Departments of Pediatrics, Cell Biology, and Physiology, Washington University School of Medicine, Division of Gastroenterology and Nutrition, St. Louis Children's Hospital, St. Louis, Missouri 63110
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ABSTRACT |
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There is still relatively limited information
about mechanisms of gene expression in enterocytes and mechanisms by
which gene expression is regulated during enterocyte differentiation.
Using the human intestinal epithelial cell line Caco-2, which
spontaneously differentiates from a cryptlike to a villouslike
enterocyte, we have previously shown that there is a marked increase in
transcription of the well-characterized
1-antitrypsin
(
1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements
and trans-acting DNA-binding proteins
responsible for expression of the
1-AT gene in Caco-2 cells
during differentiation. Footprint analysis and electrophoretic mobility
shift assays showed that hepatocyte nuclear factor-1
(HNF-1
),
HNF-1
, and HNF-4 from nuclear extracts of Caco-2 cells specifically
bound to two regions in the proximal promoter of the
1-AT gene. Cotransfection
studies showed that HNF-1
and HNF-4 had a synergistic effect on
1-AT gene expression. RNA blot analysis showed that HNF-1
and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1
binding activity increase coordinately with
1-AT mRNA
levels during differentiation of Caco-2 cells. Finally, overexpression
of antisense ribozymes for HNF-1
in Caco-2 cells resulted in a
selective decrease in endogenous
1-AT gene expression. Together,
these results provide evidence that HNF-1
and HNF-4 play a role in
the mechanism by which the
1-AT
gene is upregulated during enterocyte differentiation in the model
Caco-2 cell system.
enterocyte differentiation; hepatocyte nuclear factor-1; hepatocyte nuclear factor-4
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INTRODUCTION |
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THERE IS STILL RELATIVELY little information about
specific cis-acting elements and
trans-acting DNA binding proteins that are responsible for gene expression in enterocytes. A 20-nucleotide element in the gene for the intestinal fatty acid binding protein acts
as a suppressor of gene activation in crypt enterocytes and Paneth
cells (44). A 22-nucleotide element in the sucrase-isomaltase (SI)
gene, which binds caudal-related gene Cdx2, appears to be involved in
the expression of SI in enterocytes (47, 52). Two elements further
upstream in the 5'-flanking region of the SI gene, which bind
hepatocyte nuclear factor-1 (HNF-1
) and HNF-1
, are also
involved in expression of SI in enterocytes (52, 56). The HOXC11
homeodomain protein interacts with a 15-nucleotide element in the
promoter of the lactase gene to direct its transcription in enterocytes
(31). Another recent study has shown that a 9-nucleotide element in the
gene for intestinal trefoil factor, which binds a distinct nuclear
transcription factor, is involved in expression in intestinal goblet
cells (36).
There is also some limited information about mechanisms of expression
for genes that are expressed in both enterocytes and hepatocytes. A
specific element in the -fetoprotein gene binds HNF-1 in both
hepatocytes and enterocytes (55). A
cis-acting element in the
apolipoprotein B gene is involved in its expression in both hepatocytes
and enterocytes (38). Expression of apolipoprotein CIII in hepatocytes
and enterocytes involves a cis-acting
element, which binds apolipoprotein regulatory protein 1, Ear3/COUP-TF, and HNF-4 (30). There is even less information in the literature about
cis-acting elements and
trans-acting DNA-binding proteins involved in the regulation of gene expression that accompanies differentiation of enterocytes. One transcription factor, Cdx2, originally identified as a regulator of SI gene expression, appears to
play a defined role in the differentiation program of enterocytes (47).
In previous studies we have shown that the
1-antitrypsin
(
1-AT) gene, which is well
known as a product of hepatocytes, is also expressed in human
enterocytes in vivo (32) and in human enterocyte-like cell lines (20,
32, 33, 41). Moreover, in the human enterocyte-like cell line Caco-2
there is a marked increase in
1-AT gene expression during
spontaneous differentiation from the cryptlike to the villouslike
enterocyte (41). Because the structure and function of the
1-AT gene have been well
characterized, we have now used it as a model gene for studies designed
to identify specific mechanisms for gene expression in undifferentiated
enterocytes as well as during the differentiation program of the
enterocytes. The expression and regulation of the
1-AT gene in the enterocyte are
also of interest because previous data have raised the possibility that
1-AT is part of a local
response to injury and/or inflammation at the epithelial surface of the
intestine (20, 33).
1-AT is an ~55-kDa serum
glycoprotein that inhibits the destructive neutrophil proteases
elastase, cathepsin G, and proteinase 3 (reviewed in Ref. 39). It is
the archetype of a family of serum proteins, which are called serine
protease inhibitors or SERPINS (reviewed in Ref. 24). It is also a
classical positive acute phase protein in that plasma concentrations
increase three- to fivefold during the host response to tissue injury
and/or inflammation. Inherited deficiency of
1-AT is associated with
destructive lung disease and emphysema, and a subgroup of deficient
individuals develops liver disease (reviewed in Ref. 49).
Plasma 1-AT is predominantly
derived from the liver as shown by studies of changes in
1-AT allotypes after orthotopic
liver transplantation (1, 23). There is abundant synthesis of
1-AT in hepatoma cell lines and
hepatocytes in primary culture, and
1-AT mRNA is extremely abundant
in hepatocytes in human liver as determined by in situ hybridization
analysis (32). There is also evidence for extrahepatic sites of
synthesis, notably that in blood monocytes and tissue macrophages (40)
as well as in enterocytes.
Several things are known about the mechanism by which
1-AT gene expression is
regulated in enterocytes. Our previous studies have shown that the
increase in
1-AT gene
expression during differentiation of Caco-2 cells can be in large part
accounted for by an increase in rate of transcription (20). Second,
expression of the
1-AT gene in
undifferentiated and differentiated Caco-2 cells is regulated by
cytokines in a manner that recapitulates the expression of the
1-AT gene in the intestinal
epithelium during inflammation in vivo and is characteristic of the
physiological acute phase response (20, 33). Transcription is initiated
at the downstream "hepatocyte" specific transcription initiation
site during basal and differentiated expression of
1-AT in Caco-2 cells but is initiated at both the downstream and the upstream "macrophage" specific transcription initiation sites during modulated
[interleukin-6 (IL-6)-activated] expression in Caco-2 cells
(20) and in human intestinal epithelial cells during inflammation in
vivo (32).
In the current study we examined the possibility of identifying the
cis-acting element(s) and
trans-acting DNA-binding protein(s) responsible for 1-AT gene
expression in Caco-2 cells, whether these were different from those
responsible for
1-AT gene
expression in hepatoma Hep G2 cells and whether we could identify a
mechanism or mechanisms responsible for increased transcription during differentiation.
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EXPERIMENTAL PROCEDURES |
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Plasmid constructs and site-directed mutagenesis.
Progressive deletions of the human
1-AT promoter region from
1951 to
2 were generated by PCR amplification using a
human genomic
1-AT clone
hAAT7zf (kindly provided by Dr. K. Ponder, St. Louis, MO) as template.
These PCR fragments were subcloned into the
Kpn
I/Hind III site of the pGL3 basic
luciferase reporter vector (Promega) to generate
1-AT promoter-luciferase fusion plasmids. The internal site-directed mutations within the potential HNF-1 and HNF-4 binding sequences of this fragment were prepared by
overlap extension PCR (22) using gene-specific oligonucleotide primers
that flank the region to be altered. All PCR fragments were subjected
to DNA sequence analysis (48) to show that the designated mutations had
been made, that the remaining sequence was identical to the original
template, and that the proper orientation had been maintained.
Cell culture, DNA transfections, and luciferase assay.
The Hep G2, HeLa, and Caco-2 cells were grown as previously described
(32). The promoter-luciferase plasmids were cotransfected with the
indicated amount of HNF-1, HNF-1
, and/or HNF-4 expression vectors. A pGL3-basic and pGL3-control vector containing SV40 promoter
and enhancer sequences were used as controls in all transfection experiments, and the pSV-
-gal vector was included as an internal control to monitor and normalize for transfection efficiency. Cells
(1-1.5 × 106) were plated on 60-mm tissue
culture dishes and incubated for 24 h before transfection. Duplicate
dishes were transfected using the calcium phosphate method. Cells were
shocked with 15% glycerol 24 h after transfection and harvested 48 h
after transfection. For transfection of HeLa cells the Tfx-20 reagent
from Promega was used. Luciferase activity was detected on the Turner
Designs luminometer (model TD-120/20, Promega).
Preparation of double-strand oligonucleotides and labeling.
The wild-type and mutant oligonucleotides were synthesized on an
Applied Biosystems DNA synthesizer in the Nucleic Acid Chemistry Laboratory, Biotechnology Center, Washington University School of
Medicine (St. Louis, MO). The potential HNF-1-binding sequence corresponds to the 1-AT
promoter sequence from
86 to
50
(5'-ATAACTGGGGTGA-CCTTGGTTAATATTCACCAGCAG-3'). The
potential HNF-4-binding sequence corresponds to the sequence from
125 to
96
(5'-ATCCAGCCAGTGGACTTAGCCCCGTTTG-3') with respect to the
downstream transcriptional start site. 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.
Nuclear extracts and DNase I footprint analysis.
Nuclear extracts of Caco-2 and Hep G2 cells were prepared according to
the protocol described by Dignam et al. (15). Nuclear extracts from
HeLa cells (HeLa Scribe nuclear extract, in vitro transcription grade)
were purchased from Promega. A fragment of the human
1-AT gene that spans
nucleotides
157 to
2 was generated by PCR and subcloned
into Hind
III/Pst I site of the pL-TG vector (kindly provided by Dr. G. U. Ryffel, Karlsruhe, Germany). This fragment was labeled with 32P by a
3'-end filling reaction. For DNase I footprint analysis several
different concentrations of nuclear extracts were resuspended in buffer
so that a 20-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 poly(dI-dC), and 2% polyvinyl alcohol. Nuclear
extract dialysis buffer and BSA were used as negative controls. After a
preincubation at room temperature for 15 min, labeled probe [~8 × 104 counts/min
(cpm)] was added. The reaction was mixed well and then incubated
for another 20 min at room temperature. DNase I (Worthington
Biochemical, Freehold, NJ) was added to a final concentration 1-10
ng of enzyme per reaction. After a 1- or 2-min incubation at room
temperature, the reaction was stopped by adding 1 volume of DNase I
stop solution containing 20 mM EDTA, 200 mM NaCl, 1% SDS, 10 µg of
yeast tRNA, and 40 µg of proteinase K followed by extraction with
phenol-chloroform and ethanol precipitation. The samples were analyzed
by electrophoresis on 6% polyacrylamide-8 M urea gels, and
autoradiography was performed for 12 h at
70°C.
Electrophoretic mobility shift assays.
Complementary oligonucleotide probes for the HNF-1 and HNF-4 sequences
in the 1-AT promoter and mutant
oligonucleotide probes were labeled with
[32P]dCTP by the
3'-end filling reaction and 2 × 104 cpm were incubated for
20-40 min at room temperature with nuclear extracts from Hep G2,
Caco-2, and HeLa cells. The nuclear extracts were prepared exactly as
previously described. The binding reaction was performed using the same
protocol that was used for DNase I footprint analysis except that lower
concentrations of nuclear proteins were used and a 10-µl reaction
volume was used. Unlabeled oligonucleotide in 10-, 50- and 100-fold
molar excess was used in designated experiments. Rabbit polyclonal
anti-HNF-1
TC 284 (kindly provided by M. Yaniv and M. Pontoglio,
Paris, France) and anti-HNF-4 (kindly provided by I. Talianidis, Crete)
were used in designated experiments by incubation with nuclear extracts for 5 min at 22°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.
RNA blot analysis. Total cellular RNA was isolated from cell lines by guanidine isothiocyanate extraction and ethanol precipitation (8). Poly(A)+ RNA was isolated using oligo(dT) cellulose column chromatography (43). RNA was then subjected to agarose-formaldehyde electrophoresis and transferred to nylon filters (50) for hybridization with 32P-labeled DNA probes.
Construction of antisense ribozymes for HNF-1.
Overlap PCR methods of Zillman and Robinson (60) were used to generate
two hammerhead antisense ribozymes for HNF-1
, the F1(1) and F1(2)
PCR products. In each case a GUC codon was targeted [nucleotides
378-380 for F1(1) and 455-457 for F1(2)] and
35-flanking nucleotides were included on each side to optimize ribozyme
activity according to previous studies (12). The HNF-1
plasmid HA
(2), kindly provided by M. Yaniv (Paris, France), was used as template. Results of transient transfections indicated that the F1(1) antisense ribozyme had the highest inhibitory activity so it was used for the
stable transfection studies. For F1(1) the first primer set was
CGCTGTGGGATGTTGT and added a Xho I
restriction site at the 5' terminus. The second primer set was
TTCTGCAGGAGGACCCG and added a BamH I
restriction site at the 5' terminus. The target was reacted separately with these two primer sets. The PCR products were then gel
purified and reacted with the two primer sets to give the final
ribozyme-encoding cassette. This cassette was ligated into the
Xho I and
BamH I sites in the multiple cloning
site of plasmid pcDNA3 (Invitrogen, San Diego, CA). This plasmid was
expressed in Caco-2 cells by transient and stable transfection
protocols. For transient transfection, Caco-2 cells at 80% confluence
were cotransfected with the antisense ribozyme HNF-1
expression
plasmid and the
1-AT promoter
(
137 to
2)-luciferase reporter plasmid using
lipofectamine (GIBCO BRL, Gaithersburg, MD) and the conditions described by the manufacturer's instructions. After 64 h, luciferase activity was measured. For stable transfection, Caco-2 cells at 80%
confluence were transfected with the antisense ribozyme HNF-1
expression plasmid alone and the lipofectamine reagent. After 48 h,
transfected cells were subjected to selection in G418.
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RESULTS |
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Localization of cis-acting elements
within upstream flanking region responsible for expression of the
1-AT gene in Caco-2 cells
and Hep G2 cells.
To determine whether there were
cis-acting regulatory elements within
the upstream flanking region for expression of the
1-AT gene in Caco-2 cells and
whether these differed from those responsible for expression in Hep G2
cells, we constructed seven
1-AT-luciferase fusion plasmids
differing in the length of the
1-AT 5'-flanking sequence upstream of the luciferase coding sequence
(Fig. 1). We examined the expression of these
plasmids in undifferentiated Caco-2 cells as well as Hep G2 cells. The
results show that deleting 451 nucleotides from
1951 to
1500 results in a small drop in expression in Caco-2 cells.
Deletion of another 509 nucleotides from
1500 to
991
results in an increase in expression in Caco-2 cells. There is a
substantial drop in expression in these cells on deletion of 330 nucleotides from
991 to
661. There is no effect on
expression in Caco-2 cells of deleting 391 nucleotides from
661
to
270 but significant drops occur on deletion of 133 nucleotides from
270 to
137 and deletion of 135 nucleotides from
137 to
2. The results therefore indicate
that there are cis-acting elements for
expression in Caco-2 cells between
1951 and
1500,
1500 and
991,
991 and
661,
270 and
137, and
137 and
2. The results also indicate that
there are cis-acting elements for
expression in Hep G2 cells between
1500 and
991, between
490 and
270, between
270 and
137, and
between
137 and
2. Analysis of the percent change in
luciferase activity (Fig. 1C) suggests that there are at least three major differences between Caco-2
and Hep G2 cells: negative element(s) between
1500 and
991 and positive elements in regions from
991 to
661 and
137 to
2. We decided to first examine the
more proximal region,
137 to
2, in more detail.
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Binding sites for nuclear proteins from Caco-2 cells within proximal
upstream region of the 1-AT
gene.
We first used DNase I footprint analysis to examine the possibility
that nuclear proteins from Caco-2 bound to the region of the
1-AT promoter spanning
nucleotides
137 to
2 (Fig.
2). The results showed that
nuclear proteins from Caco-2 protected a region within the promoter
that corresponded to nucleotides
47 to
80. The protection
was concentration dependent and greater for nuclear proteins from
"differentiated" Caco-2 cells (day
9) than from undifferentiated Caco-2 cells
(day 1). This region contains a
sequence highly homologous to the consensus sequence for binding HNF-1
and is also protected by nuclear extracts from liver cells (3, 7, 9,
12, 14, 34). The increase in protection in differentiated Caco-2 cells
corresponds to the increase in expression of
1-AT in differentiated Caco-2
cells (41). There is also protection, although to a significantly
lesser extent, in a region that corresponds to nucleotides
96 to
102. This region contains part of a potential HNF-4 binding
sequence within the
1-AT
promoter.
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Expression of the HNF-1 and HNF-4 genes during
differentiation of Caco-2 cells.
Next we used RNA blot analysis to examine steady-state levels of
HNF-1
, HNF-4, and
1-AT mRNA
in undifferentiated and differentiated Caco-2 cells compared with Hep
G2 and HeLa cells (Fig. 4). The results
show that HNF-1
and HNF-4 are not expressed in HeLa cells, but there
is a similar amount of HNF-1
and HNF-4 mRNA in Hep G2 and
undifferentiated Caco-2 cells. There is a marked increase in HNF-1
and HNF-4 mRNA levels in differentiated Caco-2 cells that corresponds
temporally and in magnitude to that for
1-AT mRNA levels. The increase
cannot be attributed to RNA loading as shown by the absence of any
change in steady-state levels of glyceraldehyde-3-phosphate
dehydrogenase (GADPH) mRNA levels. Taken together with the other
results of this study, these data provide evidence that the increase in
1-AT gene expression during differentiation of Caco-2 cells correlates with an increase in HNF-1
and HNF-4 mRNA levels and an increase in the binding of HNF-1
to the
1-AT promoter.
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Evidence for a synergistic effect of HNF-1 and HNF-4
on
1-AT gene expression in
Caco-2 cells.
Next we cotransfected undifferentiated Caco-2 and Hep G2 cells with the
1-AT promoter (
137 to
2)-luciferase reporter plasmid and HNF-1
, HNF-1
, and HNF-4
expression plasmids. The luciferase activity was normalized to
-galactosidase activity conferred by a cotransfected pSV-
-gal
plasmid (Fig. 5). The results show that
HNF-1
and HNF-4 mediate concentration-dependent increases in
luciferase activity, but the effect of HNF-1
on Caco-2 cells is
negligible (Fig. 5A). In each case
the effect of HNF-1
and HNF-4 reaches a plateau when 2 µg are
transfected. At this concentration HNF-1
mediates an ~15.9-fold
and HNF-4 an ~5.7-fold increase in luciferase activity (Fig.
5A). HNF-1
and HNF-4 also mediate increases in luciferase activity in Hep G2 cells (Fig.
5B). HNF-1
mediates an
~3.5-fold and HNF-4 an ~1.5-fold increase in luciferase activity,
but HNF-1
has no significant effect. Thus there is a lower magnitude
in the effect of HNF-1
and HNF-4 on this promoter in Hep G2 cells.
This difference is unlikely to be due to differences in efficiency of
transfection because the data were normalized to
-galactosidase
activity conferred by the cotransfected pSV-
-gal plasmid and the
galactosidase activity of transfected Caco-2 cells was very similar to
that in Hep G2 cells in these experiments (data not shown). It is also
unlikely to be due to different relative levels of endogenous HNF-1
or HNF-4 expression because RNA blot analysis indicates that the
steady-state level of HNF-1
and HNF-4 mRNA are similar in
undifferentiated Caco-2 and Hep G2 cells (see Fig. 4). Interestingly,
the basal luciferase activity of the promoter was over 20-fold higher
in Hep G2 cells than Caco-2 cells. These data suggest the possibility
that there are differences in endogenous cofactors or other
transcription factors in the two cell lines. There was no significant
basal luciferase activity for the promoter in transfected HeLa cells
(data not shown).
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Effect of antisense hammerhead ribozyme for HNF-1 on
1-AT gene expression in
Caco-2 cells.
To provide further evidence for the role of HNF-1
and for the
synergistic effects of HNF-4 and HNF-1
on the increase in
1-AT gene expression during
differentiation of Caco-2 cells, we examined the effect of antisense
ribozymes for HNF-1
. First, we generated two hammerhead antisense
ribozymes (Fig.
8A).
Next, we examined the effect of the two constructs on expression from the
1-AT promoter (
137
to
2) reporter plasmid in Caco-2 cells after transient
transfection. The F1(1) anti-sense ribozyme had a more potent
inhibitory activity (Fig. 8B). If
mediated a concentration-dependent decrease in luciferase activity. At
the highest concentration used there was a reduction to
40%
levels present in cells transfected with the reporter alone. The F1(1)
antisense ribozyme in the pcDNA3 plasmid was then used to establish
stable transfected Caco-2 cell lines. Total cellular RNA was harvested
from untransfected Caco-2 cells, Caco-2 cells transfected with the
pcDNA3 plasmid (Caco-2-pcDNA3) alone and Caco-2 cells transfected with
the ribozyme (Caco-2-ribozyme) 1 and 7 days after reaching confluence.
RNA blot analysis (Fig. 8C) shows
that there is a marked decrease in steady-state levels of
1-AT mRNA on
day 1 and day
7 in the Caco-2-ribozyme cell line, but no change in
1-AT mRNA levels in the control
Caco-2-pcDNA3 cell line. The decrease is not due to a general effect as
shown by the results of RNA blot analysis with the GADPH cDNA probe (Fig. 8C,
right). If anything there is
slightly more GADPH mRNA in the Caco-2-ribozyme cell line. These data
therefore provide further evidence for the role of HNF-1
in
1-AT gene expression in Caco-2
cells and for the increase in
1-AT gene expression during
differentiation of Caco-2 cells. However, because there is still a
small increase in
1-AT mRNA
levels in the Caco-2-ribozyme cell line on day
7 compared with day 1,
the effect of HNF-1
may not completely explain the increase in
1-AT gene expression during differentiation of Caco-2 cells.
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DISCUSSION |
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1-AT is the major circulating
inhibitor of destructive neutrophil proteases such as elastase,
cathepsin G, and proteinase 3. Because it is also an acute-phase
reactant it is thought to provide proteinase inhibitor activity during
the host response to inflammation and tissue injury. Liver is the
predominant site of synthesis of plasma
1-AT, and
1-AT has been studied as a "liver-specific" gene. These studies have shown that the
cis-acting elements required for
expression of the human
1-AT
gene in liver are localized to the 5'-flanking region of the gene
(14). In fact, there appears to be three regions, distal elements at
488 to
356 and
261 and
208 and a proximal
element at
137 to
37 (14). The minimal proximal element
has been shown to be sufficient for liver-specific transcription in
vivo in cultured hepatoma cells compared with HeLa cells and in vitro
with rat liver nuclear extracts compared with nuclear extracts from
spleen (9, 12, 34). Five different
trans-acting factors from nuclear
extracts of liver have been shown to bind to this proximal element:
HNF-1
/LFB1, HNF-4/LFA1, HNF-1
/vHNF1/LFB3, LFB2 and LFC
(34). HNF-1
/LFB1 and HNF-4/LFA1 appear to be
particularly important in expression of the human
1-AT gene. Two distinct regions
within the proximal element bind these two transcription factors (53).
In fact, substitution of five nucleotides at
77 to
72
disrupts binding of HNF-1
and dramatically reduces expression of the
human
1-AT gene in the liver of
transgenic mice (53). Substitution of four nucleotides at
118 to
115 disrupts binding of HNF-4 but does not alter expression of the
human
1-AT gene in liver of
adult transgenic mice. The latter mutation does result in a reduction in expression of human
1-AT in
the liver during embryonic development.
The human 1-AT gene is also
expressed in extrahepatic tissues and cell types. Expression of this
gene in small intestinal epithelial cells has been studied by in situ
hybridization analysis of normal and inflamed human intestine and by
metabolic labeling studies in human intestinal epithelial cell lines.
The results of these studies indicate that
1-AT is synthesized by
enterocytes, that it is secreted at both poles of the enterocytes, that
its expression increases progressively during differentiation from crypt to villous tip, and that its expression is increased by inflammation in vivo and by inflammatory cytokines in cell culture. Thus the results raise the possibility that
1-AT expression in intestinal
epithelial cells plays a role in the local response to
inflammation/injury at the mucosal surface and is a target of the
enterocyte differentiation program.
In this study we examined the possibility of identifying the
cis-acting elements and
trans-acting proteins that direct
expression of 1-AT in
enterocytes, whether they are different from those involved in
expression in hepatocytes and how they mediate the increase in
transcription during differentiation. We first examined the proximal
upstream flanking region of the
1-AT gene, which appears to be
involved in liver
1-AT gene
expression. The results show that HNF-1
and HNF-4 have a synergistic
effect on
1-AT gene in
Caco-2-derived enterocytes. HNF-1
and HNF-4 also have a synergistic
effect on
1-AT gene expression
in Hep G2-derived hepatocytes, but it is considerably lower in magnitude.
The results of the current study also suggest that HNF-1 and HNF-4
play an important role in the increase in transcription of the
1-AT gene during enterocyte
differentiation. First, there is an increase in protection of the HNF-1
and HNF-4 regulatory elements from DNase I digestion during
differentiation of Caco-2 cells (Fig. 2). Second, there is an increase
in HNF-1
binding activity in nuclear extracts from differentiated
Caco-2 cells (Fig. 3). Third, there is a marked increase in HNF-1
and HNF-4 mRNA levels that correlates temporally with the increase in
1-AT mRNA levels during
differentiation of Caco-2 cells (Fig. 4). Interestingly, Tripodi et al.
(53) have reported that expression of human
1-AT gene in the gut is
abolished by mutation of the HNF-1
or HNF-4 binding site in
transgenic mice. This result indicates that expression of the
1-AT gene in the model cell
culture system Caco-2 in general recapitulates the expression of this
gene in intestine in vivo. Fourth, overexpression of antisense
ribozymes for HNF-1
in stable transfected Caco-2 cell lines resulted
in a marked decrease in endogenous
1-AT gene expression (Fig. 8).
HNF-1 was one of the first "liver-specific" transcription
factors that was characterized (5, 6, 10, 18, 21, 26). As with many
other transcription factors, which were originally believed to be
tissue- and or cell-specific, subsequent studies of HNF-1
have shown
that its expression is not restricted to liver cells. Its expression
has been demonstrated in intestine, stomach, pancreas, kidneys, and
lung (reviewed in Refs. 14, 18, and 54). Indeed, it has been shown to
regulate transcription of a number of genes in intestine.
HNF-4 is a member of the steroid hormone receptor superfamily (46).
Binding sites for this molecule are found in many genes expressed in
liver cells, and these binding sites are required for expression after
transfection in hepatoma cells (45). However, HNF-4 mRNA and protein
can be detected in intestine, kidney, and pancreas in addition to
liver. Expression in pancreas may be particularly important because
mutations in the HNF-1 and HNF-4 genes appear to be associated with
maturity-onset diabetes of the young (27, 57, 58). Recent studies have
suggested that HNF-4 also plays a role in early development and in
differentiation (16, 25, 51). Results of the current study indicate
that HNF-4 regulates
1-AT gene
expression in intestinal epithelial cells during differentiation, but
it does not appear that the contribution of HNF-4 to this synergistic
effect is due to binding to its own regulatory element in the
1-AT promoter (Fig.
7B). Instead, the results raise the possibility that there is an interaction between HNF-4 and HNF-1
, which enhances HNF-1
binding to its own regulatory element and/or that the collaborative action of HNF-4 and HNF-1
induces
trans-activation of the proximal
promoter of
1-AT by another
endogenous transcription factor. Indeed HNF-4 has recently been shown
to directly bind to HNF-1
, and this interaction has been implicated
in the mechanism by which HNF-1
negatively regulates HNF-4-dependent
genes in hepatoma cells (26). Although the results indicate that the HNF-1 binding sequence in the proximal promoter is crucial for the
synergistic effect of HNF-4 and HNF-1
, it is not entirely clear why
there is no increase in HNF-4 binding activity during differentiation
of Caco-2 cells (Fig. 3A) despite
the increase in HNF-4 mRNA levels over the same interval.
This may reflect competition for the HNF-4 binding site by members of
the HNF-3 family of transcription factors expressed endogenously.
Recent studies by Duncan et al. (17) suggest that HNF-3
and HNF-3
compete in opposite ways for the regulatory activity of HNF-4 and
HNF-1
(17). In preliminary studies we have also found that HNF-3
and HNF-3
have opposite and marked effects on regulatory activity of
the proximal promoter of the
1-AT gene (unpublished data).
These studies also indicate that other
cis-acting elements in the
1-AT gene and
trans-acting factors are involved in
its expression in enterocytes and in its upregulation during enterocyte differentiation. There is still a small increase in
1-AT gene expression during
differentiation of the Caco-2 cell line which overexpresses the
antisense ribozyme for HNF-1
(Fig. 8). Moreover, there appears to be
a positive element, or elements, between nucleotides
991 to
661 in the 5'-flanking region of the
1-AT gene, which has a much
greater effect on expression in Caco-2 cells than in Hep G2 cells (Fig.
1). Based on the known sequence in this region and comparison to
consensus sequences for known
cis-acting regulatory elements, there
are many candidates for this particular regulatory effect on the
1-AT gene, and thus further
detailed studies will be necessary for its elucidation.
Several genes that are expressed in both enterocytes and hepatocytes
have been studied for mechanism of transcriptional activation. A
proximal promoter with an HNF-1 binding sequence and HNF-1 itself appears to be involved in -fetoprotein gene expression in
enterocytes and hepatocytes (55). Moreover, a substitution in an
HNF-1-binding site upstream of the
-fetoprotein gene which results
in tighter binding of HNF-1 is associated with hereditary persistence
of
-fetoprotein expression (29). Expression of apolipoprotein B in
Caco-2 and Hep G2 cells appears to involve a similar
cis-acting element and three nuclear
proteins, but there is a different amount of the three proteins in
Caco-2 compared with Hep G2 cells (38). Distal regulatory
elements may also have different effects on apolipoprotein B expression
in Caco-2 compared with Hep G2 (37), and elements as far upstream as 70 kb may be necessary for expression in the intestine in vivo (35).
Expression of apolipoprotein CIII also involves a similar
cis-acting regulatory element in Caco-2 and Hep G2 cells (30). This element binds apolipoprotein AI
regulatory protein 1, Ear3/COUP-TF, and HNF-4 in both Caco-2 and Hep G2 cells.
There are also a number of similarities in the mechanism by which the
1-AT gene is activated in
Caco-2 cells and that of the enterocyte-specific gene SI. HNF-1
activates transcription of SI in Caco-2 cells (56). HNF-1
binds to
the SI promoter but does not affect its transcription (56). The
expression of both
1-AT and SI
increases during differentiation of Caco-2 cells in culture and
enterocytes in vivo, implicating HNF-1
in a more general role in the
differentiation program of enterocytes. However, there is a dramatic
increase in SI gene expression late in Caco-2 cell differentiation that
does not occur for
1-AT,
suggesting that additional SI-specific mechanisms determine its
activation during the most terminal phases of enterocyte
differentiation. Expression of both
1-AT and SI in enterocytes is
modulated by inflammation and specifically IL-6, with
1-AT gene expression increasing
and SI gene expression decreasing in the presence of IL-6 in cell
culture or in the presence of inflammation in vivo (20, 33, 41, 59).
There are also some similarities in mechanisms of expression of the
1-AT gene and the
enterocyte-specific gene lactase in Caco-2 cells. Lactase gene
expression in Caco-2 cells is activated by the synergistic effect of
HNF-1
and the HOXC11 homeodomain protein (31).
It was also interesting to note that HNF-1 did not bind to the
endogenous
1-AT promoter in
HeLa cells, which do not express the
1-AT gene. HNF-1
did bind to
the
1-AT promoter but only weakly. There was, however, induction of the
1-AT-reporter when HeLa cells
were cotransfected with HNF-1
, or with both HNF-1
and HNF-4, but
not with HNF-4 alone. Thus HeLa cells have all the machinery necessary
to activate a naked
1-AT
reporter but lack the critical role played by expression of the
HNF-1
gene. Previous studies have shown that extinction of both
HNF-4 and HNF-1
gene expression is also associated with the absence
of
1-AT gene expression in
fibroblasts (4, 19).
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Joyce Williams and Barbara Hermann for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
The studies were supported by the National Institutes of Health Grants DK-45085 and HL-37784, as well as a Burroughs Wellcome Experimental Therapeutics Scholar Award.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. H. Perlmutter, Dept. of Pediatrics, Washington Univ. School of Medicine, One Children's Place, St. Louis, MO 63110.
Received 25 August 1998; accepted in final form 4 February 1999.
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