Cell-specific involvement of HNF-1
in
1-antitrypsin gene expression in human respiratory
epithelial cells
Chaobin
Hu and
David H.
Perlmutter
Departments of Pediatrics and Cell Biology and Physiology,
Washington University School of Medicine, and Division of
Gastroenterology and Nutrition, St. Louis Children's Hospital, St.
Louis, Missouri 63110
 |
ABSTRACT |
The
synergistic action of hepatocyte nuclear factor (HNF)-1
and HNF-4
plays an important role in expression of the
1-antitrypsin (
1-AT) gene in human
hepatic and intestinal epithelial cells. Recent studies have indicated
that the
1-AT gene is also expressed in human pulmonary
alveolar epithelial cells, a potentially important local site of the
lung antiprotease defense. In this study, we examined the possibility
that
1-AT gene expression in a human pulmonary
epithelial cell line H441 was also directed by the synergistic action
of HNF-1
and HNF-4 and/or by the action of HNF-3, which has been
shown to play a dominant role in gene expression in H441 cells. The
results show that
1-AT gene expression in H441 cells is
predominantly driven by HNF-1
, even though HNF-1
has no effect on
1-AT gene expression in human hepatic Hep G2 and human
intestinal epithelial Caco-2 cell lines. Expression of
1-AT and HNF-1
was also demonstrated in primary
cultures of human respiratory epithelial cells. HNF-4 has no effect on
1-AT gene expression in H441 cells, even when it is
cotransfected with HNF-1
or HNF-1
. HNF-3 by itself has little
effect on
1-AT gene expression in H441, Hep G2, or
Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1
in
1-AT gene
expression in a cell line and primary cultures derived from human
respiratory epithelium.
protease inhibitors; pneumocytes; tissue-specific gene expression
 |
INTRODUCTION |
1-antitrypsin
(
1-AT) is an ~55-kDa serum glycoprotein that inhibits
the destructive neutrophil proteases elastase, cathepsin G, and
proteinase 3 (28). It is the archetype of a family of serum proteins, many of which are serine protease inhibitors and are
therefore called serpins (6, 18). Lack of elastase
inhibitory activity in the lung is thought to be responsible for the
predisposition of
1-AT-deficient individuals to
destructive lung disease/emphysema (20). Moreover,
functional inactivation of
1-AT by active oxygen intermediates released during cigarette smoking is believed to play a
role in pulmonary emphysema in
1-AT-sufficient
individuals (20). A subgroup of individuals with the
classical form of
1-AT deficiency are predisposed to
liver injury and hepatocellular carcinoma presumably because of the
hepatotoxic effect of the mutant
1-AT molecule within
liver cells (28).
Plasma
1-AT is predominantly derived from the liver, as
shown by studies of changes in
1-AT allotypes after
orthotopic liver transplantation (1, 16). Synthesis of
1-AT is abundant in human hepatoma cell lines and in
human hepatocytes in primary culture, and
1-AT mRNA is
extremely abundant in hepatocytes in human liver, as determined by in
situ hybridization analysis (26). There is also evidence
for extrahepatic sites of synthesis, including blood monocytes, tissue
macrophages, and intestinal epithelial cells (26, 29). Our
recent studies have indicated that expression of the
1-AT gene in hepatocytes and enterocytes is
predominantly driven by the synergistic action of hepatocyte nuclear
factor (HNF)-1
and HNF-4 (17). HNF-1
plays an
important role in the increase in
1-AT gene expression
that accompanies differentiation of enterocytes from crypt to villous
tip. HNF-1
, in the absence or presence of HNF-4, has no effect on
1-AT gene expression in hepatocytes or enterocytes
(17).
Several recent studies have indicated that
1-AT is also
synthesized in human pulmonary airway epithelial cells, including the
Calu-3 and A549 cell lines and normal bronchial epithelial cells in
primary culture (9, 34). Previous studies have shown
1-AT mRNA in bronchial and bronchiolar epithelial cells
of human fetal lung by in situ hybridization analysis
(22). Expression of
1-AT by airway
epithelial cells could potentially constitute an important component of
the antiprotease defense system locally within the lung.
In this study, we used the human pulmonary epithelial cell line H441 as
well as nuclear extracts and RNA from human respiratory epithelial
cells in primary culture to examine the possibility that
1-AT gene expression in pulmonary epithelial cells is
directed by the synergistic action of HNF-1
and HNF-4 and/or by the
action of more pneumocyte-specific transcription factors such as HNF-3, which have been shown to play an important role in gene expression in
pneumocytes (3-5, 30).
 |
MATERIALS AND METHODS |
Plasmid constructs.
Progressive deletions of the human
1-AT promoter region
from
1951 to
2 were generated by PCR amplification using the human genomic
1-AT clone hAAT7zf (kindly provided by Dr. K. Ponder, St. Louis, MO) as template. These PCR fragments were subcloned into the KpnI/HindIII site of the pGL3 basic
luciferase reporter vector (Promega, Madison, WI) to generate
1-AT promoter-luciferase fusion plasmids. The Mut-2
plasmid, which is an
1-AT (
137 to
2)
promoter-luciferase chimera with a mutation in the HNF-1-binding site,
has been previously described (17). Our previous
electrophoretic mobility shift assay (EMSA) studies showed a marked
decrease in binding of HNF-1
and HNF-1
to this plasmid compared
with the plasmid without alterations in the HNF-1-binding region
(17). The Mut-3 plasmid, which contains a mutation in
HNF-3 binding, was generated for these studies. For this plasmid, we
altered two nucleotides in the HNF-3-binding site at
101 to
93
(5'-TGTTTGCTC-3') within the
1-AT (
137 to
2)-promoter luciferase chimera. The highly conserved thymidines at
101 and
97 were converted to adenines. Results of EMSA indicated
that these mutations disrupted binding to nuclear proteins in extracts
from Hep G2, Caco-2, and H441 cells (data not shown).
Murine HNF-1
and HNF-1
expression plasmids (25)
provided by Dr. T. C. Simon (St. Louis, MO) were subcloned into
the pSG5 plasmid (Stratagene, La Jolla, CA). Full-length HNF-4 cDNA
subcloned into the pMT2 expression vector (31) was
provided by Dr. J. Rottman (St. Louis, MO). HNF-3
and HNF-3
expression plasmids pPac HNF-3
and pPac HNF-3
(12)
were provided by Dr. G. Suske (Marburg, Germany). The pRL-TK vector was
purchased from Promega.
Cell culture, DNA transfections, and luciferase assay.
The Hep G2, HeLa, and Caco-2 cells were grown as previously described
(26). H441 cells were purchased from American Type Culture
Collection and maintained in RPMI 1640 medium with 2 mM L-glutamine, sodium bicarbonate (0.5 g/l), 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% fetal bovine serum. This cell line is derived from a human pulmonary papillary adenocarcinoma. It expresses surfactant proteins A and B. Electron-microscopic analysis shows the
presence of multilamellar bodies and cytoplasmic structures resembling
Clara cell granules in these cells (27). The
promoter-luciferase plasmids were cotransfected with the indicated
amount of HNF-1
, HNF-1
, HNF-3
, HNF-3
, and/or HNF-4
expression vectors. A pGL3-basic vector was used as a negative control
and a pGL3-control vector containing SV40 promoter and enhancer
sequences was used as a positive control in all transfection
experiments. The pRL-TK vector was also included in all transfections
as an internal control for transfection efficiency as monitored by the
Promega dual-luciferase reporter assay system. Cells (1-1.5 × 106) were plated on 60-mm tissue culture dishes or
six-well plates and incubated for 24 h before transfection.
Duplicate or triplicate dishes were transfected using the FuGENE 6 transfection reagent or the calcium phosphate-DNA coprecipitation
method. For the calcium phosphate method, the cells were shocked with
15% glycerol 24 h after transfection. Cells were harvested
48 h after transfection. Luciferase activity was detected on the
Turner Designs luminometer (model TD-120/20, Promega).
Preparation of double-strand oligonucleotides and labeling.
Oligonucleotides were synthesized on an Applied Biosystems DNA
synthesizer in the Nucleic Acid Chemistry Laboratory, Biotechnology Center, Washington University School of Medicine. The potential HNF-1-binding sequence corresponds to the
1-AT promoter
sequence from
87 to
52
(5'-ATAACTGGGGTGA-CCTTGGTTAATATTCACCAGCAG-3'). The
potential HNF-4-binding sequence corresponds to the sequence from
128
to
93 (5'-ATCCAGCCAGTGGACTTAGCCCCGTTTG-3') with respect to the
downstream transcriptional start site. The HNF-3 consensus sequence
corresponds to 5'-GCCCATTGTTTGTTTTAAGCC-3', and the HNF-4 consensus
sequence corresponds to 5'-GGAAAGGTCCAAAGGGCGCCTTG-3'. The
oligonucleotides were purified by denaturing PAGE, annealed at 65°C
for 1 h, allowed to cool slowly at room temperature, and then
labeled using [
-32P]dCTP and the Klenow fragment of
DNA polymerase I. Radiolabeled oligonucleotides were separated
from free nucleotide by nondenaturing PAGE.
EMSA.
Complementary oligonucleotide probes for the HNF-1 and HNF-4 sequences
in the
1-AT promoter as well as consensus HNF-1, HNF-3, and HNF-4 sequences were labeled with [32P]dCTP by the
3'-end filling reaction, and 2 × 104 counts/min were
incubated for 20-40 min at room temperature with nuclear extracts
from Hep G2, Caco-2, H441, and human respiratory epithelial cells
(25). The nuclear extracts from H441, Hep G2, and Caco-2
cells were prepared according to the protocol described by Dignam et
al. (11). Nuclear extracts from HeLa cells (HeLa Scribe
nuclear extract, in vitro transcription grade) were purchased from
Geneka Biotechnology (Montreal, PQ, Canada). Nuclear extracts from
human respiratory epithelial cells in primary culture, also prepared by
the method of Dignam et al., were kindly provided by Dwight Look (St.
Louis, MO). Nuclear extracts were resuspended in buffer so that a
10-µl reaction volume contained a final concentration of 60 mM KCl,
25 mM HEPES, pH 7.6, 1 mM dithiothreitol, 0.1 mM EDTA, 7.5% glycerol,
1 µg of poly(dI-dC), and 2% polyvinyl alcohol. Unlabeled
oligonucleotide in molar excess was used in designated experiments.
Nuclear extract dialysis buffer and BSA were used as negative controls.
Rabbit polyclonal anti-HNF-1
TC 284 (kindly provided by M. Yaniv and
M. Pontoglio, Paris, France), anti-HNF-1
[kindly provided by G. Ryffel, Essen, Germany (35)], anti-HNF-3
and HNF-3
(kindly provided by R. H. Costa, Urbana, IL), and anti-HNF-4 (kindly provided by I. Talianidis, Crete) were used in designated experiments by incubation with nuclear extracts for 2 h at 4°C before addition of radiolabeled oligonucleotide probe. The products were analyzed on 5% polyacrylamide-2.5% glycerol gels cast in 0.5×
Tris-borate-EDTA.
RT-PCR for analysis of RNA levels.
Total RNA from H441, Hep G2, and Caco-2 cells was isolated with
the RNeasy kit (Qiagen, Hilden, Germany) and then digested with
RQ1 RNase-free DNase (Promega). Poly(A)+ RNA was isolated
using oligo(dT) cellulose column chromatography (32).
Poly(A)+ RNA from human respiratory epithelial cells was
kindly provided by Drs. Theresa Joseph and Dwight Look. The RNA was
then subjected to RT-PCR using the Access RT-PCR system (Promega). The
primers for amplification were based on previous studies (10,
12): 5'-GAAAGCAACGGGAGATCCTCCGAC-3' (sense) and
5'-CCTCCACTAAGGCCTCCCTCTCTTCC-3' (antisense) for HNF-1
,
5'-GTAGACAGTAGGGGCTC-3' (sense) and 5'-GGGGAATCCTTTAAACGG-3' (antisense) for HNF-3
, 5'-GCCTGAGCCGCGCTCGGGAC-3' (sense)
and 5'-GGTGCAGGGTCCAGAAGGAG-3' (antisense) for
HNF-3
, 5'-CTTCCTTCTTCATGCCAG-3' (sense) and
5'-ACACGTCCCCATCTGAAG-3' (antisense) for HNF-4,
5'-TCACGTCTAGAACAGTGAATCGAC-3 (sense) and
5-GTGGGCTGCAGTACCAGCTCAACC-3' (antisense) for
-AT, and 5'-AGGGCTGAGTGTTCTGGGATTTC-3' (sense) and
5'-GGTTACGGCAGCACTTTTATTTTT-3' (antisense) for
-actin. Minimal
modifications were applied to the manufacturer's instructions for
the conditions: annealing was done at 60-65°C depending on
melting temperature of oligonucleotides, the concentration of
MgSO4 was 1 mM, and 35 cycles were used for amplifications. PCR products were separated on 1% agarose gels and
visualized by ethidium bromide staining.
 |
RESULTS |
Expression of
1-AT in the human pulmonary epithelial
cell line H441.
RT-PCR analysis showed the presence of
1-AT RNA in the
H441 cell line as well as in Hep G2 and Caco-2 cells (Fig.
1). This data indicated that we could use
the H441 cell line as a model for expression of the
1-AT
gene in pulmonary epithelium.

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Fig. 1.
Expression of 1-antitrypsin
( 1-AT) in H441, Hep G2, and Caco-2 cells. Total cellular
poly(A)+ RNA from H441, Hep G2, and Caco-2 cells was
subjected to RT-PCR analysis for 1-AT and -actin.
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To determine whether there were cis-acting regulatory
elements within the upstream flanking region for expression of the
1-AT gene in H441 cells and whether these differed from
those responsible for expression in Caco-2 and Hep G2 cells, we used
seven
1-AT-luciferase fusion plasmids differing in the
length of the
1-AT 5'-flanking sequence upstream of the
luciferase coding sequence (Fig. 2). We
examined the expression of these plasmids in H441, Caco-2 and Hep G2
cells. The results show that the general localization of cis-acting elements in H441 cells is similar to that in
Caco-2 and Hep G2 cells. There is a drop in expression on deletion from
991 to
661,
490 to
270,
270 to
137, and
137 to
2,
indicating the presence of cis-acting elements in these
regions. The major positive elements were in the two most proximal
regions. We decided to first examine the most proximal region,
137 to
2, in H441 cells in more detail. Previous studies showed that this
region contains most of the elements responsible for tissue-specific regulation of transcription (17 and references therein). Moreover, all
the potential HNF-1-, HNF-4-, and HNF-3-binding sequences are found in
this proximal promoter region.

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Fig. 2.
Expression in H441, Hep G2, and Caco-2 cells of
1-AT-luciferase fusion plasmids carrying progressive
deletions of 5'-flanking sequences. Left: schematic drawings
of deletions. Right: results, reported as relative
luciferase activity, which represents percent maximal activity compared
with activity of the plasmid carrying the longest 5'-flanking sequence,
assumed to give 100% luciferase activity. Cells were cotransfected
with 1-AT promoter-luciferase fusion plasmids (7.5 µg)
and the pRL-TK control plasmid (0.1 µg). For H441 cells, the FuGENE 6 transfection reagent was used. The calcium phosphate-DNA
coprecipitation method was used for Hep G2 and Caco-2 cells. After
48 h, firefly and Renilla luciferase activities were
measured and are reported as means ± SD for 3 separate
determinations.
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Binding of nuclear proteins from H441 cells to the proximal
1-AT promoter.
Because our previous studies indicated that HNF-1 and HNF-4 and
cis-acting HNF-1- and HNF-4-binding elements in the proximal upstream flanking region of the
1-AT gene play a major
role in expression of
1-AT in one extrahepatic site,
intestinal epithelium, we first used EMSA to determine whether HNF-1
and HNF-4 are expressed in H441 cells and whether they bind to the
HNF-1 (
87 to
52)- or HNF-4 (
128 to
93)-binding regions of the
proximal
1-AT promoter (Fig.
3). The results show that the
HNF-1-binding region (
87 to
52) forms a single complex with nuclear
extracts from H441 cells compared with two complexes present in Hep G2,
undifferentiated Caco-2 (day 1), and differentiated Caco-2
(day 4) cells. Our previous studies showed that the more
slowly migrating complex corresponds to HNF-1
and the more rapidly
migrating complex corresponds to HNF-1
(17). These
previous studies also showed an increase in the relative proportion of
HNF-1
compared with HNF-1
during differentiation of Caco-2 cells.
The single complex present in H441 cells appears to correspond to
HNF-1
. These data also show a marked increase in the relative
proportion of HNF-1
compared with HNF-1
in Hep G2 compared with
Caco-2 cells and a trace of the HNF-1
complex in HeLa cells that do
not express
1-AT.

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Fig. 3.
Trans-acting nuclear proteins from H441 cells,
Hep G2 cells, undifferentiated Caco-2 cells [day 1 (d1)],
differentiated Caco-2 cells [day 4 (d4)], and HeLa cells
that bind to the hepatocyte nuclear factor (HNF)-1 ( 87 to 52)- and
HNF-4 ( 128 to 93)-binding regions of the proximal
1-AT promoter and to the HNF-3 and HNF-4 consensus
sequences as assessed by electrophoretic mobility shift assay (EMSA).
Nuclear extracts were reacted with radioactive probe, and reaction
products were subjected to PAGE. Relative migration of specific
DNA-binding proteins is indicated on left for HNF-1 ,
HNF-1 , HNF-3, and HNF-4.
|
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The HNF-4-binding region (
128 to
93) forms a single complex with
Hep G2 and Caco-2 cells but does not form a complex with nuclear
proteins from H441 or HeLa cells (Fig. 3). This complex is much more
abundant in Caco-2 than in Hep G2 cells and decreases during
differentiation of Caco-2 cells. Because it comigrates with the complex
formed with the HNF-4 consensus sequence and migrates faster than the
complex formed with the HNF-3 consensus sequence, this complex is
probably formed by HNF-4, not HNF-3.
The HNF-3 consensus sequence forms a single complex in H441, Hep G2,
Caco-2, and HeLa cells. These data indicate that HNF-3 is present in
H441, Hep G2, and Caco-2 cells but does not bind to the only two
potential sites for HNF-3 binding in the proximal promoter of the
1-AT gene. Together, these initial EMSA studies suggest
that HNF-1
, but not HNF-3, binds to the proximal
1-AT promoter in H441 cells. There is no evidence for HNF-4 in H441 cells.
To determine the specificity of each of these complexes, EMSA was done
in the absence or presence of unlabeled specific and unrelated
oligonucleotide competitors and antibodies. The single complex formed
in H441 cells with the HNF-1-binding region of the
1-AT
promoter (
87 to
52) is blocked by unlabeled
87 to
52
oligonucleotide but not by unlabeled glucocorticoid response element
(GRE) oligonucleotide (Fig.
4A). It is also blocked by antibody to HNF-1
(Fig. 4B). Our previous studies showed
that the more slowly migrating complex formed with the HNF-1-binding region of the
1-AT promoter in Hep G2 and Caco-2 cells
is supershifted by antibody to HNF-1
(17). In Fig.
4B, we also examined nuclear extracts from primary cultures
of human respiratory epithelial cells and found a single complex that
was supershifted by antibody to HNF-1
. These results indicated that
HNF-1
also binds to the proximal
1-AT promoter in
human airway epithelium in vivo. It is not clear to us at this time why
the single complex in human respiratory epithelial cells is slightly
slower in migration than that in H441 cells.

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Fig. 4.
Specificity of trans-acting nuclear proteins
from H441, Hep G2, Caco-2, and human respiratory epithelial (Hu resp
epi) cells in primary culture that bind to the HNF-1 ( 87 to 52)-
and HNF-4 ( 128 to 93)-binding regions of the proximal
1-AT promoter and to the HNF-3 consensus sequence as
assessed by EMSA. Reactions were carried out and analyzed as described
in MATERIALS AND METHODS. A: 5 µg of nuclear
extracts were preincubated with 2 µl of anti-HNF-1 or
anti- 1-AT antibody before the reaction with labeled
probe was initiated. Unlabeled 1-AT HNF-1
oligonucleotide or unlabeled irrelevant glucocorticoid response element
(GRE) oligonucleotide was added to separate reactions in 20-, 40- and
100-fold molar excesses. B: 1 µl of anti-HNF-1 antibody
was used. C: unlabeled 1-AT HNF-4
oligonucleotide was added to separate reactions in 20- and 50-fold
molar excess, and unlabeled irrelevant GRE probe was added in 50-fold
molar excess. D: 1 µl of anti-HNF-3 , anti-HNF-3 , and
anti- 1-AT antibody was used. Unlabeled HNF-3 consensus
and irrelevant GRE oligonucleotides were added to separate reactions in
20-, 40-, and 100-fold molar excess. Arrows, primary band shift.
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Next, we examined the specificity of complexes formed with the
HNF-4-binding region of the
1-AT promoter. The single
complex formed with this region in Hep G2 and Caco-2 (18)
cells is blocked by unlabeled
128 to
93 oligonucleotide,
but not by unlabeled GRE oligonucleotide, and is supershifted by
antibody to HNF-4 (Fig. 4C). A complex is not formed with
this region by nuclear proteins from H441 cells.
The single complex formed by nuclear proteins from H441 cells with the
HNF-3 consensus sequence is blocked by antibody to HNF-3
and
antibody to HNF-3
but not by antibody to
1-AT (Fig. 4D). It is also blocked by unlabeled HNF-3 but not by
unlabeled GRE oligonucleotide. This shows the identity and specificity
of the complex formed with the HNF-3 consensus sequence by HNF-3 in
H441 cells. Together with the results in Fig. 1, these data show that
HNF-3 is present in H441 cells but does not bind to the only potential
HNF-3-binding sites in the proximal
1-AT promoter.
Role of HNF-1 and HNF-4 in expression of
1-AT in
H441 cells.
H441 cells were compared with Hep G2 and Caco-2 cells for luciferase
activity after cotransfection of the
1-AT promoter
(
137 to
2)-luciferase reporter plasmid and HNF-1
expression
plasmid (Fig. 5A). The results
show that HNF-1
mediates a concentration-dependent increase in
luciferase activity in H441, but not in Hep G2 or Caco-2, cells. In
contrast, HNF-1
mediates increases in luciferase activity in all
three cell types, albeit to a significantly greater extent in H441 than
in the other two cell types (Fig. 5B). The role of HNF-1 in
expression of
1-AT in H441 cells was also shown by
comparing the luciferase activity of the
1-AT promoter
(
137 to
2)-luciferase reporter plasmid with that of the
corresponding Mut-2 plasmid, mutated in the HNF-1-binding site. There
was a marked decrease in the luciferase activity of the Mut-2 plasmid (data not shown).

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Fig. 5.
Effect of HNF-1 and HNF-1 on expression of the
1-AT gene in H441, Caco-2, and Hep G2 cells using the
1-AT proximal promoter ( 137 to 2)-luciferase fusion
plasmid. Cells were cotransfected with 1-AT ( 137 to
2)-luciferase reporter plasmid (7.5 µg) and pRL-TK plasmid (0.1 µg) in the absence or presence of HNF-1 (A) or HNF-1
(B) expression plasmids in the amounts indicated. H441 cells
were transfected with the FuGENE 6 reagent, and Hep G2 and Caco-2 cells
were transfected by the calcium phosphate-DNA coprecipitation
method. After 48 h, firefly and Renilla
luciferase activities were measured. Relative luciferase activity
represents normalized luciferase activity of each condition compared
with normalized luciferase activity in cells transfected with the
1- AT promoter alone. Values are means of triplicate
determinations at each point.
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Next, we examined the effect of HNF-1
and HNF-4 together. Our
previous studies showed that HNF-1
and HNF-4 have a synergistic effect on
1-AT expression in Hep G2 and Caco-2 cells
(17). The results in Fig. 6
are very similar to those in Fig. 5, showing that HNF-1
alone
mediates an increase in luciferase activity in all three cell types but
HNF-1
alone mediates a significant increase only in H441 cells.
HNF-4 by itself only mediates a two- to threefold increase in the three
cell types. The combination of HNF-1
and HNF-4 has an additive
effect on luciferase activity in Hep G2 cells, a marked synergistic
effect in Caco-2 cells, but neither additive nor synergistic effects in
H441 cells. The combination of HNF-1
and HNF-4 is also synergistic
in Caco-2 cells but neither additive nor synergistic in Hep G2 or H441
cells. These data show that HNF-1
plays a unique role in
1-AT expression in H441 cells and that HNF-4 has minimal
activity in H441 cells in the absence or presence of HNF-1
or
HNF-1
.

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Fig. 6.
Effect of HNF-1 together with HNF-4 on expression of
the 1-AT gene in H441, Caco-2, and Hep G2 cells. Cells
were cotransfected with the 1-AT ( 137 to
2)-luciferase reporter plasmid (7.5 µg) and pRL-TK plasmid (0.1 µg) in the absence or presence of HNF-1 expression plasmid (0.4 µg), HNF-1 expression plasmid (0.4 µg), HNF-4 expression plasmid
(0.4 µg), or a combination of these expression plasmids. Experimental
protocol was otherwise identical to that described Fig. 5 legend.
Values are means ± SD for triplicate determinations of each
condition.
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Effects of HNF-3 on
1-AT gene expression in H441
cells.
Previous studies indicated that HNF-3 plays a dominant role in gene
expression in H441 and pulmonary epithelial cells (3-5, 28). Moreover, HNF-3 has been shown to interact with HNF-1 and HNF-4 and to bind to a cis-acting sequence element that
overlaps with the element that binds HNF-1 and HNF-4 (8,
13-15, 22, 23, 33). Therefore, we examined the role of
HNF-3 in
1-AT gene expression in H441 compared with Hep
G2 and Caco-2 cells (Fig. 7). The results
show that HNF-3 by itself has no effect on luciferase activity in H441
cells. Moreover, HNF-3 does not mediate a significant effect on
1-AT gene expression in H441 cells when added together
with HNF-1
or HNF-1
. HNF-3 also has no effect by itself on
luciferase activity in Hep G2 and Caco-2 cells but does mediate a
modest upregulatory effect when added together with HNF-1
in Hep G2
and Caco-2 cells. The lack of effect could not be attributed to
saturation by endogenous HNF-3 in the nuclear extract, because there
was no significant reduction in luciferase activity in H441 cells
transfected with the
1-AT (
137 to
2)-luciferase
promoter mutated in the HNF-3-binding site compared with the same
plasmid without the mutation (data not shown). These data indicate that
HNF-3 has no effect on
1-AT gene expression in pulmonary
epithelial cells. HNF-3 does have an additive effect with HNF-1
in
Hep G2 and Caco-2 cells, even though it does not bind to the proximal
1-AT promoter, implying that this effect involves an
interaction with HNF-1
or with a cofactor necessary for HNF-1
activity.

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Fig. 7.
Effect of HNF-3 together with HNF-1 on expression of the
1-AT gene in H441, Hep G2, and Caco-2 cells. The
protocol was identical to that described in Fig. 5 legend. HNF-3 and
HNF-3 plasmids were used at 0.1 µg, and HNF-1 and HNF-1
plasmids were used at 0.2 µg.
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Expression of
1-AT and HNF-1
in primary cultures
of human respiratory epithelial cells.
To examine the possibility that HNF-1
is also involved in expression
of the
1-AT gene in another respiratory epithelial cell
system, we used RT-PCR analysis of human respiratory epithelial cells
in primary culture (Fig. 8). The results
show the presence of
1-AT and HNF-1
RNA in the
cultured cells, as well as in H441, Hep G2, and Caco-2 cells. The
results also show HNF-3
and HNF-3
, but not HNF-4, RNA in primary
cultures, demonstrating that the repertoire of these transcription
factors expressed in H441 cells faithfully reflects respiratory
epithelium in vivo. Taken together with data from the EMSA (Fig.
4B), these results provide further evidence for the unique
involvement of HNF-1
in
1-AT gene expression in
respiratory epithelial cells.

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|
Fig. 8.
Expression of 1-AT, HNF-1 , HNF-3 ,
HNF-3 , and HNF-4 in cultured human respiratory epithelial cells.
Total cellular RNA from H441, Hep G2, and Caco-2 cells and
poly(A)+ RNA from primary cultures of human respiratory
epithelial cells were subjected to RT-PCR analysis for
1-AT, HNF-1 , HNF-3 , HNF-3 , HNF-4, and
-actin.
|
|
 |
DISCUSSION |
Although
1-AT has always been considered a
hepatic-derived plasma protein, there is abundant evidence that it
is synthesized in extrahepatic sites by mononuclear phagocytes and
epithelial cells (reviewed in Ref. 28). Interestingly,
extrahepatic synthesis of
1-AT is particularly
characteristic of the human compared with the mouse and rat. In fact,
early studies of transgenic mice, engineered to express human
1-AT by using as transgene a genomic fragment
encompassing the coding region and most of its 5'-flanking region,
showed expression of human
1-AT, but not endogenous
mouse
1-AT, in many extrahepatic tissues
(19). More recent studies have shown that
1-AT is synthesized in human airway epithelial cells in
primary culture, cell lines, and human lung tissue in situ (9,
21, 22). Airway epithelium is likely to be a particularly important site of synthesis for
1-AT, because it is the
major physiological inhibitor of neutrophil elastase, cathepsin G, and proteinase 3, neutrophil proteases that can degrade the connective tissue matrix of the lung in vivo, and because
1-AT
deficiency predisposes to destructive lung disease/emphysema
(20). However, the detection of
1-AT gene
expression in a cell line and primary cultures derived from respiratory
epithelium here and in previous studies (9, 22) does not
by itself indicate that this expression is physiologically relevant.
Even the detection of
1-AT mRNA in human respiratory
epithelium by in situ hybridization analysis (21) does not
ensure that a physiological function for
1-AT derived
from this source as opposed to the liver, in which expression levels
are much higher. Previous studies showed that the allotype of
1-AT in plasma converts to that of the donor after
orthotopic liver transplantation (1, 16), indicating that
plasma
1-AT is predominantly derived from liver. Whether
extrahepatic expression is physiologically relevant will be
definitively addressed only if there is correction of a functional
abnormality by tissue-specific transgenic expression of
1-AT in an
1-AT-knockout mouse.
In this study, we examined the possibility that expression of
1-AT in human pulmonary epithelial cells involved
transcriptional mechanisms similar to those at play in intestinal
epithelial cells and/or hepatocytes. Our previous studies showed that
the synergistic action of HNF-1
and HNF-4 plays a prominent role in
1-AT transcription in enterocytes and hepatocytes and in
the mechanism by which the
1-AT gene is upregulated
during differentiation of enterocytes from crypt to villous tip
(26). Previous studies from other laboratories have
implicated HNF-3 and thyroid transcription factor-1 in gene expression
in pulmonary epithelium (3-5, 30). To our surprise,
the results showed that
1-AT gene expression in
pulmonary epithelial cells is predominantly driven by HNF-1 and not at
all by HNF-3. Even more surprising, HNF-1
is responsible for
activating
1-AT gene expression in pulmonary epithelial
cells, even though it has no effect on the
1-AT gene in
intestinal epithelial cells or hepatocytes. HNF-1
is highly
homologous to HNF-1
, particularly in the NH2-terminal
DNA-binding region, but diverges within the COOH-terminal activation
domain (7). There is relatively limited information about
the function of HNF-1
, except that it is expressed in
dedifferentiated cells and somatic cell hybrids that have lost the
ability to express liver-specific genes and lack the transcription factors HNF-1
and HNF-4 (2). HNF-1
is also known to
be expressed in some tissues that do not express HNF-1
, including
thymus, testis, ovary, and lung (2, 7), but this is the
first report that we can find in the literature of a specific
transcriptional role for HNF-1
in one of these tissues. It is also
noteworthy that HNF-1
activates the
1-AT gene, a gene
characteristic of the differentiated hepatocyte, in lung cells, but not
in hepatocytes or enterocytes. These data imply that there are cell
type-specific mechanisms for the action of HNF-1
and militate
against it being merely a part of dedifferentiation from the hepatic
phenotype. The fact that HNF-4 does not appear to mediate a synergistic
effect with HNF-1
on
1-AT gene expression in lung
cells, even though it does mediate a synergistic effect with HNF-1
in liver and intestinal cells, also implies cell type specificity in
the role of HNF-1
on the
1-AT gene in lung cells.
The results also indicate that HNF-3
and HNF-3
do not directly
activate
1-AT gene expression in lung, intestinal, or
liver cells. This is somewhat surprising, because HNF-3 appears to play a major role in gene expression in differentiated pulmonary epithelial cells, and there are two potential HNF-3-binding sites within the
proximal promoter of the
1-AT gene. We did find,
however, that HNF-3
and HNF-3
have an additive effect with
HNF-1
on
1-AT gene expression in Hep G2 and Caco-2
cells. Previous work has shown that HNF-3 interacts with HNF-1
in
regulation of the liver-specific trans-activation of
aldolase-
, but in this case the interaction is antagonistic
(14). Because HNF-3
and HNF-3
do not bind to the
proximal promoter of the
1-AT gene, their additive
effect must involve interaction with HNF-1
or a cofactor necessary
for transcriptional activation by HNF-1
.
 |
ACKNOWLEDGEMENTS |
The authors are indebted to M. Maksin for preparation of the manuscript.
 |
FOOTNOTES |
The studies were supported in part by National Institutes of Health
Grants HL-37784 and DK-52526.
Address for reprint requests and other correspondence: D. Perlmutter, Dept. of Pediatrics, University of Pittsburgh School of
Medicine, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213-2583 (E-mail:
perldav{at}chplink.chp.edu).
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.
10.1152/ajplung.00271.2001
Received 25 September 2001; accepted in final form 21 October 2001.
 |
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