(Received for publication, April 18, 1997)
From the University of Pennsylvania School of
Medicine, Institute for Environmental Medicine, Philadelphia,
Pennsylvania 19104-6068, the
Department of Pediatrics,
University of British Columbia, Vancouver, British Columbia, Canada
V5Z 4H4, and the ¶ Department of Pediatrics, University of
Pennsylvania School of Medicine, Children's Hospital of
Philadelphia, Philadelphia, Pennsylvania 19104-4318
The lung-specific surfactant protein B (SP-B) is
essential for surfactant function and normal respiration. We
investigated the role of thyroid transcription factor-1 (TTF-1) and
hepatocyte nuclear factor 3 (HNF3) in the down-regulation of SP-B gene
expression by phorbol ester in pulmonary adenocarcinoma H441 cells.
Responsiveness to 12-O-tetradecanoylphorbol-13-acetate
(TPA) localized to the SP-B proximal promoter (140/
65 bp) and
specifically to binding sites for TTF-1 and HNF3, which act as
cell-specific enhancers of SP-B expression. Treatment of cells with TPA
(10 nM) caused a time-dependent decrease in
both TTF-1 and HNF3 in nuclear extracts and accumulation of both
factors in the cytoplasm as assessed by electromobility shift, Western,
Southwestern, and immunofluorescence assays. Treatment did not alter
the mRNA content or DNA binding activity for either transcription
factor. We conclude that down-regulation of SP-B gene expression by
phorbol ester involves cytoplasmic trapping and loss of TTF-1 and HNF3
from the nucleus. This mechanism of action is independent of AP-1 and
other transcription factors known to be influenced by phorbol
ester.
Surfactant protein B (SP-B),1 an 8-kDa hydrophobic protein secreted by epithelial cells of the lung, enhances the formation and stability of the surface active phospholipid film which prevents collapse of lung alveoli during respiration. Expression of SP-B is essential for the surface tension lowering properties of pulmonary surfactant and for normal respiratory function. Absence or inactivation of SP-B in animal models results in respiratory distress and/or respiratory failure, and inherited deficiency of SP-B is a lethal disorder of infants (1).
Expression of the SP-B gene is limited to the lung and is developmentally regulated at both transcriptional and post-transcriptional levels (2, 3). Recent studies of the SP-B gene promoter have identified important roles for two transcription factors in the cell selective expression of the gene (4-6). Thyroid transcription factor-1 (TTF-1), a member of the NKx2n family of homeodomain transcription factors, binds to sites in both the proximal and more distal SP-B promoter and transactivates the promoter in vitro. TTF-1 also transactivates expression of two other surfactant protein genes, SP-A and SP-C (7, 8), a clara cell-specific protein (CCSP) (9), as well as the thyroid-specific thyroglobulin, thyroperoxidase, and thyrotropin receptor genes (10-12). Consistent with its proposed roles in lung organogenesis and epithelial cell differentiation, expression of TTF-1 in developing lung has a temporal-spatial distribution pattern similar to that of SP-B and in adult lung is expressed only in type II cells and subsets of nonciliated bronchiolar (clara) cells (13).
The other known transcription factor affecting expression of the SP-B
gene is hepatocyte nuclear factor 3 (HNF3), which is a member of the
forkhead winged helix family of transcription factors. HNF3 binds to
the proximal region of the human SP-B promoter adjacent to the TTF-1
binding sites and enhances promoter activity (5). The HNF3 family,
consisting of isoforms, ,
, and
, also act as enhancers for
the lung-specific CCSP gene (14, 15) as well as numerous liver-specific
genes (16).
In liver and thyroid, respectively, HNF3 and TTF-1 have been implicated
in the response of tissue-specific genes to hormones and other agents.
Down-regulation of hepatic cholesterol 7-hydroxylase by insulin and
phorbol ester localizes to the promoter region, which contains a HNF3
binding sequence (17), and an intact HNF3 binding site is necessary for
glucocorticoid regulation of IGFBP-1 (18). In thyroid cells,
down-regulation of thyroid-specific genes by thyrotropin, cAMP, v-Ras
activation, interferon-
, or phorbol ester is associated with a
decrease in either TTF-1 content (19, 20) or binding activity (10, 21,
22). At present there is no information regarding the possible role of
TTF-1 and/or HNF3 in hormonal modulation of lung-specific proteins.
Development of the fetal lung, including synthesis of surfactant
components, is regulated by a variety of hormones and other factors
(23). Expression of the SP-B gene is increased by glucocorticoids and
activators of cAMP and inhibited by transforming growth factor-, tumor necrosis factor-
, insulin, and phorbol ester (2, 24-27). The
mechanism of action of 12-O-tetradecanoylphorbol-13-acetate (TPA) on SP-B gene expression was initially examined in pulmonary adenocarcinoma NCI H441 cells, and evidence was obtained for
post-transcriptional regulation mediated by cis-acting
elements in the 3
-untranslated region (24, 28). Subsequent studies
found that TPA decreased the transcription rate of the SP-B gene in
both H441 cells and fetal lung (29), and a deletional study of the SP-B
gene promoter localized TPA responsiveness to the proximal promoter
region (30). This region (
140/+7 bp) of the promoter does not contain
AP-1 sites which are known to mediate many effects of TPA through
binding of the nuclear protooncogenes fos and
jun. Since the TPA-responsive region of the promoter
includes the binding sites for both TTF-1 and HNF3, which are known to
activate SP-B gene transcription, we proposed that the inhibitory
effects of phorbol ester were mediated through one or both of these
transcription factors.
In the present report we have further localized TPA responsiveness to the TTF-1 and HNF3 cis-acting elements of the SP-B promoter and demonstrate that TPA treatment causes a loss of both transcription factors from the nucleus and their accumulation in the cell cytoplasm. Preliminary results from this study have been reported in abstracts (31, 32).
Cell culture media, antibiotics, and fetal calf serum were obtained from the Cell Center Facility, University of Pennsylvania. Restriction and other DNA enzymes were purchased from Promega Corp. (Madison, WI) or from Pharmacia Biotech Inc. TPA, hormones, and other biochemicals were obtained from Sigma. Reinforced nitrocellulose membrane (Duralose) and pBluescript were purchased from Stratagene (La Jolla, CA). NCI H441, HeLa, and A549 cells were obtained from American Type Culture Collection (Rockville, MD). Radioisotopes and enhanced chemiluminescence detection system were ordered from NEN Life Science Products.
AntibodiesPolyclonal monospecific anti-SP-B, which
recognizes both precursor and mature forms of human SP-B, was prepared
in rabbits using an organic lipid extract of bovine surfactant
(Surfactant TA; Ross Laboratories, Columbus, OH) as described
previously (33) and obtained from M. Beers (University of Pennsylvania,
Philadelphia). Polyclonal anti-TTF-1 antibody was a gift from P. Minoo
(University of Southern California, Los Angeles, CA). Antibodies to
human HNF3, HNF3
, and HNF3
were obtained from J. E. Darnell (Rockefeller University, New York).
A 600-bp 5 end human SP-B cDNA probe
was obtained by digesting full-length cDNA with BamHI
(34). A 500-bp TTF-1 probe was excised by EcoRI digestion
from a full-length mouse TTF-1 cDNA obtained from R. Di Lauro
(Naples, Italy). A 620-bp rat HNF3 cDNA, which shares >90%
homology with the human counterpart and contains the homeodomain region
of HNF3
, was excised by digesting Pl-H3
(gift of W. S. Chen,
Rockefeller University, New York) with EcoRI and
BamHI. Randomly labeled radioactive probes were generated using [32P]dCTP and Ready To Go DNA labeling kit from
Pharmacia.
Plasmid RSV-CAT contains the Rous sarcoma virus
(RSV) long terminal repeat (LTR) promoter enhancer expressing the
bacterial chloramphenicol acetyltransferase (CAT) gene (35). The
promoterless pOOCO contains the CAT gene linked to SV40 polyadenylation
signal and small t intron in pUC 18 vector (36). Plasmid pOTCO harbors herpes simplex thymidine kinase (TK) promoter fragment (109 to +55
nucleotides) inserted into the BglII site of pOOCO (37). The
plasmid 6RL expressing lacZ under the RSV-LTR
promoter-enhancer was used as control for transfection efficiency in
experiments with plasmids containing CAT (38). We have previously
described preparation of the plasmid
-1039/+431CO
n containing SP-B genomic DNA
linked to CAT (30).
A series of SP-B 5-flanking sequences (
404/
35,
404/
255,
217/
141,
140/
65 and
217/
53) were prepared by polymerase chain reaction amplification and confirmed by sequencing (Table I).
These DNA fragments were linked to pOTCO. TTF-1, HNF3, and AP-2
oligonucleotides were synthesized commercially (Integrated DNA
Technologies, Inc. Coralville, IA) and were purified by Bio-Gel columns. Double-stranded (ds) TTF-1 oligonucleotides were designed with
5
HindIII and 3
SalI compatible ends, while
HNF3 ds oligonucleotides were made with 5
SalI and 3
XbaI compatible ends (Table I). After annealing, the ds
oligonucleotides were cloned into compatible ends in pOTCO. For the
TTF-1/HNF3 construct, the two oligonucleotides were linked followed by
ligation into the HindIII/XbaI sites of pOTCO.
The presence and orientation of inserts were confirmed by sequencing
recombinant plasmids using a Sequenase kit (U. S. Biochemical
Corp.).
|
The NCI H441 and A549 cells were grown in Dulbecco's modified Eagle's medium and HeLa cells were grown in modified Eagle's medium. Both media were supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and fungizone (2.5 µg/ml). H441 cells were plated 1-2 days prior to treatment with combinations of dexamethasone (50 nM), TPA (0.1-30 nM), and 8-bromo-cAMP (0.1 mM) plus isobutylmethylxanthine (0.1 mM). For experiments involving dexamethasone plus TPA treatment, cells were induced first with dexamethasone (50 nM) for ~24 h. In time course experiments cells were treated with TPA for different time periods (1, 2, 4, 6, 8, and 24 h) prior to harvest.
Transient TransfectionsTest plasmid DNA (2.5 µg) was
mixed with 1 µg of internal control plasmid 6RL and coprecipitated by
the CaCl2 procedure (37). Total DNA in each treatment,
including vector alone, was kept constant at 5 µg with the addition
of Bluescript plasmid. Coprecipitates were added directly to cells and
incubated for 18-20 h at 37 °C. Subsequently cells were washed
twice with phosphate-buffered saline, RPMI medium with 10% fetal
bovine serum added, and incubation continued for 48-54 h. To test the
effect of TPA on expression of different test plasmids, cells were
grown in 10 nM TPA following transfection. Total protein,
-galactosidase, and CAT assays were determined in cells as described
(30). CAT activity was normalized to
-galactosidase activity for
comparisons between different plasmid constituents in untreated
cultures. Since TPA treatment increases the expression of
-galactosidase, comparisons of CAT activity in the presence and
absence of TPA used non-normalized data.
Total RNA was
extracted from H441 cells grown in TPA + dexamethasone by the acidic
phenol-guanidinium isothiocyanate method (39) and poly(A)+
RNA was isolated using the PolyATtract® mRNA isolation system (Promega Corp.). Either 20 µg of total RNA or 2 µg of
poly(A)+ RNA was used for Northern blot analysis. Contents
of SP-B, TTF-1, HNF3, and -actin mRNAs were determined by
hybridizing at 42 °C with labeled cDNA probes. For dot blot
hybridization analysis, serial dilutions of RNA were immobilized on
membranes using a 96-well dot blot apparatus and hybridized as above.
Blots were exposed to DuPont reflection film with intensifying screens
for 1-4 days. The autoradiograms were scanned using a densitometer (Hoeffer GS300, Hoeffer Scientific Instruments, San Francisco, CA) and
relative densities calculated from the linear portions of the
dose-response curve for each RNA sample.
Nuclear
extracts were prepared from cells following the method of Dignam
et al. (40) with minor modifications. Briefly, H441 cells
(~108) were washed twice in cold phosphate-buffered
saline and resuspended in 1 ml of cold buffer A (10 mM
HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride,
0.1% Nonidet P-40). After 10 min on ice the lysate was centrifuged at
500 × g for 3 min. The supernatant was designated as
the cytoplasmic fraction and was stored at 80 °C at 10 mg/ml. The
pellet was resuspended in buffer A and incubated on ice for 10 min
followed by centrifugation at 500 × g for 5 min to
obtain a purified nuclear pellet. Nuclear extract was isolated by
resuspending the pellet in 25 µl of buffer B (25 mM
HEPES, pH 7.6, 1 mM KCl, 20% glycerol, 0.1 mM
EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mM each of leupeptin and pepstatin) with
frequent vortexing for 15 min at 4 °C. After centrifuging at
20,000 × g for 20 min, the supernatant was dialyzed
against 25 mM HEPES, pH 7.6, 1 mM KCl, 20%
glycerol, 0.1 mM EDTA, 0.5 mM DTT, and extracts were stored at
80 °C as 1 µg/µl aliquots.
A modified
method of Hennighausen and Lubon (41) was adopted to identify
DNA-protein interactions. The end-labeled probe (10,000 cpm) was
incubated with 2 µg of nuclear protein in a reaction mix with binding
buffer (0.5 µg of poly(dI-dC), 0.5 µg of sonicated salmon sperm
DNA, 10 mM HEPES, pH 7.6, 80 mM KCl, 1 mM EDTA, 1 mM EGTA, and 6% glycerol) and
incubated on ice for 20 min. For cytoplasmic EMSA experiments the same
amount of probe was incubated with 20 µg of cytoplasmic protein with
binding buffer. For supershift analysis, the probe and nuclear extract
were incubated for 15 min on ice in a total volume of 25 µl and for
an additional 15 min at 22 °C after addition of 1 µl of antibody
(TTF-1, HNF3, HNF3
, or HNF3
, diluted 1:10 or 1:100) or
preimmune antisera. For competitor analysis cold probes (1 ×, 10 ×,
and 100 ×) were added along with labeled probe, nuclear extract, and
binding buffer and incubated for 15 min on ice. Products were analyzed
on low ionic strength gels (4.5 mM Tris borate, 1 mM EDTA, pH 8.0), 5.0% Long Ranger (FMI Bioproducts,
Rockland, ME) with 0.25% glycerol and autoradiography performed. To
quantitate the amount of probe in a retarded complex, the area on the
gel with a retarded band was excised and counts/min determined in a
scintillation counter.
The 76- and 77-bp
fragments of the SP-B gene 5-flanking sequence corresponding to
140/
65 and
217/
141 bp upstream of the transcription start site
and the commercially synthesized double-stranded oligonucleotides
designated TTF-1, HNF3, and AP-2 (Table I) were used as probes for
EMSA. Mixtures of complementary oligonucleotides were heated initially
to 95 °C for 5 min and were annealed by incubating serially at
65 °C, 37 °C, and on ice for 10 min each. 1-2 pmol of DNA or
oligonucleotides were end-labeled with [
-32P]ATP using
T4 polynucleotide kinase (Promega Corp.) and purified using Sephadex
G-50 columns.
SouthWestern blot analysis was done following the modified method of Singh et al. (42). Nuclear extracts were dialyzed at 4 °C in excess volume of dialysis buffer without glycerol for 6 h with two to three buffer changes. Twenty-five µg of protein of nuclear extracts were separated on 12% SDS (TTF-1 and AP-2) or 4.5-14% gradient native (HNF3) PAGE, and proteins were transferred electrophoretically to Duralose membrane as described (33). Membranes were blocked in 3% milk powder in Tris-buffered saline and denatured with serial dilutions (1.5, 0.75, 0.375, 0.18, 0.09 M) of guanidium chloride in HEPES, pH 7.0, buffer. Denatured membranes were blocked again for 30 min and incubated overnight with end-labeled TTF-1, HNF3, or AP2 ds oligonucleotides in binding buffer (0.2 M Tris, pH 7.2, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml of poly(dI-dC), and 1 µg of sonicated salmon sperm DNA). Blots were washed and exposed to x-ray film with intensifying screen for 4 to 5 days.
ImmunolocalizationH441 and HeLa cells were plated on 10-cm
culture dishes at ~30-35% confluence; after 1 day the cells were
treated with 10 nM TPA for 24 h. Cells were fixed in
methanol for 20 min at 20 °C, washed twice with phosphate-buffered
saline, and immunostained with rabbit polyclonal anti-SP-B, -TTF-1,
-HNF3, or -HNF3
antibodies (1:500 dilution) or non-immune serum
(1:250) overnight at 4 °C, followed by CY3-conjugated goat
anti-rabbit IgG secondary antibody treatment for 2 h at 22 °C.
Stained cells were observed on an inverted microscope with a
fluorescent light source and photographed.
A cytoplasmic fraction
was recovered from H441 cells, and 25 µg of protein were separated by
SDS-PAGE, and proteins were transferred electrophoretically to Duralose
membranes. Immunoblotting was performed with antibodies to HNF3,
HNF3
(1:1000) and HNF3
(1:500), and goat anti-rabbit IgG antisera
conjugated with horseradish peroxidase (1:10,000). Immunoreactive
products were detected on a x-ray film using enhanced chemiluminescence
according to the manufacturer's directions (NEN Life Science
Products). Bands were quantitated by scanning densitometer.
In all experiments we included two to three replicates, and the experimental results were confirmed in two or more experiments. Data analysis was performed with Statview 512+ (Abacus Concepts, Calabasas, CA) and analyzed by Student's t test or analysis of variance, compared by Fisher Exact Test. Data are expressed as mean ± S.E. except where otherwise noted.
We have shown previously that treatment of
transfected H441 cells with TPA inhibited the expression of CAT
reporter gene driven by the SP-B gene promoter fragment 1039/+431
(30); similar responsiveness was found with lacZ as a
reporter gene.2 Deletional
studies using SP-B-CAT constructs localized TPA responsiveness to the
proximal promoter (30), however, studies of TPA inhibition with further
deletions of the 5
-flanking region were not possible because of low
basal SP-B promoter activity. We therefore used the heterologous TK
minimal promoter to test TPA responsiveness of linked SP-B DNA elements
that did not contain the SP-B gene TATA box (
33/
30).
To establish TPA responsiveness of SP-B DNA fragments linked to a
heterologous promoter, we initially performed TPA dose-response studies. H441 cells were grown with different concentrations of TPA for
48 h after transfecting with SP-B(1039/+431)CAT or
with SP-B(
404/
35)TK-CAT. Inhibition was comparable with
the two constructs; half-maximal inhibition occurred at ~0.5 nM TPA, and CAT activity was 10% of control at 10 nM TPA (Fig. 1A).
Cell specificity of the TPA response was examined in H441 and A549 cells, both derived from lung adenocarcinomas, and HeLa cells. After transfection with TK-CAT, TPA treatment (10 nM, 24 h) decreased CAT activity in H441 cells, increased activity in A549 cells, and had no effect in HeLa cells (Fig. 1B). Compared with these control results, transfection with plasmids containing the SP-B DNA fragment resulted in TPA inhibition of CAT activity in H441 and A549 cells but did not affect the activity in HeLa cells. These findings are consistent with the previous observation of lung cell line specificity of SP-B promoter activity (30) and localization of cell-specific enhancer elements in the proximal sequence (5, 30).
To localize more precisely the region for TPA responsiveness, we
transfected H441 cells with plasmids containing different fragments of
SP-B proximal promoter, generated by polymerase chain reaction, linked
to TK-CAT (Fig. 1C). In these experiments TPA reduced the
strength of TK promoter alone (TK-CAT) to ~50% of control. In cells
transfected with plasmids containing TK promoter plus SP-B fragments
404/
35,
140/
65, and
217/
53, TPA reduced CAT activity to
<12% of control, similar in magnitude to the inhibition observed with
the SP-B promoter (
1039/+431-CAT). TPA did not decrease CAT
expression for constructs containing the SP-B sequences
404/
255 and
217/
141. These results indicate that the TPA responsive element(s)
in the SP-B gene reside within bp
140/
65 and that TPA
responsiveness occurs independent of promoter function consistent with
the expected properties of a regulated enhancer element.
The proximal 5-flanking region of SP-B gene contains 2 TTF-1 binding sites and an HNF3 binding site that function as cell line-specific enhancer elements (5). To determine whether TPA acts at
these sites, we transfected H441 cells with plasmids containing the two
TTF-1 binding sites (
112/
89) and/or the HNF3 binding site
(
89/
72) linked TK-CAT (see Table I).
Constructs containing both the HNF3 and TTF-1 binding sites increased
CAT activity ~10-fold compared with TK-CAT alone, comparable with the
enhancer response observed with SP-B fragment
140/
65 (Fig.
2A). Promoter activity of
TK-CAT was enhanced 4-5-fold with plasmids containing either the TTF-1
or HNF3 binding sites. These findings suggest that the TTF-1 and HNF3
elements influence SP-B gene transcription independently and
additively, consistent with data using site-directed mutagenesis of the
promoter (5).
Responsiveness of the TTF-1 and HNF3 sequences to TPA is shown in Fig.
2B. TPA treatment reduced CAT activity by 90% in cells transfected with plasmids containing enhancer sequences compared with
~25% decrease in cells transfected with TK-CAT alone. We observed a
similar reduction in CAT activity (~85%) in cells transfected with
the SP-B(
1039/+431)CAT construct. Thus, TTF-1 and HNF3
sites appear to be similarly sensitive to TPA.
We used EMSA to investigate the effect of TPA on binding
activity of nuclear factors for the SP-B promoter. Transcriptional down-regulation of SP-B could result from binding of a TPA-induced inhibitory protein and/or from reduced binding of existing
transcription factors. Nuclear extracts were isolated from treated and
untreated H441 cells, and EMSA was carried out using as a probe the
SP-B(140/
65) DNA fragment, which is TPA-responsive by
transfection analysis. We observed three retarded complexes using
nuclear extracts from cultured cells (Fig.
3A). These bands were not
affected by treatment of cells with dexamethasone and/or cyclic AMP,
hormones which increase SP-B gene expression, but were consistently
reduced after TPA treatment (Fig. 3A). Prior treatment of
nuclear extracts with proteinase K or 10% SDS, or preincubation at
65 °C for 10 min, completely eliminated the retarded bands (data not
shown), suggesting that the binding factors are heat-labile proteins.
EMSA was also performed with labeled upstream (
217/
141 Fig.
3B) and downstream (
57/
38, see Fig. 5A) SP-B
DNA fragments; single bands were observed with both probes, and these
did not change with TPA treatment. Thus, the EMSA data indicate that
the TPA effect is associated with loss of proteins binding to the
140/
65 region of the SP-B gene.
Retarded Bands on EMSA Are TTF-1 and HNF3
We determined the
identity of the TPA-responsive proteins in the EMSA analysis using
competition (Fig. 4A) and
supershift (Fig. 4B) approaches. For competitor analysis,
end-labeled SP-B (140/
65) probe was incubated with 1 ×, 10 ×, and
100 × excess of unlabeled ds oligonucleotides corresponding to
TTF-1 or HNF3 binding sites. Excess TTF-1 oligonucleotide eliminated
the lowest of the three retarded bands, while the upper two bands were
either reduced or eliminated with the HNF3 oligonucleotide (Fig.
4A). These results tentatively identified the bottom band as
a TTF-1-DNA complex and the top two bands as HNF3-DNA complexes.
To immunologically identify the proteins in retarded bands, labeled
SP-B (140/
65 bp) probe was incubated with nuclear extract followed
by an additional incubation with non-immune or specific antiserum (Fig.
4B). No supershifted band was observed on EMSA with the
addition of preimmune serum (Fig. 4B, non-immune serum) or
with HNF3-
antibody (not shown). The smallest of the retarded complexes was partially supershifted with TTF-1 polyclonal antibody at
both 1:10 and 1:100 dilutions (Fig. 4B, TTF-1). With HNF3
antibody, intensity of the middle band was reduced and two supershifted bands were observed; a partial shift occurred at 1:100 antibody dilution, and a complete shift was seen at 1:10 dilution (Fig. 4B, HNF3
). The higher supershifted band that was observed
with the greater concentration of HNF3
antibody may be an
heterodimeric complex of HNF3
and
proteins. Complete shift of
the top retarded band was observed with HNF3
antibody at both
concentrations (Fig. 4B, HNF3
). Thus, competitor and
supershift analyses identify the TPA-responsive bands on EMSA as TTF-1
and HNF3.
To further characterize the effect of TPA we used the TTF-1 and HNF3 oligonucleotides (Table I) as probes in EMSA experiments. We found two retarded bands with TTF-1 oligonucleotide (Fig. 5A, TTF-1) as observed previously (5); intensity of both bands was reduced significantly by 8 h of TPA treatment and was almost eliminated by 24 h. HNF3 oligonucleotide probe also produced two retarded bands, perhaps representing dimers of HNF3 isoforms (Fig. 5A, HNF3). HNF3 binding activity in both bands decreased significantly in nuclear extracts after 8 h of TPA treatment and was usually not detected after 24 h.
For comparison we also determined the binding activity of the same
nuclear extracts for the AP-2 oligonucleotide probe, representing bp
57/
38 of SP-B promoter downstream of the TTF-1 and HNF3 binding sites. EMSA with the AP-2 probe produced one strong retarded band (Fig.
5A, AP-2) and two additional retarded bands with longer exposure (not shown). None of the retarded bands with AP-2 probe were
affected by TPA treatment of cells.
To quantitate the reduction in TTF-1 and HNF3 binding activities, we excised the retarded bands from gels and counted the radioactivity. TPA treatment caused a similar, time-dependent decrease in both binding activities (Fig. 5B). The reduction was statistically significant after 8 h of TPA treatment and was <10% of control at the 24 h time point. These findings suggest that transcriptional down-regulation of SP-B by TPA results from reduced binding of TTF-1 and HNF3 transcription factors to the SP-B proximal promoter.
We confirmed the changes in DNA binding activity by SouthWestern
analysis. Nuclear extracts were isolated from H441 cells grown in TPA
for different time periods. Proteins were separated on a 12% SDS-PAGE,
transferred to membrane, and probed with radiolabeled TTF-1
oligonucleotide, which bound to a single band of expected size (~39
kDa). The autoradiographic signal decreased significantly after 8 h of TPA treatment and was very weak after 24 h (Fig. 6, TTF-1). For analysis of
HNF3, proteins were separated on a non-denaturing gel. HNF3
oligonucleotide probe bound to three distinct bands with untreated
nuclear extract (Fig. 6, HNF3), and reduced binding of probe
to all three HNF3 bands was observed with 6 h of TPA treatment.
Apparent differences between control and early TPA time points in this
gel were not reproducible and probably resulted from the variability in
protein loading noted by Coomassie staining. As a control, blots were
also hybridized with AP-2 oligonucleotide probe. A 52-kDa band was
observed, which was not significantly altered by TPA treatment (Fig. 6,
AP-2).
To examine content of the transcription factors, we carried out Western
blot analyses of nuclear extract. A satisfactory signal was not
achieved with TTF-1 antibody. Gels probed with antibodies to HNF3
and HNF3
revealed bands of ~51 and ~36 kDa, respectively, which
decreased in intensity in nuclear extracts from cells treated with TPA
for 8 and 24 h compared with control (data not shown). Thus,
decreased HNF3 DNA binding activity after TPA treatment is due to
reduced content of HNF3 proteins in nuclear extracts. Loss of
transcription factor from nuclei could result from either decreased
expression of the factor or a change in intracellular localization.
Experiments were carried out to address these two possibilities.
To
determine the effect of TPA on expression of TTF-1 and HNF3 genes, we
isolated total and poly(A)+ RNA from H441 cells grown in 10 nM TPA for different time periods. On Northern analysis,
probing with TTF-1 cDNA produced a single band of 2.6 kilobases,
which did not change in intensity with TPA treatment for 24 h
(Fig. 7A, TTF-1). Likewise a
single 2.8-kilobase, TPA-insensitive band was identified on probing
with HNF3 cDNA (Fig. 7A, HNF3). Using dot blot
hybridization analysis we found the expected reduction in SP-B mRNA
levels by 8 h of TPA treatment, whereas TTF-1 and HNF3 mRNA
levels remained unchanged over 24 h (Fig. 7B). Thus,
decreased nuclear content of TTF-1 and HNF3 is not due to reduced
expression of the genes.
TTF-1 and HNF3 Accumulate in the Cytoplasm of TPA-treated Cells
EMSA was performed with cytoplasmic fraction to examine the
effect of TPA treatment on TTF-1 and HNF3 DNA binding activities in the
cytoplasm. As with the nuclear extracts, two retarded bands were
observed with TTF-1 probe (Fig. 8,
TTF-1). A single retarded band was found with HNF3 probe on
short exposure of gels to films (Fig. 8, HNF3), and a second
band of lower mobility was seen after longer exposure (not shown). Low
levels of TTF-1 and HNF3 binding activities were present in the
cytoplasmic fractions from control cells, and activities of both
transcription factors were consistently increased with 2 h of TPA
treatment (Fig. 8). These results could reflect a change in either DNA
binding activity or content of the proteins.
To examine localization of immunoreactive HNF3, TTF-1, and SP-B in
intact H441 cells, we performed immunostaining using fluorescently labeled secondary antibodies (Fig. 9).
Control, untreated H441 cells stained with HNF3 (Fig. 9B)
or TTF-1 (Fig. 9F) antibodies showed strong fluorescence in
the nucleus and minimal cytoplasmic signal. After 24 h of TPA
treatment there was little nuclear fluorescence and the appearance of
cytoplasmic fluorescence, which was distributed primarily in a
perinuclear fashion for both HNF3
(Fig. 9D) and TTF-1
(Fig. 9H); similar results were found for HNF3
(not
shown). In general, fluorescence was stronger with HNF3 stained cells compared with TTF-1 stained cells. Using SP-B antibody a bright orange
fluorescence was observed in the cytoplasm of most control cells (Fig.
9J), which was nearly eliminated in cells treated with TPA
for 48 h (Fig. 9L). Staining of H441 cells with
non-immune serum did not produce any fluorescence (Fig. 9N).
Similarly, no fluorescence with either TTF-1 or HNF3 antibodies was
noticed in HeLa cells (not shown), confirming other reports that these cells lack both transcription factors (5). No effect of TPA treatment
on cell structure was observed by phase microscopy (left panels of Fig. 9).
Finally, we carried out Western analysis of cytoplasmic proteins using
TTF-1 and HNF3 antibodies. Similar to findings with nuclear extracts,
staining of cytoplasmic Western blots with TTF-1 antibody did not
result in satisfactory results. Blots probed with HNF3 and HNF3
antibodies identified an expected 51-kDa band and a weak band at 36 kDa, respectively; intensity of both of these bands increased with time
of TPA treatment (Fig. 10). Probing of
Western blots with HNF3
identified a single protein band of 47 kDa,
which did not change during prolonged TPA treatment. The identity of
the higher molecular weight bands observed with HNF3
and HNF3
antibodies is not known. These findings by protein blotting are in
agreement with the EMSA and Southwestern data, and together the results
suggest that TPA reduces SP-B gene transcription by causing cytoplasmic
trapping of TTF-1 and HNF3.
Previous studies of SP-B gene expression in H441 cells established the strong inhibitory effect of phorbol esters (24, 27), and we recently found that this effect involved transcriptional down-regulation mediated by regulatory elements in the proximal promoter region (29, 30). In the current study we provide evidence that TPA causes a loss of nuclear TTF-1 and HNF3, which are trans-activators of SP-B gene expression, secondary to cytoplasmic trapping of the proteins without alteration of their mRNA contents or DNA binding activities. Regulation of gene expression by coordinate effects on the intracellular distribution of two transcription factors represents a previously undescribed mechanism for the action of phorbol esters.
By deletional analysis and use of heterologous promoter constructs the
TPA responsive region of the SP-B gene was localized to bp 140/
65
upstream of transcription start site. AP1 sites, which mediate many
transcriptional responses of PKC activation, lie outside this region of
the SP-B gene, suggesting that c-Fos and c-Jun are not involved in this
TPA effect. This region of the promoter does contain binding sites for
TTF-1 and HNF3, which act as lung cell specific enhancers of SP-B gene
expression (4, 5). We initially explored two possibilities of TPA
action: 1) induction of a repressor protein binding to this region and 2) decreased binding of existing transcription factors. Down-regulation of gonadotropin-releasing hormone by TPA, for example, involves induction of a DNA·protein complex with kinetics consistent with repressor action (43). Using EMSA we were unable to detect new protein
binding to the
140/
65-bp region after TPA treatment. The three
retarded bands observed with this probe were identified as TTF-1 and
HNF3 by supershift and oligonucleotide competition approaches. The
second possibility was supported by the observation that all three
shifted bands decreased in intensity after TPA treatment.
Several lines of evidence from our study, examining both DNA binding
activity and immunoreactive protein, indicated that TPA caused a loss
of TTF-1 and HNF3 from the nucleus and accumulation in the cytoplasm.
The increase in cytoplasmic TTF-1 and HNF3 (by 2 h) preceded loss
of the factors from the nucleus, consistent with failure of nuclear
translocation as the primary event. Transport of proteins from the
cytoplasm to the nucleus is an important regulated event for many
nuclear proteins including selected transcription factors (44), and for
many proteins a sequence of basic amino acids serves as a nuclear
localization signal (45). Nuclear import can be modulated by the
phosphorylation of sites adjacent to the localization signal, either
inhibiting nuclear transport (e.g. lamin B2, protein kinase
A catalytic subunit, and viral Jun) or increasing translocation as
described for lamin A (22, 46-48). Activity of a localization signal
can also be regulated by protein-protein interaction, either blocking
import as in the case of IB
binding to NF-
B or conferring
nuclear localization as occurs with chaperones (49-51). Regulated
control of nuclear localization is important during the cell cycle, in
developmental processes such as occurs with MyoD during mesodermal
induction and with inflammatory responses mediated through NF-
B (49, 52). Nuclear localization of HNF3
has been mapped to the winged helix DNA binding domain (53), but a localization signal has not been
reported for TTF-1.
Although not directly addressed in the current study, it is likely that
changes in the phosphorylation state of TTF-1 and/or HNF3 are involved
in the response of these transcription factors to TPA treatment.
Zannini et al. (54) mapped seven serine phosphorylation sites in TTF-1 and observed phosphorylation with PKC in
vitro; however, effects of PKC on DNA binding and/or
transactivation activity were not examined. Other studies found that
TTF-1 DNA binding activity was phosphorylation-dependent
and that inactivation by phosphatase action was reversed by treatment
with protein kinase A and cAMP (10, 11). The modification of TTF-1
activity by v-Ras also appears to involve phosphorylation as indicated
by the finding that v-Ras activation excludes the protein kinase A
catalytic subunit from the nucleus (22). The HNF3 and
isoforms
are phosphorylated in HepG2 cells, but a role in nuclear localization
or transcriptional activity has not been determined (53). With regard
to TPA effects in lung cells, further studies are required to determine
whether the phosphorylation state of TTF-1 and HNF3 is altered and
whether this represents a direct or indirect effect of PKC.
A consistent finding in the current study was the parallel effect of TPA on TTF-1 and HNF3. The nature of the linkage between these two transcription factors is not known. It is possible that both proteins undergo the same alteration in phosphorylation state mediated by PKC, sharing a common mechanism for nuclear translocation. Alternatively, only one of the transcription factors could be the target of PKC-mediated phosphorylation with nuclear transport of the other factor dependent on TTF-1/HNF3 heterodimerization. Finally, PKC could mediate phosphorylation of a separate protein, which would serve to either inhibit or promote nuclear transport. It is important to note, however, that the parallel responses for the two factors does not reflect a general change in transcription factor binding to the proximal promoter, since we found no change with TPA treatment in protein binding to DNA regions just upstream and downstream of the TTF-1/HNF3 binding sites.
Our finding of TPA-induced cytoplasmic trapping of TTF-1 is supported by data from studies in thyroid cells (22). Exposure of a differentiated thyroid cell line to 16 nM TPA for 3 days reduced TTF-1 binding activity in nuclear extracts as well as the activity of transfected thyroglobulin promoter. This response was reversible and recovery of TTF-1 binding activity was not inhibited by the presence of cycloheximide. By contrast, treatment of cells with a high concentration of TPA (350 nM), which down-regulates PKC, caused relatively little loss of TTF-1 binding activity, consistent with the proposal that activation of PKC is responsible for the loss of nuclear TTF-1 binding activity. Although intracellular distribution of TTF-1 was not examined in this study, the response to TPA is consistent with cytoplasmic trapping as we have observed. It is likely that cytoplasmic trapping of TTF-1 and/or HNF3 in response to TPA also operates in regulation of other genes that are activated by these transcription factors. In the lung, TTF-1 is an important transactivating factor for expression of SP-A and SP-C in type II cells and CCSP in bronchiolar clara cells (7-9). Furthermore, TPA inhibits both SP-A (24, 27, 29) and SP-C gene expression acting at the level of transcription.3 In hepatic cells, expression of T4 binding globulin, apolipoprotein A-1, and CYP7A genes are inhibited by TPA and responsiveness mapped to promoter regions containing HNF binding sites (17, 55, 56). The possible role of altered intracellular distribution of transcription factors in the regulation of these and potentially other genes awaits further investigation.
We examined the three isoforms of HNF3 with regard to TPA
responsiveness. Using specific antibodies, we identified the and
isoforms of HNF3 in nuclear extracts by both supershift and Western
analysis; content of both isoforms increased in the cytoplasmic fraction after TPA treatment, but there was no change in levels of
HNF3
. Previous studies also identified HNF3
as the isoform binding to human and rabbit SP-B promoter (5, 15, 57); however,
Bohinski et al. (5) reported that only the
isoform was
expressed in H441 cells. The molecular weights of immunoreactive HNF
proteins in the current study were consistent with the stated specificity of the antibodies that we used. Thus, additional analysis of HNF3 isoforms is warranted.
The SP-B gene also contains three additional TTF-1 binding sequences
further upstream in the 5-flanking region (
439/
331), which
recently were shown to both bind TTF-1 and to enhance heterologous promoter activity (6). Since inhibition of SP-B promoter activity by
TPA was equivalent for plasmid constructs with or without this upstream
region (30), the proximal TTF-1 sites appear to be primary. It is
possible, however, that TTF-1 binds to the upstream sites with a higher
affinity than for the proximal sites and that the residual promoter
activity found in the presence of TPA represents the enhancer effect of
TTF-1 at the distal sites.
The physiologic relevance of TPA down-regulation of surfactant protein
gene expression may relate to lung growth and differentiation as well
as the effects of inflammatory processes in the lung. Like TPA,
transforming growth factor- and platelet-derived growth factor both
activate PKC and, in the developing lung, regulate cell proliferation,
epithelial cell differentiation, and synthesis of lung matrix proteins.
In particular, transforming growth factor-
modulates pulmonary type
II cell differentiation and decreases synthesis of both surfactant
phospholipids and associated proteins (26, 58-61). Various
inflammatory mediators, including endotoxins, cytokines, and free
oxygen radicals, modulate gene expression by activating PKC, and tumor
necrosis factor-
decreases SP-A and SP-B gene expression in studies
with both H441 cells and cultured fetal lung (27).
Our findings lead us to propose the following model for TPA-mediated effects on expression of SP-B. Exposure of lung cells to low concentrations of TPA activates PKC, resulting in phosphorylation of newly synthesized and resident TTF-1 and/or HNF3 in the cytoplasm. Phosphorylation could occur directly via PKC or indirectly through a phosphorylation cascade and results in inactivation of the nuclear translocation signal on one or both factors. With translocation to the nucleus blocked, cytoplasmic levels of the factors increase and nuclear content decreases secondary to degradation and/or nuclear export. Transactivation of SP-B gene expression diminishes to a low level reflecting the strong enhancer role of the two factors. This model predicts that reversibility of TPA inhibition does not require new protein synthesis, and this has been observed in preliminary experiments.4 An alternative explanation for cytoplasmic trapping of the transcription factors could involve phosphorylation of proteins serving as chaperones or transport functions rather than direct phosphorylation of TTF-1/HNF3. The mechanism for cytoplasmic trapping is currently under investigation.
In summary, we have found that TPA acts post-translationally to
down-regulate SP-B gene expression by cytoplasmic trapping of both
TTF-1 and HNF3 without a loss of DNA binding activity. This mechanism
of phorbol ester regulation of gene expression is distinct from
previously described processes involving AP-1, AP-2, NF-B, and
octomer-binding protein. Cytoplasmic trapping of TTF-1 and HNF3 may
also be involved in PKC-mediated down-regulation of thyroid-specific
and liver-specific genes, respectively.
We thank Kathy Notofrancesca and Henry Shuman for assistance with immunofluorescence, Sree Angampalli and Yue Ning for technical assistance, Hannah Jones for participation in the transfection studies, and Sandra Mosiniak for preparing the manuscript. We also thank J. E. Darnell, P. Minoo, W. S. Chen, and R. Di Lauro for providing antibodies or cDNA.