(Received for publication, December 2, 1996, and in revised form, April 24, 1997)
From the Children's Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, The Children's Hospital Research Foundations, Cincinnati, Ohio 45229-3039
Thyroid transcription factor 1 (TTF-1) is a
homeodomain-containing nuclear transcription factor expressed in
epithelial cells of the lung and thyroid. TTF-1 binds to and activates
the transcription of genes expressed selectively in the respiratory
epithelium including pulmonary surfactant A, B, C and Clara cell
secretory protein. Transfection with a plasmid encoding the cyclic
AMP-dependent protein kinase (protein kinase A; PKA)
catalytic subunit, Cat-, stimulated the phosphorylation of a
TTF-1-flag fusion protein 6-7-fold in H441 pulmonary adenocarcinoma
cells. Recombinant TTF-1 was phosphorylated by purified PKA catalytic
subunit in the presence of [
-32P]ATP. PKA
catalytic subunit family members, Cat-
and Cat-
, markedly
enhanced the transcriptional activation of surfactant B gene promoters
by TTF-1 in vitro. Peptide mapping was used to identify a
PKA phosphorylation site at the NH2 terminus of TTF-1. A
17-amino acid synthetic peptide comprising this site completely inhibited the PKA-dependent phosphorylation of TTF-1
in vitro. A substitution mutation of TTF-1
(Thr9
Ala) abolished phosphorylation by PKA and reduced
transactivation of the surfactant B gene promoter. Transfection with a
plasmid encoding the cAMP regulatory element binding factor inhibited transcriptional activity of the surfactant protein B gene
promoter. Phosphorylation of TTF-1 mediates
PKA-dependent activation of surfactant protein B gene
transcription.
Pulmonary morphogenesis begins with the evagination of the foregut endoderm into the splanchnic mesenchyme. Thereafter, branching morphogenesis and differentiation of respiratory epithelial cells result in the generation of the conducting airways and alveolar gas exchange areas critical to perinatal adaptation to air breathing. Respiratory function depends upon surfactant lipids and proteins that are secreted into the alveolus by type II epithelial cells, reducing surface tension at the air/liquid interface and keeping the lung from collapsing at end-expiration. Various humoral factors influence the differentiation of respiratory epithelial cells and the production of surfactant proteins and lipids. Surfactant proteins A, B, and C (SP-A, SP-B, and SP-C, respectively)1 play critical roles in the organization and function of surfactant phospholipids and are expressed in a highly tissue-specific manner, their expression increasing markedly during the latter part of gestation (for review, see Ref. 1). SP-B, a 79-amino acid amphipathic peptide, plays an important role in the formation of lamellar bodies and tubular myelin and is critical to lung function after birth (2). Mice and humans with genetic defects in the SP-B gene succumb from respiratory failure in the immediate postnatal period (3, 4). In the fetal lung, SP-B synthesis increases markedly during late gestation and is induced by glucocorticoids and cAMP (5, 6).
The specificity of surfactant protein gene expression in respiratory
epithelial cells is mediated at least in part by the binding of thyroid
transcription factor 1 (TTF-1) to cis-acting elements in the
5-flanking region of its target genes (7, 9). Transcription of SP-A
(8), SP-B (7, 9), SP-C (10), and Clara cell secretory protein (CCSP; 7)
is markedly activated by TTF-1. TTF-1 is a homeodomain-containing
phosphoprotein of the Nkx family and is expressed in the developing
brain, thyroid, and lung (11). TTF-1 also plays a critical role in
pulmonary morphogenesis. Gene-targeted deletion of the mouse TTF-1 gene (titf1) caused severe pulmonary hypoplasia (12). TTF-1 also plays a role in gene expression in the thyroid gland, activating thyroperoxidase and thyroglobulin gene transcription (23, 24, 32).
Because of the role of TTF-1 in surfactant protein gene expression and
previous studies supporting the role of cyclic AMP in lung epithelial
differentiation and function, the present study was designed to assess
the potential interactions between cAMP-dependent pathways
and the activity of TTF-1.
The action of cAMP is initiated by binding of hormones or other signaling molecules to cell surface receptors stimulating the activity of adenylyl cyclase in the plasma membrane, activating protein kinases that phosphorylate regulatory proteins, including nuclear transcription factors (13-15). A number of intracellular signaling pathways have been elucidated including protein kinase A (PKA), protein kinase C (PKC), and Ras/Raf/MEK/ERK, Rac/MEKK/SEK/JNK, JAK/STAT (for review, see Ref. 16). The potential role of these intracellular regulatory cascades in respiratory epithelial cell gene expression has not been discerned at present.
The actions of PKA on gene transcription are mediated by
phosphorylation of various transcription factors including CREBs, CREM,
and other CRE binding proteins (ATFs) that influence gene transcription
(for review, see Refs. 14 and 17). Although cAMP is known to stimulate
respiratory epithelial cell differentiation and surfactant protein
expression (6), the intracellular pathways mediating the effects of
cAMP or gene transcription in the lung are unknown. In the present
work, the PKA catalytic subunits Cat- and Cat-
, but not CREB,
strongly stimulated the human SP-B promoter in lung epithelial cells.
Cat-
phosphorylated TTF-1 and synergistically stimulated human SP-B
promoter when cotransfected with TTF-1, into H441 pulmonary
adenocarcinoma and HeLa cells.
Human SP-B gene promoter-luciferase reporter
constructs SP-B-218 and SP-B-500 were generated as described previously
(18). PKA catalytic subunit expression vector Cat-, Cat-
and
inactive form Cat-
m were obtained from Dr. R. A.
Maurer, Oregon Health Sciences Institute (19, 20). Rc-TTF-1 expression
vector was obtained from Dr. R. Di Lauro, Stazione Zoologica Anton
Dohrn, Italy. CREB expression vector was obtained from Dr. M. R.
Montminy, Salk Institute, La Jolla, CA. pCR3 vector was purchased from
Invitrogen (San Diego). Rc-HNF-3
, Rc-HNF-3
and Rc-HFH-8
expression vectors were obtained from Dr. R. H. Costa from University
of Illinois at Chicago.
The wild type TTF-1-flag expression construct (TTF-1 wt) was made as
follows. Two primers were synthesized and used for polymerase chain
reaction amplification using TTF-1 expression plasmid from Dr. R. Di Lauro as a template. The upstream primer
(5-GCCACCATGTCGATGAGTCCAAAGCACACGACT-3
) contains a Kozak sequence for
efficient translation and 21-mer matching the 5
end of TTF-1 cDNA
sequence. The downstream primer (3
AACGAAATACCAGCCTGGACCCTGATGTTCCTGCTGCTACTGTTCACTATT5
)
contains a 21-mer matching the 3
end of TTF-1 cDNA sequence and a
in-frame flag sequence (underlined sequence) before two stop codons. A mutant TTF-1-flag construct consists of a Thr9
Ala
substitution; TTF-1 Thr9
Ala was made essentially the
same except the upstream primer has the sequence
5
-GCCACCATGTCGATGAGTCCAAAGCACACGGCTCCGTTCTCAGTGTCTGAC-3
(the underlined G changes Thr to Ala). The polymerase chain
reaction product of TTF-1 with the Kozak and flag sequences was
subcloned directly into the pCR3 TA cloning vector (Invitrogen) to
generate pCR3/TTF-1-flag expression construct. The orientation and DNA sequence of the constructs were confirmed by DNA sequencing.
TTF-1-his-tag fusion protein construct for bacterial expression was
made previously (21). The TTF-1 cDNA was directionally cloned into
the pET 21b bacterial expression vector (pET System, Novagen Inc.,
Madison, WI). BL21(DE3) Escherichia coli was transformed, and expression of recombinant TTF-1 was induced by the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 2 mM. Nickel-nitrilotriacetic acid
affinity purification was performed under denaturing conditions.
H441 cell culture, DNA transfection, and luciferase assay were performed as described previously (18). Mean values and S.D. are presented except where described. Statistical differences were assessed using analysis of variance or paired t test where appropriate.
H441 Cell Radiolabeling, Immunoprecipitation, and Western BlotH441 cells were seeded in a 25-mm flask at a density of
1 × 106 cells/flask. The next day, 2 µg of
pCR3/TTF-1-flag and 2 µg of PKA catalytic subunit plasmid DNA
(Cat-) were added with Lipofectin and cultured overnight. The medium
was then changed to RPMI with 10% fetal calf serum and the cells
cultured for 2 days at 37 °C. Cells were washed once and incubated
with serum-free, phosphate-free medium for 45 min. Two ml of
phosphate-free medium containing 10% dialyzed calf serum (Life
Technologies, Inc.) with 0.3 mCi of 32PO4/flask
was then added. After a 3-h incubation, the radioactive medium was
removed. Cells were rinsed and incubated for 5 min with 1 ml of cell
lysis buffer (100 mM NaCl, 50 mM Tris buffer, pH 7.4, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate) containing the phosphatase inhibitors (10 mM sodium orthovanadate and 10 mM NaF) and
protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 5 mM phenylmethylsulfonyl fluoride).
Lysed cells were disrupted further by aspiration through a 21-gauge
needle. Supernatants were first incubated with 10 µl of mouse serum
(Sigma) and 5 µl of 0.5 mg/µl of DNase I (Boehringer Mannheim)
overnight at 4 °C. The next day, 30 µl of protein A/G plus-agarose
(Santa Cruz Biotechnology) was added and the incubation continued for
90 min at 4 °C. Samples were centrifuged and the supernatants
incubated overnight with 10 µl of flag M2 monoclonal antibody (Kodak)
or 5 µl of TTF-1 monoclonal antibody at 4 °C on a rotator. The
next day, 20 µl of protein A/G plus-agarose was added and the
incubation continued for 90 min. The supernatant was discarded and
agarose complex centrifuged, washed three times with cell lysis buffer,
and washed once with buffer containing 150 mM NaCl, 50 mM Tris, pH 7.5, and 5 mM EDTA. Samples were
boiled in sample loading buffer, centrifuged, and the supernatant
proteins separated by sodium dodecyl sulfate-gel electrophoresis as
described by Laemmli (22). The intensity of protein phosphorylation was
visualized after exposure of the dried gels to x-ray film and
quantitated by PhosphorImaging.
Western blot analysis of nuclear protein extracts from TTF-1 wt and
TTF-1 (Thr9 Ala) transfected H441 cells was performed
using the Phast Gel system (Pharmacia Biotech Inc.).
TTF-1-his-tag
protein (1 µg), TTF-1 homeodomain polypeptide (1 µg) obtained from
Dr. Di Lauro, or synthetic TTF-1 peptides (30 µg) made by Genemed
Inc. (San Francisco), were incubated with 1 µl (22 units) of purified
PKA catalytic subunit (Calbiochem) in the presence of 40 µCi of
[-32P]ATP and reaction buffer (20 µM
ATP, 1 mM CaCl2, 20 mM
MgCl2, 4 mM Tris, pH 7.5). After incubation at
37 °C for 30-60 min, the phosphorylated products were either
separated on a 10% polyacrylamide gel (protein) followed by
autoradiography or spotted on a phosphocellular disc and counted by
liquid scintillation.
Transcription of the human SP-B gene is conferred by
cis-acting elements located 500 to +41 from the start of
transcription and is dependent upon the binding of TTF-1 to distinct
sites within this region (7, 9). To assess the role of
cAMP-dependent protein kinases on SP-B gene transcription,
plasmid expression vectors expressing two forms of PKA catalytic
subunit, Cat-
and Cat-
, were cotransfected into H441 cells with
the SP-B promoter fragment, SP-B-500-luciferase. Cat-
and Cat-
stimulated SP-B gene transcription (Fig. 1A).
Activation of SP-B-500-luciferase was dose-dependent, and
the inactive form of PKA catalytic subunit, Cat-
m, had
no effect on the activity of SP-B-500-luciferase. Cat-
was
consistently more active than Cat-
. The Cat-
construct was therefore utilized for subsequent studies.
To define further the region of the SP-B promoter which was responsive
to PKA, SP-B-218-luciferase (containing 218 to +41) was also tested.
Activity of SP-B-218-luciferase was markedly stimulated by PKA Cat-
(Fig. 1B), supporting the concept that DNA elements
responding to Cat-
resided within
218 to +41 of the human SP-B
gene, a region containing cis-acting elements activated by
TTF-1 (7). When the mutant SP-B-218 promoter (SP-B-218 TT; Ref. 7)
lacking TTF-1 binding activity was used, no stimulation was observed by
PKA Cat-
(Fig. 1B). DNA homology search revealed no CRE
consensus elements within this region of the SP-B promoter, suggesting
that the stimulatory effects of PKA Cat-
on SP-B-218-luciferase were
not mediated by direct activation of CRE elements in the SP-B gene.
Importantly, direct cotransfection of H441 cells with the expression
plasmid RSV-CREB inhibited transcription of SP-B-500-luciferase in H441
cells (data not shown).
To
further identify transcription factors regulating the activity of the
SP-B promoter, the effects of TTF-1, HNF-3, HNF-3
, and HFH-8
expression vectors on the activity of SP-B-218-luciferase were assessed
in H441 cells (Fig. 2). Cotransfection with a vector expressing TTF-1 consistently activated SP-B-218-luciferase
(6-7-fold), whereas HNF-3 family members were less active in H441
cells. Stimulation of SP-B-218-luciferase by TTF-1 (~6 fold) was
similar to that observed after transfection with Cat-
(5-fold) in
H441 cells. Addition of 8-bromo-cAMP to H441 cells increased
SP-B-500-luciferase activity approximately 2-fold, 24 h after
treatment (data not shown).
PKA Catalytic Subunit-stimulated Phosphorylation of TTF-1 in H441 cells
To assess whether TTF-1 was phosphorylated by PKA catalytic subunit, a TTF-1-flag construct (pCR3/TTF-1-flag) was generated. A flag sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was inserted into the COOH terminus of the rat TTF-1 polypeptide producing a TTF-1 fusion peptide that was used to immunoprecipitate and distinguish endogenous TTF-1 from the flag-TTF-1 produced by the pCR3/TTF-1-flag construct. pCR3/TTF-1-flag transactivated SP-B-500-luciferase in H441 cells, demonstrating that the TTF-1-flag fusion peptide retained its biological activity (data not shown).
To test whether PKA increased the phosphorylation of TTF-1,
pCR3/TTF-1-flag was cotransfected with either Cat- or
Cat-
m into H441 cells (Fig. 3). After
radiolabeling with 32Pi, TTF-1 from the cell
lysates was immunoprecipitated with the flag antibody. Cat-
markedly
increased the phosphorylation of TTF-1-flag, the protein migrating in
approximately the same position as endogenous phosphorylated TTF-1.
Cat-
enhanced TTF-1-flag phosphorylation approximately 6-7-fold
compared with Cat-
m in H441 cells. Phospho-TTF-1-flag
consisted of two bands (Mr = 40,000-43,000),
supporting the concept that TTF-1 was phosphorylated at multiple sites
or that multiple forms of TTF-1 were present.
PKA Catalytic Subunit-phosphorylated TTF-1-his-tag in Vitro
Bacterial expressed TTF-1-his-tag protein was purified by
Ni-column chromatography. Purified TTF-1-his-tag fusion protein and
TTF-1 homeodomain polypeptide were incubated with the purified PKA
catalytic subunit in the presence of [-32P]ATP and
separated on a 10% polyacrylamide gel. As shown in Fig. 4, PKA catalytic subunit directly phosphorylated the
TTF-1 fusion protein (Fig. 4, lane c), but not the TTF-1
homeodomain polypeptide, indicating that the PKA phosphorylation site
resided outside the homeodomain of TTF-1. To identify the site of PKA
phosphorylation, a set of synthetic peptides comprising TTF-1
phosphorylation sites was generated.
Identification of TTF-1 Peptide Containing the PKA Phosphorylation Site
Di Lauro and colleagues identified several phosphorylation
sites on TTF-1 (23). Four peptides were synthesized covering these
phosphorylation sites. The peptides were incubated with the purified
PKA catalytic subunit in the presence of [-32P]ATP.
Only peptide 1 (residues 1-17) was phosphorylated by the PKA catalytic
subunit (Fig. 5A). In competition
experiments, peptide 1 abolished TTF-1-his-tag protein phosphorylation
by PKA (Fig. 5B, lane d). These results
demonstrated that the PKA phosphorylation site was located near the
NH2 terminus of TTF-1 in a region associated with
transactivation activity (24).
TTF-1 (Thr9
Peptide 1 contains the sequence
Arg-His-Thr-Thr-Pro consistent with a consensus sequence for
phosphorylation by PKA (14). A PKC consensus sequence Ser-Pro-Arg-His
that shares Arg-His with the PKA site, is located immediately proximal
to the NH2 terminus of the putative PKA site (Fig.
6A). A mutant peptide 1 (peptide 1m1), in
which Thr9 was changed to Ala (Arg-His-Thr-Thr-Pro Arg-His-Thr-Ala-Pro), was synthesized and tested as substrate for PKA
and PKC in vitro. As shown in Fig. 6B, the
phosphorylation of mutant peptide 1m1 by PKA was completely abolished,
indicating that TTF-1 Thr9 is phosphorylated by PKA.
Phosphorylation of the mutant peptide 1m1 by PKC was fully
retained.
TTF-1(Thr9
A mutant form of TTF-1 (Thr9 Ala)
was generated by mutation of the TTF-1 cDNA (see "Materials and
Methods"). Whereas TTF-1 wt stimulated the activation of the SP-B
promoter (SP-B-218), TTF-1 (Thr9
Ala) was considerably
less active in stimulating transcription of SP-B-218-luciferase (Fig.
7). Differences in the activity of TTF-1 wt and TTF-1
(Thr9
Ala) were not related to differences in the level
of TTF-1 peptides produced by the constructs. Western blot analysis of TTF-1 wt and TTF-1 (Thr9
Ala)-transfected H441 cells
demonstrated equal amounts of TTF-1 fusion proteins (data not shown).
Immunofluorescent staining by anti-flag monoclonal antibody showed that
TTF-1 wt and TTF-1 (Thr9
Ala) were nuclear localized
and strongly expressed in the transfected cells (data not shown).
TTF-1(Thr9
To
assess the role of phosphorylation of TTF-1 Thr9 on
PKA-dependent activation of SP-B-218 promoter, the
phosphorylation mutant TTF-1 (Thr9 Ala) was transfected
into H441 cells and failed to mediate Cat-
-dependent
stimulation of SP-B-218-luciferase in H441 cells (Table
I).
|
Respiratory adaptation at birth depends upon the regulated synthesis and secretion of surfactant lipids and proteins into the alveolar spaces, reducing surface tension at the air/liquid interface. The synthesis of surfactant lipids and proteins is developmentally regulated, increasing during advancing gestation and stimulated by a variety of humoral factors including glucocorticoids and cAMP. The present findings demonstrate that PKA activated both TTF-1 phosphorylation and TTF-1-dependent SP-B gene transcription, suggesting that the effects of cAMP on surfactant protein B gene expression may be mediated, at least in part, by the modulation of the activity of TTF-1 on the SP-B transcription, rather than by binding and activating CRE.
TTF-1 is expressed throughout the conducting and peripheral respiratory tract during lung development (11, 25). In human lung, after birth, TTF-1 is detected in nuclei of subsets of epithelial cells, both in the conducting airway and in type II epithelial cells (25, 26). In the lung, TTF-1 expression overlaps with that of SP-A, SP-B, SP-C, and CCSP, but its distribution is more extensive than that of the surfactant proteins or CCSP (27-29). Although TTF-1 strongly influences the transcription of each of these genes, each has a distinct temporal and spatial pattern of expression, supporting the concept the activity of TTF-1 may be further modified to confer the distinct regulatory features typical of each gene. The marked increase in surfactant protein synthesis occurring in late gestation is not directly related to changes in the level of TTF-1 mRNA (30), providing a strong inference that the activity of TTF-1 on its target genes may be further modulated. The present work supports the hypothesis that the stimulatory effects of cyclic AMP on surfactant homeostasis may be influenced, at least in part, by phosphorylation of TTF-1, which, in turn, enhances its activity on SP-B gene transcription.
Effects of cAMP on gene transcription are mediated by the activation of
PKAs; subsequent protein phosphorylation may alter the activity of
regulatory proteins including nuclear transcription proteins. A variety
of nuclear transcription proteins are targets of PKA. Some bind to CREs
that are present in target genes. Many genes that are regulated by PKA
contain the CRE consensus sequence TGACGTCA (17). We were unable to
identify CRE sequences fitting this consensus sequence in the
5-flanking regions of mouse SP-A and human or mouse SP-B and SP-C
genes. Likewise, expression of recombinant CREB failed to activate SP-B
gene transcription from the SP-B-500-luciferase construct in H441
cells. SP-A gene expression is also enhanced by cAMP and is not
mediated by CREB/ATF family members (31). In the present work, SP-B
transcription was markedly stimulated by cotransfection with TTF-1, and
this activity was further induced by cotransfection with Cat-
and
-
, suggesting that the activation by the catalytic subunit of PKA
may be mediated by enhancing the activity of TTF-1 on the SP-B
promoter.
TTF-1 phosphorylation was markedly stimulated by cotransfection with
Cat-, but not by the inactive form, Cat-
m. These
findings are consistent with observations in thyroid carcinoma cells in
which cAMP stimulated TTF-1 phosphorylation (32). In those studies, the
binding activity of TTF-1 to the thyroglobulin promoter was enhanced by
phosphorylation of TTF-1. Although a number of phosphorylation sites
were identified in the TTF-1 polypeptide sequence, the phosphorylation
sites involved in activation of gene transcription have not been
identified, although phosphorylation of TTF-1 is not required for
binding to DNA (23). In this report, peptide mapping and site-specific
mutagenesis demonstrated that PKA-dependent phosphorylation
of TTF-1 near the NH2 terminus (Thr9) mediates
the activation of SP-B gene transcription in pulmonary adenocarcinoma
cells.
cAMP influences a variety of cellular functions in the developing lung,
stimulating surfactant synthesis and secretion, surfactant protein gene
expression, and fluid secretion from respiratory epithelial cells. The
numbers of -adrenergic receptors and the activity of adenylate
cyclase in the lung increase dramatically during late gestation (33,
34) and are further influenced by hormonal factors that stimulate lung
cell differentiation (35). In late gestation, the numbers of
-adrenergic receptors in the lung increase in association with the
increased surfactant production and secretion required for perinatal
respiratory adaptation. The present findings support the concept that
the effects of cAMP on distal respiratory epithelial function may be
mediated, at least in part, by the activity of TTF-1, which modulates
surfactant protein gene transcription. Whether other effects of cAMP on
respiratory epithelial cell function in the perinatal lung are mediated
by cAMP-dependent activation of TTF-1 remains to be
clarified.
We thank Dr. Richard A. Maurer for PKA
catalytic subunit expression vectors; Dr. Marc R. Montminy for CREB
expression vectors; Dr. Roberto Di Lauro for TTF-1 expression vector;
and Dr. Robert H. Costa for HNF-3, HNF-3
, and HFH-8 expression
vectors. We thank Dr. Hong Du for helping with Western blot analysis,
Dr. Sui Lin for helping with phosphoprotein immunoprecipitation, and Manely Ghaffari for technical support.