From the Departments of Biochemistry and Obstetrics-Gynecology, the Cecil H. & Ida Green Center for Reproductive Biology Sciences, the University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038
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ABSTRACT |
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Surfactant protein (SP)-A gene
transcription is stimulated by factors that increase cyclic AMP. In the
present study, we observed that three thyroid transcription factor-1
(TTF-1) binding elements (TBEs) located within a 255 base pair region
flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for
maximal cyclic AMP induction of bSP-A2 promoter activity. We found that
TTF-1 DNA binding activity was increased in nuclear extracts of
pulmonary type II cells cultured in the presence of cyclic AMP. By
contrast, the levels of immunoreactive TTF-1 protein were similar in
nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into
immunoprecipitated TTF-1 protein also was markedly increased by cyclic
AMP treatment. Moreover, exposure of nuclear extracts from cyclic
AMP-treated type II cells either to potato acid phosphatase or alkaline
phosphatase abolished the cyclic AMP-induced increase in TTF-1
DNA-binding activity. Interestingly, the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA), known to
activate protein kinase C, also enhanced incorporation of
[32P]orthophosphate into TTF-1 protein; however, the DNA
binding activity of TTF-1 was decreased in nuclear extracts of
TPA-treated type II cells. Expression vectors encoding TTF-1 and the
catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon
SP-A2 gene linked to human growth hormone, as reporter) containing
TBEs, or with a reporter gene construct containing three tandem TBEs
fused upstream of the bSP-A2 gene TATA box and the transcription
initiation site. Coexpression of TTF-1 and PKA-cat increased fusion
gene expression 3-4-fold as compared with expression of TTF-1 in the
absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was
suppressed by cotransfection of a dominant negative form of PKA
regulatory subunit RI. We suggest that a PKA-induced increase of
TTF-1 phosphorylation and TBE binding activity mediates cyclic
AMP-induced expression of the SP-A gene in lung type II cells.
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INTRODUCTION |
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Pulmonary surfactant, a phospholipid-rich, developmentally regulated lipoprotein synthesized exclusively by type II pneumonocytes, acts to reduce alveolar surface tension, thereby preventing alveolar collapse upon exhalation of air (1). Four lung-specific proteins have been found to be associated with surfactant: surfactant protein (SP)1-A, SP-B, SP-C, and SP-D. These appear to serve important roles in surface activity, surfactant phospholipid reutilization, and immune function within the alveolus (2).
Expression of the gene encoding SP-A, the major surfactant-associated protein, occurs primarily in alveolar type II cells and to a lesser extent in bronchiolar epithelial (Clara) cells (3, 4). SP-A gene expression is developmentally regulated in fetal lung in concert with surfactant phospholipid synthesis (5, 6); gene transcription is initiated only after ~70% of gestation is completed in all mammalian species thus far studied (6, 7). SP-A expression is subject to multifactorial regulation; we have observed that cyclic AMP and glucocorticoids have major regulatory effects (6). In studies using rabbit, human, and baboon fetal lung in organ culture, SP-A mRNA and protein levels were found to be augmented by cyclic AMP analogues and by agents that increase the levels of intracellular cyclic AMP (8-10). Cyclic AMP also enhances the rate of type II cell differentiation and enlargement of prealveolar ducts (9). SP-A is encoded by a single copy gene in rabbits (5), rats (11), dogs (12), and mice (13), while in baboons (14) and humans (15, 16), there are two highly similar SP-A genes (the SP-A1 and SP-A2 genes). In human fetal lung in culture, expression of the SP-A2 gene is considerably more responsive to the stimulatory effects of cyclic AMP than is SP-A1 (17).
To understand the molecular basis for type II cell-specific and cyclic AMP regulation of SP-A gene expression, we have utilized differentiated type II cells in primary culture transfected with reporter gene constructs containing 5'-flanking sequences from the rabbit and human SP-A genes. Two E-box motifs were identified within the 5'-flanking sequence of the rabbit SP-A gene that are required for basal and cyclic AMP-induced expression of SP-A promoter activity (18). These elements were found to bind the basic helix-loop-helix-zipper factors USF1 (19) and USF2.2 Another cis-acting element, termed CRESP-A, which displays sequence similarity to both a palindromic cyclic AMP-responsive element (CRE) and a nuclear receptor half-site, also was found to be required for cyclic AMP induction of SP-A promoter activity in transfected type II cells (20-22). Characterization of CRESP-A indicated that this sequence does not bind the transcription factor CRE-binding protein, CREB, but rather may serve as a binding site for a member of the nuclear receptor superfamily that binds DNA as a monomer (22). A GT box element, located proximal to the transcription initiation site, also was found to be essential for basal and cyclic AMP regulation of the human SP-A2 gene. This element was found to bind Sp1, as well as a 55-kDa protein distinct from Sp1 (23). These findings suggest that basal and cyclic AMP induction of SP-A promoter activity are mediated by the cooperative interaction of transcription factors bound to at least three different response elements.
Thyroid transcription factor-1 (TTF-1, also named thyroid enhancer-binding protein or NKx 2.1), is a homeodomain transcription factor that has been found to be involved in the tissue-specific regulation of three different thyroid-specific genes, namely thyroglobulin, thyroperoxidase, and thyrotropin receptor (24-28). TTF-1 expression is restricted to the developing thyroid gland, lung epithelia, and restricted areas of the developing brain (24, 29). The importance of TTF-1 in development was underscored by the findings of targeted deletion of the TTF-1 gene; homozygous TTF-1 null mice were still-born and lacked lung parenchyma, thyroid, and anterior pituitary glands (30). TTF-1 was found to bind to DNA sequences located in the 5'-flanking regions of the mouse SP-A (31), SP-B (32), SP-C (33), and Clara cell-specific protein (CC10) (34) genes and to promote transactivation of heterologous reporter genes containing the 5'-flanking sequences from these surfactant protein genes in nonpulmonary cells.
It remains to be determined how the actions of TTF-1 to influence morphogenesis of thyroid, pituitary, and lung during early embryogenesis are regulated differently from those actions to enhance expression of specific genes at later stages of fetal development and postnatally. It was our objective, in the present study, to define the role of TTF-1 in the cyclic AMP induction of SP-A gene transcription. In type II cell transfection studies, we observed that TTF-1 binding elements (TBEs) in the 5'-flanking sequence of the baboon SP-A2 (bSP-A2) gene are crucial for cyclic AMP induction of bSP-A2 promoter activity. We also found that cyclic AMP treatment of cultured type II cells promotes increased TTF-1 phosphorylation and an associated increase in TBE binding activity. Furthermore, cotransfection of cyclic AMP-dependent protein kinase (PKA) with TTF-1 in a lung adenocarcinoma cell line caused an increase in the capacity of TTF-1 to transactivate the SP-A2 promoter. We therefore suggest that the cyclic AMP-mediated increase in TTF-1 DNA binding and transcriptional activity serves a major role in the cyclic AMP induction of SP-A gene expression in lung type II cells.
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MATERIALS AND METHODS |
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Plasmids and Antiserum--
A full-length baboon TTF-1 cDNA
(~1.2 kilobase pairs) isolated from a 92-day gestational age fetal
baboon lung cDNA library was ligated to
EcoRI/SalI-digested pCMV5 vector to produce the TTF-1 expression vector, pCMV5/TTF-1. PKA expression vectors, RSV/PKA-cat-, RSV/PKA-cat-
, and the mutated RSV/PKA-cat-
m
(35), were kindly provided by Dr. Richard A. Maurer (Oregon Health
Sciences University). An expression vector containing the mutant form
of PKA regulatory subunit RI
under the control of the mouse
metallothionein-1 promoter (MT-RI
-mut) (36), was kindly provided by
Dr. Stanley McKnight (University of Washington, Seattle).
bSPA2
255:hGH was constructed by polymerase chain reaction
amplification of a DNA fragment containing 255 bp of the 5'-flanking
DNA and 40 bp of the first exon of the bSP-A2 gene, followed by
ligation to the first exon of the hGH structural gene in
pACsk20GH, which contains the left 17% of human adenovirus
5 genome and the promoterless hGH structural gene. To generate fusion
genes containing TBE mutations, oligonucleotides were made containing
mutations in each of the three TBEs of the bSP-A2 gene (TBE1, TBE2, and
TBE3); in each case, the TBE was replaced with a restriction enzyme
site sequence. The binding sequence in TBE1 was changed to an
EcoRI site, that in TBE2 was changed to a KpnI
site, and that in TBE3 was changed to an XbaI site. These
oligonucleotides where then used in polymerase chain reaction with
oligonucleotides corresponding to the 5'- or 3'-ends of the bSP-A2
genomic region from
255 to +40; bSP-A2 genomic clone was used as
template. Polymerase chain reaction fragments were digested with
appropriate endonucleases and cloned into pACsk20GH. To
construct (TBE)3SP-A:hGH, double-stranded oligonucleotides corresponding to sequences between
185 and
165 of the bSP-A2 gene
5'-flanking DNA containing TBE1 (5'-GTGCTCCCCTCAAGGGTCCTA-3') were
self-ligated to form a concatamer of three TBE1 repeats and inserted
into the HindIII site of bSP-A2
50:hGH, where
50 bp of the 5'-flanking DNA and 40 bp of the first exon of the bSP-A2 gene were fused to the first exon of the hGH structural gene in pACsk20GH.
Lung Type II Cells and Cell Lines-- Lung tissues from mid-gestation human abortuses were maintained in organ culture in serum-free Waymouth's MB752/1 medium (Life Technologies, Inc.) containing Bt2cAMP for 3 days to promote type II cell differentiation. Phenotypically differentiated type II cells are not detected in mid-gestation human fetal lung. We previously observed that type II cells spontaneously differentiate when the human fetal lung is placed in organ culture in serum-free medium and that the rate of type II cell differentiation is enhanced by treatment with cyclic AMP analogues (9). After culture, the tissues were digested with collagenase for preparation of lung type II cells as described (20, 37). The human lung adenocarcinoma cell line A549 (ATCC CCL 185) was maintained in Waymouth's MB752/1 medium containing fetal bovine serum (10%, v/v).
Generation of Recombinant Adenoviruses-- To generate recombinant adenoviruses, 293 cells, a permissive human embryonic kidney cell line, were cotransfected with recombinant pACsk20GH containing bSP-A2:hGH fusion genes and with pJM17; the latter contains the entire adenovirus genome plus insertion of a 4.3-kilobase pair plasmid. The pJM17 itself is too large to be packaged into viral particles. Infectious viral particles are formed upon in vivo recombination of the plasmids to produce a recombinant viral genome of packageable size. Viral DNA was analyzed for the presence of the fusion genes by restriction endonuclease digestion and DNA sequencing. The recombinant viruses were titered in 293 cells at least twice to ensure the accuracy of the titer.
Expression of SP-A Fusion Genes in Transfected Type II Cells-- Type II cells plated at a density of 5-9 × 106 cells/60-mm dish were maintained overnight in Waymouth's MB 752/1 medium containing 10% fetal bovine serum. The cells were then washed twice with medium and incubated for 1 h with 1 × 106 recombinant viral particles, resulting in a multiplicity of infection of 0.1-0.2. In this manner, the same number of cells (1 × 106) were infected in each experiment. The medium was then aspirated and replaced with fresh medium in the absence or presence of Bt2cAMP (1 mM). Media from transfected cells were collected every 24 h and assayed for hGH by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from lung type II cells as described previously
(38). Protein concentrations were determined by a modified Bradford
assay (Bio-Rad). Double-stranded oligonucleotides containing TBE1
(underlined) (5'-GTGCTCCCCTCAAGGGTCCT-3'),
CRESP-A (underlined)
(5'-GAGCGGGTGACCTCAGCCCT-3'), or the GT box from the human
SP-A2 gene (underlined) (5'-TCTCAGGGGTGGGGAAGAA-3') were
end-labeled using [-32P]ATP (ICN, Costa Mesa, CA) and
used as probes. Nuclear proteins were incubated with the radiolabeled
DNA probes for 20 min at room temperature in reaction buffer (20 mM Hepes, pH 7.6, 75 mM KCl, 0.2 mM
EDTA, 20% glycerol) and 1 µg of poly(dI-dC)-poly(dI-dC) (Pharmacia)
as nonspecific competitor. Protein-DNA complexes were resolved on 5%
nondenaturing polyacrylamide gels and visualized by autoradiography. To
determine whether TTF-1 DNA binding is phosphorylation-dependent, nuclear extracts (10 µg) were
incubated either with 0.5 units of potato acid phosphatase (type III;
Sigma) or 1 unit of alkaline phosphatase (Boehringer Mannheim) for 5 min at room temperature as described (39). As a control, phosphatases were boiled for 15 min before incubation with nuclear extracts. Phosphatase reactions were stopped with 1 mM sodium
vanadate and NaF. The phosphatase-treated nuclear extracts were
incubated with 32P-labeled TBE1 oligonucleotides,
fractionated on a 5% nondenaturing polyacrylamide gel, and visualized
by autoradiography.
Metabolic Labeling and Immunoprecipitation-- Human fetal lung type II cells were maintained either in control medium or in medium containing Bt2cAMP (1 mM) for 5 days. Parallel dishes of cells were incubated in control medium for 4 days and in medium containing TPA (10 nM) for an additional 24 h. For 32P labeling, the type II cells were washed twice with Tris-buffered saline solution and incubated in phosphate-free Dulbecco's modified Eagle's medium (Life Technologies) for 1 h. [32P]orthophosphate (NEN Life Science Products) was then added to achieve a concentration of 0.5 mCi/ml, and cells were further incubated in appropriate medium for 2 h. After washing twice with cold Tris-buffered saline, cells were lysed in 1 ml of RIPA buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 5 mM sodium fluoride, and 5 mM sodium vanadate). Cells were disrupted completely by scraping and shearing through a 27-gauge needle. After pelleting the cellular debris by centrifugation for 10 min at 4 °C, aliquots of lysates containing 1 × 107 cpm were precleared with 50 µl of protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C. Precleared lysates were then incubated with 5 µl of TTF-1 antiserum or the corresponding preimmune serum. The immune complexes were recovered upon incubation with 40 µl of protein A/G PLUS-agarose for 1 h at 4 °C. Pellets were washed twice with RIPA, twice with high salt RIPA (RIPA containing 1 M NaCl), and twice more with RIPA. For [35S]methionine labeling, type II cells which had been cultured for 5 days under conditions described above were incubated in methionine-free DMEM (Life Technologies) for 30 min; [35S]methionine was then added to achieve a final concentration of 0.125 mCi/ml. Cells were lysed, and TTF-1 was immunoprecipitated as described for 32P-labeled cells. The immunoprecipitates isolated from the 32P-labeled and [35S]methionine-labeled cells were resuspended in 2 × SDS-sample buffer, resolved on a 12.5% SDS-polyacrylamide gel, and visualized by autoradiography.
Transient Transfection--
Transient transfections were carried
out using A549 cells maintained in Waymouth's MB752/1 medium (Life
Technologies) containing 10% fetal bovine serum. The plasmids used
consisted of either 4 µg of bSP-A2255:hGH or
(TBE)3SP-A:hGH fusion genes, together with 2 µg of either
an expression vector containing the entire TTF-1 open reading frame
(pCMV5/TTF-1) or the empty expression vector (pCMV5) and 2 µg of
either an expression vector containing the whole coding sequence of
PKA-cat (RSV/PKA-cat) or the empty expression vector (RSV), with or
without 2 µg of an expression vector containing a mutant form of PKA
regulatory subunit RI
(MT-RI
-mut); 1 µg of RSV/
-Gal was used
as the internal control. Prior to transfection, the plasmids were
combined with 30 µg of DOTAP (Boehringer Mannheim) in Hanks'
balanced salt solution (pH 7.4) and incubated at room temperature for
20 min. A549 cells grown to logarithmic phase (50-70% confluence) on
60-mm diameter dishes were washed twice with phosphate-buffered saline.
Plasmid-DOTAP mixtures in 2 ml of Waymouth's MB 752/1 medium without
serum were then added to the cells. The cells were then incubated for
24 h at 37 °C and washed with phosphate-buffered saline.
Waymouth's MB 752/1 medium (1 ml) was then added to each dish, and the
cells were incubated at 37 °C for another 24 h. Media were then
collected and assayed for hGH content by radioimmunoassay (Nichols
Institute). Variations in transfection efficiency were corrected
by normalizing hGH to
-galactosidase activity.
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RESULTS |
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TTF-1 Binding Elements Are Essential for Cyclic AMP Induction of
SP-A Promoter Activity in Type II Cells--
In our initial studies of
the regulation of the bSP-A2 gene, we observed that 255 bp of bSP-A2
5'-flanking DNA was sufficient to mediate high basal and cyclic AMP
induction of bSP-A2 promoter activity in transfected type II cells. By
DNase I footprinting and EMSA, three TBEs were characterized within the
255- bp 5'-flanking region.3 Whereas, TBE1
manifested the strongest DNase I footprint, the findings of EMSA have
clearly indicated that TBE2 and TBE3 also serve as specific TTF-1
binding sites.3 The
positions and sequences of these TBEs are shown in Fig.
1A. To analyze the functional
significance of the TBEs in mediating basal and cyclic AMP induction of
bSP-A2 promoter activity in type II cells, the three TBEs were
individually mutated within the context of the 255-bp bSP-A2
5'-flanking region. Fusion genes were constructed composed of 255 bp of
bSP-A2 5'-flanking DNA with or without TBE mutations plus 40 bp of the
first exon, linked to the human growth hormone (hGH) structural gene,
as reporter. These fusion genes were then incorporated into the genome
of a replication-defective human adenovirus 5 for highly efficient and
reproducible transfer by infection (20, 37). Equal amounts of
recombinant adenoviruses were introduced into primary cultures of rat
fetal lung type II cells incubated in the absence or presence of
Bt2cAMP. The concentration of hGH that accumulated in the
culture medium was determined by radioimmunoassay. As shown in Fig.
1B, mutagenesis of TBE1 caused a marked reduction of
bSP-A2255:hGH fusion gene expression in transfected type
II cells. Moreover, the cyclic AMP induction of fusion gene expression
also was markedly decreased. Mutagenesis of TBE2 and TBE3 caused a
moderate decrease in basal and cyclic AMP-induced fusion gene
expression. These findings suggest that the integrity of TTF-1 binding
elements is essential for maximal basal and cyclic AMP-induced bSP-A2
promoter activity in type II cells and that TBE1 plays the most
critical role in this regard.
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TTF-1 DNA Binding Activity of Type II Cell Nuclear Extracts Is Increased by Bt2cAMP Treatment-- To study the mechanism(s) whereby TTF-1 mediates cyclic AMP induction of SP-A promoter activity in type II cells, we first utilized EMSA to analyze the effects of cyclic AMP on TTF-1 DNA binding activity in nuclear extracts from cultured type II cells. An oligonucleotide containing TBE1 was radiolabeled and incubated with equivalent amounts of nuclear proteins from type II cells cultured for 5 days in the absence or presence of 1 mM Bt2cAMP. This incubation time was utilized because we previously observed that SP-A gene expression is markedly increased in type II cells incubated for 5 days in medium containing Bt2cAMP, as compared with cells maintained in control medium (37). The DNA-protein complexes were separated from free probe on a nondenaturing polyacrylamide gel (Fig. 2A). As can be seen, type II cell nuclear protein-TBE1 complex formation was markedly increased in nuclear extracts from type II cells cultured in the presence of Bt2cAMP as compared with those of type II cells cultured in control medium. The finding that formation of the protein-DNA complex was abolished by co-incubation with antiserum raised against TTF-1 indicates that the binding complex contains TTF-1 (data not shown). To determine whether the increased TTF-1·TBE1 complex formation was due to increased TTF-1 protein expression, equivalent amounts of nuclear proteins from type II cells cultured in the absence or presence of Bt2cAMP were analyzed for TTF-1 content by immunoblotting (Fig. 2B). Despite the increased TTF-1·TBE1 complex formation, TTF-1 protein levels were relatively unaffected by Bt2cAMP treatment. Therefore, the increased TTF-1·TBE1 complex formation in Bt2cAMP-treated type II cells was due to increased TTF-1 DNA binding activity, not to increased levels of TTF-1 protein.
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TTF-1 Phosphorylation Level Is Increased in Type II Cells Cultured in the Presence of Bt2cAMP-- To determine whether phosphorylation of TTF-1 was associated with the increased TTF-1 DNA binding activity in Bt2cAMP-treated type II cells, we analyzed whether cyclic AMP altered the rate of TTF-1 phosphorylation. Type II cells were maintained in the absence or presence of Bt2cAMP for 5 days and then cultured for 2 h either with [32P]orthophosphate or [35S]methionine. The radiolabeled cell lysates were immunoprecipitated with anti-TTF-1 or preimmune serum. As can be seen in Fig. 4, incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 was markedly increased in type II cells cultured in the presence of Bt2cAMP (lane 2) as compared with type II cells cultured in control medium (lane 1). By contrast, incorporation of [35S]methionine into immunoprecipitated TTF-1 was relatively unaffected by cyclic AMP treatment (lanes 3 and 4), indicating that the rate of TTF-1 synthesis was similar in Bt2cAMP-treated type II cells as compared with that of type II cells cultured in control medium. Therefore, the increased TTF-1 DNA binding activity in Bt2cAMP-treated type II cells is associated with an increase in TTF-1 phosphorylation. The broad band in the immunoprecipitation of 35S-labeled type II cell lysates may in part represent differentially phosphorylated forms of TTF-1. The faster migrating component of the band also may comprise some nonspecific interactions with the antiserum, since a portion of this was observed in immunoprecipitations using preimmune serum (data not shown).
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TTF-1 Binding Activity Is Abolished by Phosphatase Treatment-- To determine whether increased TTF-1 phosphorylation is responsible for the increase in TTF-1 DNA binding activity in Bt2cAMP-treated type II cells, we analyzed the effect of phosphatases on the TTF1·TBE1 complex formation. Nuclear extracts from type II cells cultured in the absence or presence of Bt2cAMP were exposed either to potato acid phosphatase or alkaline phosphatase for 5 min as described (39) prior to EMSA. In parallel, nuclear extracts were treated with heat-inactivated phosphatases as controls. As can be seen in Fig. 5, treatment either with potato acid phosphatase (lanes 4 and 5) or alkaline phosphatase (lanes 8 and 9) markedly reduced TTF-1·TBE1 complex formation in nuclear extracts from control and cyclic AMP-treated type II cells to levels less than those of nuclear extracts from control cells in the absence of phosphatase treatment. By contrast, heat-inactivated phosphatases had no effect on TTF-1 binding activity (lanes 6, 7, 10, and 11). These findings indicate that TTF-1 binding activity is phosphorylation-dependent and that increased TTF-1 phosphorylation in Bt2cAMP-treated type II cells is responsible for the increased TTF-1 DNA binding activity.
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Phorbol Ester Promotes TTF-1 Phosphorylation but Inhibits the TTF-1 DNA Binding Activity-- It was reported recently that TTF-1 was effectively phosphorylated by protein kinase C (PKC) in vitro (40). On the other hand, it also was reported that TPA, a phorbol ester known to activate PKC, reduces the levels of SP-A protein and mRNA expression in H441 lung adenocarcinoma cells (41, 42). To determine whether TTF-1 is phosphorylated by PKC in vivo, type II cells maintained in control medium for 4 days were treated with 10 nM TPA for 24 h and then labeled with [32P]orthophosphate and immunoprecipitated with anti-TTF-1. To compare the effects of TPA to those of Bt2cAMP on TTF-1 phosphorylation, type II cells were incubated for 5 days in medium containing 1 mM Bt2cAMP. The type II cells were incubated with TPA for only 24 h to avoid the effects of long term incubation to down-regulate PKC. In previous studies, it was found that preincubation of H441 cells with 50 nM of phorbol 12,13-dibutyrate for 24 h, did not alter the binding of [3H]phorbol 12,13-dibutyrate to PKC in the H441 cells (41). These published findings indicate that PKC was not down-regulated under these treatment conditions.
As shown in Fig. 6A, the incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 in TPA-treated type II cells was increased to a level comparable with that of Bt2cAMP-treated cells, suggesting that TTF-1 was effectively phosphorylated by PKC in vivo. To determine the effects of TPA-induced phosphorylation on TTF-1 binding activity, EMSA was used to analyze TTF-1 DNA binding activity in nuclear extracts from TPA-treated type II cells as compared with nuclear extracts from type II cells cultured either in control medium or in the presence of Bt2cAMP. Interestingly, TTF-1 DNA binding activity of nuclear extracts from TPA-treated type II cells was weaker than that of type II cells cultured in the control medium (Fig. 6B), while the TTF-1 protein level in nuclear extracts of TPA-treated type II cells was comparable with those of type II cells cultured either in control medium or in the presence of Bt2cAMP (data not shown). These findings suggest that TTF-1 phosphorylation by PKC inhibits the TTF-1 DNA binding activity; this may be responsible, in part, for the decreased SP-A gene expression in TPA-treated lung adenocarcinoma cells as previously reported (41, 42).
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TTF-1 Transcriptional Activity Is Increased by PKA--
To
determine whether the cyclic AMP-mediated increase in TTF-1
phosphorylation and DNA binding activity in Bt2cAMP-treated type II cells is associated with increased TTF-1 transcriptional activity, A549 cells were cotransfected with
bSP-A2255:hGH, containing three TTF-1 binding elements,
with a TTF-1 expression vector and expression vectors containing either
the
- or
-isoform of the PKA catalytic subunit; the respective
empty vectors were cotransfected as controls where appropriate. A549
cells, a human lung adenocarcinoma cell line of presumed type II cell
origin (43) were chosen, since we have found that they do not express detectable levels of endogenous TTF-1 protein (data not shown). In
previous studies, we observed that expression of rabbit and human
SP-A:hGH fusion gene constructs are not cyclic AMP-inducible in these
cells. Transcription of the reporter gene was evaluated by measuring
the hGH concentration of the culture medium. As shown in Fig.
7A, cotransfection with TTF-1
alone increased bSP-A2
255:hGH fusion gene expression
approximately 3-fold as compared with transfection with
bSP-A2
255:hGH and control vector. Cotransfection with PKA-cat-
(the
-isoform of the PKA catalytic subunit) and TTF-1 increased the activity of bSP-A2
255:hGH by 8-fold as
compared with cells transfected only with the reporter gene construct
alone; this was 2.7-fold greater than with TTF-1 in the absence of
PKA-cat. Cotransfection with PKA-cat-
(the
-isoform of the PKA
catalytic subunit) and TTF-1 increased the expression of
bSP-A2
255:hGH by 11-fold as compared with the reporter
gene construct alone; this was 3.7-fold greater than with TTF-1 in the
absence of PKA-cat. Neither PKA-cat-
nor PKA-cat-
had the effect
of altering bSP-A2
255:hGH expression in the absence of
cotransfected TTF-1 (Fig. 7A), further suggesting that the
effect of PKA is mediated by TTF-1. By contrast, a mutated form of
PKA-cat-
, PKA-cat-
m, had no effect on fusion gene expression in
cells cotransfected with TTF-1. The effect of PKA-cat also was lost
when the major TTF-1 binding site TBE1 was mutated in
bSP-A2
255:hGH (Fig. 7B). To ensure that the
effects of PKA-cat observed were due to increased TTF-1 activity, rather than to increased TTF-1 expression via PKA induction of CMV
promoter activity, the levels of TTF-1 protein were analyzed in the
transfected cells by immunoblotting. Immunoreactive TTF-1 was
undetectable in A549 cells transfected with "empty" expression vector but readily detected in cells transfected with pCMV5/TTF-1. Co-transfection with RSV/PKA-
, RSV/PKA-
, or RSV/PKA-
m
expression vectors had essentially no effect on the levels of
immunoreactive TTF-1 in the A549 cells (data not shown).
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DISCUSSION |
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We previously observed that cyclic AMP serves a major role in the induction of type II cell differentiation (8) and SP-A gene expression (8-10) in human, rabbit, and baboon fetal lung in culture. In type II cell transfection studies, we found that cyclic AMP stimulation of SP-A promoter activity is dependent upon the cooperative interaction of transcription factors bound to at least three types of regulatory elements. These include a CRE-like element (20-22), which appears to bind a member of the nuclear receptor family, an E-box (18), which binds USF1 (19) and USF2,2 and a GT box, which binds Sp1 (23) and other members of the Krüppel family.4 We have observed in type II cell transfection studies that mutagenesis of any one of these elements causes a marked reduction of basal and cyclic AMP stimulation of SP-A promoter activity (18, 20-23); however, the mechanisms whereby transcription factors binding to these elements mediate cyclic AMP responsiveness have not been determined.
In the present study, we observed that three TBEs within the
5'-flanking region of the bSP-A2 gene also are functionally required for cyclic AMP activation of the bSP-A2 promoter in lung type II cells.
Mutation of the TBEs caused a marked reduction of basal and cyclic
AMP-induced bSP-A2255:hGH fusion gene expression in
transfected type II cells. These findings suggest that cyclic AMP
stimulation of SP-A gene expression in lung type II cells also is
dependent upon the cooperative interactions of TTF-1 with transcription
factors bound these other response elements and that all of these
elements are essential for cyclic AMP induction of SP-A promoter
activity.
In studies to define the mechanism(s) whereby TTF-1 mediates cyclic AMP induction of SP-A gene expression in type II cells, we observed that TTF-1 DNA binding activity of type II cell nuclear extracts was increased by cyclic AMP treatment. By contrast, nuclear protein binding activities for CRESP-A and the GT box were unaffected by cyclic AMP. These findings indicate for the first time that cyclic AMP specifically increases TTF-1 binding activity in type II cells. Our finding that the levels of immunoreactive TTF-1 in nuclear extracts as well as the rate of incorporation of [35S]methionine into immunoisolated TTF-1 were unaffected by cyclic AMP treatment of type II cells suggests that cyclic AMP induction of TTF-1 binding activity is not mediated by changes in its nuclear localization or expression.
To begin to define the mechanisms whereby cyclic AMP increases TTF-1 DNA binding activity, we analyzed effects on TTF-1 phosphorylation in human fetal type II cells in primary culture. We observed that, in association with its effect of stimulating TTF-1 DNA binding activity, cyclic AMP treatment markedly increased the rate of 32P-phosphate incorporation into immunoisolated TTF-1. The finding that phosphatase treatment effectively abolished the cyclic AMP induction of TTF-1 DNA binding activity indicates that cyclic AMP-induced TTF-1 phosphorylation mediates the increase in binding activity for TBEs within the bSP-A2 5'-flanking sequence. While findings of previous studies (28, 39) indicated that TTF-1 DNA binding activity for the thyroglobulin and thyroperoxidase gene promoters was induced by treatment of nuclear extracts with the PKA catalytic subunit in vitro, no evidence has been presented to indicate that TTF-1-binding activity is increased by PKA-mediated phosphorylation in vivo. In fact, in studies of TTF-1 binding activity in FRTL-5 cells, it appeared that binding activity was reduced in thyrotropin-treated cells as compared with controls (28). Furthermore, the role of PKA-mediated phosphorylation in TTF-1 DNA binding activity was questioned in a later study (40) in which it was found that TTF-1 was not phosphorylated by PKA in vitro and that mutated forms of TTF-1 that could not be phosphorylated manifested normal levels of DNA binding and transcriptional activity in transfected HeLa cells. On the other hand, in a recent study, a PKA phosphorylation site near the N terminus (Thr9) of TTF-1 was identified and found to be essential for PKA activation of the SP-B promoter in H441 cells, a lung adenocarcinoma cell line of Clara cell origin (44). The findings of the present study clearly indicate that cyclic AMP treatment of primary cultures of lung type II cells under conditions that increase SP-A gene transcription causes an increase in TTF-1 phosphorylation and DNA binding activity.
In the present study, we observed that the rate of TTF-1 phosphorylation also was increased when type II cells were cultured in the presence of TPA. This is consistent with previous findings that TTF-1 may serve as a substrate for PKC-induced phosphorylation in vitro (40). However, in contrast to the inductive effects of cyclic AMP, we found that TPA treatment caused a reduction of TTF-1 DNA binding activity. Interestingly, TPA has been found to decrease SP-A protein and mRNA levels in H441 cells in a time- and dose-dependent manner (41) and to decrease the rate of SP-A gene transcription (42). Based on our findings, we suggest that the inhibitory effect of TPA on SP-A gene expression is mediated, in part, by a decrease in TTF-1 DNA binding activity. It is possible that phosphorylation of specific residues by PKC interferes with the DNA binding activity of TTF-1 in TPA-treated type II cells. It should be noted that it is not known whether the increased TTF-1 phosphorylation that we observed in the TPA-treated type II cells is directly mediated by PKC or whether it is an indirect effect via activation of another protein kinase. Since the TTF-1 protein level in nuclear extracts of TPA-treated type II cells was found to be comparable with that of type II cells cultured either in control medium or in the presence of Bt2cAMP, phosphorylation of TTF-1 does not seem to alter the nuclear localization or steady state levels of TTF-1 protein. This is in contrast to recently published findings of Kumar et al. (45), which suggest that TPA treatment of H441 cells cause cytoplasmic trapping of TTF-1, resulting in a loss of TTF-1 from the nucleus.
A549 is a lung adenocarcinoma cell line of presumed type II cell origin
(43) that lacks endogenous TTF-1.3 In the present study,
A549 cells were transfected with a reporter gene construct
(bSP-A2255:hGH) comprised of 5'-flanking sequence from
the bSP-A2 gene containing three TBEs; cotransfection of a TTF-1
expression vector caused an induction of bSP-A2 promoter activity. The
response to TTF-1 was increased further by cotransfection of the PKA
catalytic subunit. The finding that PKA-cat had no effect of increasing
bSP-A2 promoter activity in the absence of cotransfected TTF-1 and that
mutation of the major TTF-1 binding site abolished PKA induction of
TTF-1 transcriptional activity suggests that the action of PKA to
induce bSP-A2 gene expression is mediated, at least in part, through
TTF-1. To further substantiate the role of TTF-1 in PKA induction of
SP-A promoter activity, A549 cells transfected with a reporter gene
containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box
and transcription initiation site ((TBE)3SP-A2:hGH) were
cotransfected with PKA-cat and TTF-1 expression vectors. The finding
that PKA-cat enhanced transactivation of (TBE)3SP-A:hGH by
cotransfected TTF-1 indicates that the action of PKA to increase SP-A
promoter activity is mediated specifically by TTF-1 binding to TBEs in
the absence of other response elements. These findings, together with
those indicating that cyclic AMP specifically increases TTF-1 binding
activity in type II cell nuclear extracts, suggest that TTF-1 is the
cyclic AMP-responsive transcription factor in lung type II cells. It should be noted, however, that basal and TTF-1/PKA-stimulated levels of
expression of the (TBE)3SP-A2:hGH fusion gene were
considerably reduced as compared with those of the
bSP-A2
255:hGH fusion gene construct, indicating the
importance of the cooperative interaction of the TBEs with other
response elements in basal and cyclic AMP regulation of SP-A promoter
activity.
In A549 cell transfection studies, we also observed that the TTF-1
induction of bSP-A2255:hGH fusion gene expression in the
absence of cotransfected PKA catalytic subunit was prevented by
cotransfection of a dominant negative form of PKA RI
. This finding
suggests that the inductive effect of TTF-1 on bSP-A2 promoter activity
in A549 cells is dependent upon phosphorylation by endogenous PKA
activity. Interestingly, Bt2cAMP had no effect of
increasing TTF-1 transcriptional activity in A549 cells cotransfected with TTF-1 and bSP-A2
255:hGH (data not shown). These
findings suggest that A549 cells may be defective in some component of the cyclic AMP-mediated signaling pathway.
In conclusion, the findings presented in this study suggest that cyclic AMP-responsive expression of the SP-A gene is mediated by an increase in TTF-1 transcriptional activity, which is associated with increased TTF-1 phosphorylation and DNA binding activity. It appears that the PKA-mediated increase in TTF-1 phosphorylation and DNA binding activity may constitute the primary mechanism whereby cyclic AMP induces SP-A gene expression. We suggest that the increase in TTF-1 phosphorylation and DNA binding activity may, in turn, facilitate its interaction with transcription factors bound to other cis-acting elements found to be essential for cyclic AMP induction of SP-A promoter activity, including CRESP-A (20-22), the GT box (23), and E-box sequences, which bind USF1 (18, 19) and USF2,2 as well as with components of the basal transcription complex.
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ACKNOWLEDGEMENT |
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We are grateful to Margaret Smith for expert help with cell and tissue culture.
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FOOTNOTES |
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* This work was supported in part by NHLBI, National Institutes of Health Grant U10-HL-52647.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a predoctoral fellowship from the Chilton Foundation
(Dallas, TX).
§ To whom correspondence should be addressed: Dept. of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9038. Tel.: 214-648-2944; Fax: 214-648-8856.
1
The abbreviations used are: SP, surfactant
protein; bSP, baboon SP; TTF-1, thyroid transcription factor 1; PKA,
protein kinase A; PKC, protein kinase C; CRE, cyclic AMP-response
element; EMSA, electrophoretic mobility shift assay; TBE, TTF-1 binding
element; PKA-cat, catalytic subunit of PKA; RI, PKA regulatory
subunit I
; hGH, human growth hormone; Bt2cAMP, dibutyryl
cyclic AMP; TPA, 12-O-tetradecanoylphorbol-13-acetate; RIPA,
radioimmune precipitation; bp, base pair(s); CMV, cytomegalovirus;
-Gal,
-galactosidase.
2 E. Gao, Y. Wang, J. L. Alcorn, and C. R. Mendelson, unpublished observations.
3 J. Li, E. Gao, and C. R. Mendelson, unpublished observations.
4 E. Gao, L. Wang, and C. R. Mendelson, unpublished observations.
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REFERENCES |
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