each done in
duplicate. Shown at left of the graph are diagrammatic
representations of the test constructs, identified by the length in
kilobases of the SP-C 5` flanking sequences (thick line) and
showing the SP-C transcriptional start site with +1 and an arrow. D, shown is the relative CAT activity of HeLa
cells transiently transfected with the indicated deletion test
constructs in the presence (dark stippled bars) and absence (light stippled bars) of TTF-1.
Seven SP-C-CAT deletion constructs containing sequences from 13 to 0.10 kb of murine SP-C promoter were used to map regions essential for SP-C transcriptional activity and to localize the TTF-1-responsive region. Plasmids containing from 13 to 0.32 kb of 5` SP-C flanking sequence were approximately 10-fold more active than the promoterless CAT reporter plasmid in transient transfections of MLE-15 cells (Fig. 1B). Deletion of SP-C promoter sequences from 0.32 to 0.23 kb reduced basal activity to only 4-fold over background, identifying a potential stimulatory element located between -0.32 and -0.23 kb. TTF-1 stimulated CAT activity approximately 7-fold in constructs containing 4.8 or 0.8 kb of SP-C DNA and 10-fold in the test constructs with 0.32 kb of 5` flanking SP-C DNA (Fig. 1C). Further deletions resulted in two discrete reductions in TTF-1-mediated transactivation. The level of TTF-1 stimulation was reduced to 2-3-fold with the 0.23 SP-C-CAT construct and all TTF-1 response was lost in cotransfection with the 0.18 SP-C-CAT construct. These results functionally map the location of TTF-1 transcriptional control elements necessary for SP-C transcription to positions -230 to -180.
Several of the same SP-C deletion constructs were used to test the stimulation of SP-C in a non-lung cell line. CMV-TTF-1 transactivated SP-C-CAT constructs in the HeLa epithelial cell line (Fig. 1D), while the same SP-C-CAT constructs were inactive in the 3T3 fibroblast cell line (data not shown).
Figure 2: DNase I footprint analysis of the SP-C promoter. Shown is an autoradiogram representing the in vitro DNase I digestion pattern of the coding strand of the promoter proximal 320 bp of the murine SP-C sequence in the presence of nuclear extracts from 3T3 and MLE-15 cells and the nonspecific protein BSA. Triangles indicate increasing DNase I digestion. The leftmost lane, marked G, reveals the G-specific (Maxam and Gilbert) sequencing reaction products. Boxes to the right of the autoradiograph indicate the regions protected from DNase cleavage. The dark boxes represent regions strongly protected from DNase I digestion by MLE-15 extracts, as compared to the BSA control lanes, and the stippled bars the more subtle alterations in the DNase I digestion pattern at the boundaries of the footprints. HS indicates a DNase I-hypersensitive site in footprint C3. Along the left-hand side is marked the relative location of the start site (+1), minimal TTF-1 motifs (T1-T9; see Fig. 3for sequences), and the distal extent of the promoter(-320).
Figure 3: Mapping of TTF-1 cis-acting response elements. A, the sequence of the murine SP-C promoter is shown, identifying the position of the restriction sites used to generate the 0.32-, 0.23-, 0.18-, and 0.10-kb deletion constructs, as well as the fragments fused to the HSV-TK promoter. The DNase I footprints identified in Fig. 2are shown boxed and labeled C1-C5, the 70- and 40-bp TTF-1-responsive fragments are underlined, and bold sequences correspond to the minimal TTF-1 motifs (25) labeled T1-T9 in the text and 1-6 in panels B and C. The heavy box shows the position of the TATA box; the SP-C start of transcription is indicated by an arrow and +1. B, relative CAT activity of extracts from MLE-15 cells is shown for transfections with plasmids where fragments of the murine SP-C 5` flanking sequence were linked to the HSV-TK promoter. To the left are diagrammatic representations of the test constructs identified by the length in base pairs of the SP-C 5` flanking sequences (thick line) linked to the HSV-TK promoter (thin line). The minimal TTF-1 motifs are enumerated 1-6 and tandem multimerization of fragments is indicated by 2x and 3x. The bar graph shows the relative CAT activity in the presence (dark bars) and absence (white bars) of coexpressed TTF-1 and represents an average of two independent transient transfections done in triplicate, normalized for transfection efficiency, and quantitated on a PhosphorImager. C, relative CAT activity of HeLa cells transiently transfected with the indicated test constructs in the presence (dark striped bars) and absence (light striped bars) of coexpressed TTF-1.
In order to precisely map the cis-active regulatory element, the TTF-1-responsive 70-bp fragment was further subdivided into a distal 33-bp fragment (-227/-195, encompassing footprint C3 with elements T2 and T3) and a 40-bp fragment (-197/-158, encompassing C2 with elements T4-T6). In both cell lines, only the proximal 40-bp fragment conferred significant TTF-1 responsivity to the TK promoter. Oligomerization of the 70-bp and the 40-bp fragments increased the magnitude of TTF transactivation (Fig. 3B). Transactivation by the concatamers of the 70- and 40-bp subfragments was largely orientation independent (data not shown). These experiments delineated the site of TTF-1 transactivation to the -197/-158 region, a region containing only the T4-T6 consensus elements.
Figure 4: Point mutations identify specific sequences necessary for TTF-1 binding. A, sequences of the probes and competitors used in the electrophoretic mobility shift assay analyses. C2 is the 24-bp double-stranded probe corresponding to the wild type murine SP-C sequence (-186/-163) encompassing the minimal TTF-1 motifs numbers 4 and 5 (underlined). mT4 and mT5 are mutant versions of the SP-C probe with the indicated mutations (in bold letters) in motifs 4 and 5, respectively. SP-B is a 24-bp double-stranded oligonucleotide from the human SP-B promoter (-113/-90), which had been identified as binding TTF-1, and mSP-B is the multiple mutant version of the same. Putative TTF-1 consensus sequences are underlined in all cases. B-D, shown are autoradiograms of electrophoretic mobility shifts of SP-C probes containing the number 4 and 5 minimal TTF-1 motifs. Open arrows indicate the position of the free probe; closed arrows indicate the specific protein-DNA complexes. B, wild type SP-C probe (C2) is complexed with MLE-15 extracts and competed with 100-fold excess of unlabeled self (C2), and mutant (mT4 and mT5) oligonucleotides. C, wild type SP-C probe (C2) is complexed with MLE-15 extracts and competed with unlabeled self (C2), human SP-B (SPB), and mutant human SP-B (mSPB) oligonucleotides. The asterisk indicates the addition of TTF-1-specific antisera. The stippled arrowhead indicates the position of the supershifted complex. D, wild type (C2) SP-C probe forms a specific complex with purified recombinant TTF-1 homeodomain protein (rTTF1 HD). Mutant oligonucleotide probes display minimal (MT4) or no binding (MT5) to the truncated TTF-1 homeodomain peptide.
Figure 5: Point mutations identify specific sequences necessary for TTF-1 transactivation. Shown is the relative luciferase activity of extracts from MLE-15 and Hela cells transiently transfected with test constructs in which 0.32 kb of the murine SP-C promoter drives the luciferase reporter gene. Diagrammed on the left are the test constructs wherein the SP-C promoter is represented by the thick line, the SP-C transcriptional start site is indicated with an arrow, and the minimal TTF-1 motifs are enumerated 1-9. In the mutant constructs, SPCmT4 and SPCmT5, the mutant TTF-1 sites are indicated with an asterisks and specific mutations are shown in bold type beneath the diagram. The bar graphs are relative luciferase activity of extracts made from MLE-15 cells in the presence (black bars) or absence (white bars) of coexpressed TTF-1 or extracts from HeLa cells in the presence (dark striped bars) or absence (light striped bars) of coexpressed TTF-1. The bars each represent an average relative luciferase activity from at least two independent transfections each assayed in duplicate and which had been normalized for transfection efficiency.
In this report we demonstrate that the homeodomain transcription factor TTF-1 binds to and activates the murine SP-C promoter. Cotransfection of plasmids producing TTF-1 with SP-C-CAT reporter constructs delineated a minimal region of the SP-C gene that was sufficient to activate transcription in MLE-15 and HeLa cells. This region of the SP-C gene contained two sites (within nucleotides -186 to -163) that bound TTF-1 or recombinant TTF-1 homeodomain protein in vitro. Mutation of either TTF-1 motif interfered with TTF-1 binding and eliminated the ability of TTF-1 to activate transcription, demonstrating that both TTF-1 binding sites were required for the activation of the SP-C promoter.
TTF-1 expression is limited to the thyroid, brain, and lung in the adult rat and human(16) . TTF-1 transactivates the thyroglobulin, thyroperoxidase, and thyrotropin receptor genes in thyroid carcinoma cell lines and binds to a consensus 5`-CAAG-3` motif distinct from other homeobox genes(25, 26) . TTF-1 was recently identified as a transactivator of genes encoding surfactant proteins as well as a Clara cell-specific gene(18, 19) . In the lung, TTF-1 mRNA temporally precedes SP-C expression and, like SP-C, is restricted to epithelial cells at the distal tips of developing airway during branching morphogenesis. These observations support the hypothesis that TTF-1 may be a critical determinant of SP-C gene transcription.
Our analyses of the SP-C promoter and adjacent sequences have shown that the TTF-1-responsive region of the murine SP-C promoter contains two functional CAAG elements configured on opposite strands of DNA as part of a 13-bp palindrome at nucleotides -182 to -170. Analysis of other TTF-1-activated genes demonstrates that transcriptional stimulation is achieved by action at multiple TTF-1 binding sites and suggests this as a mechanism for differential expression of genes by TTF-1(27) . The similarities and differences in the arrangement and context of defined binding sites in TTF-1-responsive promoter regions from both thyroid- and lung-specific genes are summarized in Fig. 6. The TTF-1 binding site in the SP-C promoter is similar to the composite TTF-1-responsive region of the rat thyroglobulin gene where two core CAAG elements are arranged with dyad symmetry. An array of four TTF-1 binding sites was identified in the murine SP-A promoter clustered over the -166 to -117 region(18) . Electrophoretic mobility shift assay experiments with SP-A binding site probes identified differential binding affinities for TTF-1(18) . SP-B has two distinct TTF-1 binding sites, both of which are required for transactivation by TTF-1(19) , consistent with our findings for TTF-1 stimulation of SP-C transcription. The two essential TTF-1 binding sites identified adjacent to the SP-B promoter are notable in their degenerate recognition site. Thus the configuration (linear, palindromic), number, and sequence of TTF-1 binding sites are arranged in a distinct profile for each gene. In the sequence comparison of Fig. 6, we highlight a common sequence motif consisting of (G/A)(G/T)GCTCT, which is closely apposed to the core CAAG motifs of the thyroid- and lung-specific TTF-1-responsive genes. This region warrants further investigation because mutations that have altered this sequence (in conjunction with mutations in TTF-1 response elements) have dramatically reduced expression of reporter constructs(19, 27, 28) .
Figure 6: Comparison of TTF-1-responsive sequences. Shown are sequences in the vicinity of TTF-1-responsive regions from the thyroid-specific genes: rat thyroglobulin (raTg), rat thyroperoxidase (raTPO), and rat thyrotropin receptor (raTSHR), and from the lung-specific surfactant genes: murine surfactant protein A (muSP-A), murine surfactant protein B (muSP-B), and murine surfactant protein C (muSP-C). The minimal TTF-1 motifs are boxed with arrows showing relative orientation where multiple motifs are clustered; asterisks indicate specific nucleotides which had been altered (in this and others' work) and shown to effect TTF-1 responsiveness. The vertical lines show sequence identities in the TTF-1-responsive regions that extend beyond and flank the minimal TTF-1 motif.
Cooperative binding of homeodomain protein to clustered DNA binding sites has been reported for several diverse homeodomain transcription factors (29, 30) similar to the tandem binding sites found in the SP-C promoter and the other TTF-1-responsive genes. The requirement for cooperative TTF-1 binding to establish activation is supported by the consistent observation that mutation of one binding site eliminates TTF-1 transactivation. Cooperative binding may serve to stabilize transcription complexes or produce a tight regulation of the target gene due to concentration of TTF needed to fully occupy multiple sites. In addition to the unique array of composite TTF-1 binding sites for each gene, varying cellular concentrations and distinct intracellular forms of TTF-1 could account for differential regulation of surfactant protein genes by TTF-1. TTF-1 is uniformly expressed at high levels in distal epithelial cells of the branching airway during lung development (16, 17) . In the postnatal lung, TTF-1 was detected most abundantly in alveolar Type II cells and at reduced levels in subsets of differentiated bronchiolar epithelial cells(17) . TTF-1 was undetectable in Type I cells. This distribution of TTF-1 is consistent with the developmental profile and cells of the mature lung that differentially express SP-A, -B, and -C genes. TTF-1 has recently been shown to undergo sulfhydryl-dependent oligomer formation, which affects its binding properties for clustered cis-active sites (31) . These experiments suggest that within a cell, monomeric and higher order forms of TTF-1 may exist that could alter the binding and stability of TTF-1 DNA complexes. Thus, the different configurations of TTF-1 binding sites in conjunction with varied TTF-1 concentrations and oligomeric forms of TTF-1 in a cell could produce the heterogeneity of SP-A, -B, and -C expression in specific distal epithelial cells.
Factors other than the abundance of TTF-1 must
further distinguish thyroid from lung cell expression and specify Type
II cell expression of SP-C. Transcriptional control of TTF-1-regulated
genes appear to operate by distinct mechanisms in thyroid and lung. The
transcription factors HNF-3 and TTF-1 bind to adjacent sites in
the SP-B promoter and are synergistic in stimulating
transcription(19) . This is distinct from the mechanisms
operating in the thyroid where the paired box factor, PAX-8, and TTF-1
utilize overlapping recognition sites and bind in a mutually exclusive
manner to composite sites(32) . Binding of either PAX-8 or
TTF-1 is sufficient to transactivate thyroglobulin promoter constructs
in thyroid cells or in HeLa cells. The footprint analysis of the SP-C
promoter suggests that additional sites for protein-DNA interaction
flank the TTF-1 binding sites. The nature and number of these binding
factors is unknown, but we speculate that their combinatorial binding
may alter accessibility and the dynamics of TTF-1 activation. The
present work supports a model where TTF-1 plays a critical role in the
transcriptional regulation of the SP-C gene in Type II cells by
interacting with two adjacent cis-active elements (-197 to
-158), with cell selective expression of SP-C modulated by an
enhancer element located in the -320 to -230 region. These
unique combinations of TTF-1 with other factors specific to thyroid or
lung as well as distinct binding affinities of the sites for TTF-1 may
contribute to cell-selective and organ-specific expression of
TTF-1-responsive genes in the lung and thyroid.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M38314[GenBank].