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J Biol Chem, Vol. 273, Issue 45, 30024-30032, November 6, 1998
From the Retinoic acid (RA),1 a
vitamin A metabolite and a potent regulator of development and
differentiation, acts through transcriptional regulation of gene
expression. Modulation of gene expression by RA results primarily in
the induction of genes but also, in some cases, repression. Genes
induced by RA include RAR Activation of the AFP gene by RA has been reported in McA-RH8994 and F9
cell lines (8, 9). These RA effects are superimposed on a complex
transcriptional regulatory system, since high-level tissue-specific
activation of the AFP gene from mouse, rat, and human cell lines
requires three enhancer elements in addition to a minimal promoter
(12-14). Several transcription factors have been reported to be
involved in AFP gene regulation, including fetoprotein transcription
factor (15), promoter coupling factor (16), Nkx2.8 (17), HNF1
(hepatocyte nuclear factor 1) (13, 18, 19), C/EBP
(CCAAT/enhancer-binding protein) (18), NF1 (nuclear factor 1) (18), and
RXR and RAR receptors (8, 9).
HNF1 and its regulator, hepatocyte nuclear factor 4 (HNF4), appear to
play a key role in the liver-specific regulation of many genes
expressed during liver differentiation and development (for review, see
Ref. 20). HNF1 is a homeodomain protein required for the expression of
AFP and many other liver-specific genes such as fibrinogen HNF4 is an orphan receptor of the nuclear receptor superfamily (23).
HNF4 forms dimers and binds to the promoter sites of the transthyretin
(24), phosphoenolpyruvate carboxykinase (25), and HNF1 genes (26). In
the promoter of the HNF4 gene, an HNF1 transcriptional response has
been demonstrated, suggesting the presence of a regulatory loop between
HNF4 and HNF1 (27). Additionally, deletion of HNF4 from cell somatic
hybrids eliminated expression of HNF1, demonstrating that these two
genes form a network of transacting factors controlling multiple
liver-specific genes (28). HNF1 also negatively regulates its own
expression and that of other HNF4-dependent genes that lack
HNF1-binding sites. This repression is due to direct interaction
between HNF1 and HNF4 (29).
Previous studies have shown RA activation of AFP gene expression,
primarily through elements that we characterized in the nearest
upstream enhancer, at Cell Culture and Transfection--
Cell lines Hep3B and
McA-RH8994 were purchased from the American Type Culture Collection
(Rockville, MD). Cells were cultured in minimal essential medium
(Sigma) supplemented with 10% fetal calf serum. Calcium phosphate
transient transfection assays were performed essentially as described
with the modification that assays were performed in six well plates
rather than 10-cm plates (30). Reagents were scaled down accordingly.
In brief, 1 day before transfection, cells were seeded in each well at
8.45 × 104 cells. AFP-CAT plasmid constructs (3.2 µg) were mixed with 2.5 M CaCl2 and 2 × HeBS (280 mM NaCl, 50 mM Hepes, and 1.5 mM Na2PO4 at pH 7.05) and the
plasmid precipitate was applied to cells for 5 h. Cells were next
treated with 10% glycerol for 2 min, washed three times with 1 ml of
phosphate-buffered saline, and incubated with 2 ml of MEM with or
without RA (Sigma). Cells were refed with MEM with or without RA each
day for total of 2.5 days. After treatment, the cells were harvested,
and CAT activity was assayed by the phase extraction method using
[3H]chloramphenicol as substrate. For co-transfection
experiments, expression plasmids were mixed with the AFP-CAT plasmids
at a 1:19 ratio (i.e. 3.2 µg of AFP-CAT plasmid to 0.17 µg of expression plasmid). Plasmid pGEM was added to equalize the
amount of plasmid per well. The figures show simultaneous platings for
each plasmid, and each data point is the average of three separate
transfections. Each experiment described was reproduced at least three
times. The following plasmids for transfection have been previously
described: AFP-gene expression plasmids pAFP7300, pSpe Northern Blot Hybridization--
Total RNA was extracted by the
guanidinium isothiocyanate method (33). Twenty µg of total RNA per
lane was resolved by electrophoresis on a 1.2% agarose gels containing
2.2 M formaldehyde and then transferred to nylon membranes
by capillary blotting. 32P-Labeled HNF1 Gel Mobility Shift Assay--
Double-stranded oligonucleotides
to the HNF1-binding sites in the rat AFP promoter (AFP HNF1-1,
Retinoid Effects on AFP mRNA--
Preliminary studies revealed
that AFP expression in Hep3B cells was decreased by RA treatment
(all-trans-RA, t-RA as shown in the Fig.
1), in contrast to other hepatoma cell
lines where RA had the opposite effect. To analyze the RA-mediated
inhibitory effect, Hep3B cells were treated with retinoids
(10
To assess whether down-regulation of the AFP gene required on-going
protein synthesis, cycloheximide was added concurrently with RA for
48 h. Hep3B cells treated with cycloheximide (10 µg/ml) and RA
at 10
The time course of RA-mediated mRNA down-regulation on the AFP gene
was also examined. Equal numbers of cells (5 × 106)
were grown with or without RA for up to 48 h (Fig.
2a). Without RA treatment, the
level of AFP mRNA increased continuously as the cells reached
confluence. RA treatment blocked the increase and by 48 h caused
an absolute decrease in mRNA level. The inhibition was relatively
slow; it was first noted at 16 h and became evident after 24 h of RA treatment. By 48 h, there was a 10-fold difference between
RA-treated and untreated cells (Fig. 2b). Since the
inhibition was not blocked by cycloheximide, the effect was most
consistent with transcriptional inhibition of a relatively stable
mRNA with a long half-life. It has been reported that RA
(10 The Proximal AFP Promoter Mediates RA-dependent
Down-regulation--
The AFP 5'-flanking region has three enhancers
that have been shown to stimulate expression of the AFP promoter in an
additive fashion, as well as a complex promoter (31). A region between enhancer 1 and the promoter has been reported to be involved in repression of the AFP gene and has been termed a DNA silencer region
(39-41). Accordingly, RA-mediated down-regulation of AFP gene
expression might involve upstream elements, the promoter, or
interaction among them. Transfection analysis was employed to verify
that RA effects were transcriptional and to localize the gene controls
that mediate the effects.
For determination of general regions that might be critical for
RA-induced down-regulation, a series of AFP-CAT plasmids derived from
the 7.3 kb upstream of the AFP transcription start site was utilized
(Fig. 3a). Expression of
plasmid pAFP7300, which contained the full 7.3-kb upstream AFP gene
regulatory region, showed 2.6-fold inhibition by RA (Fig.
3b). Plasmids p1483
To further localize the negative effect, a series of AFP promoter-CAT
constructs were examined in which enhancer elements were maintained but
with progressive deletion of the promoter region (Fig.
4a). These contained deletions
to HNF1 and HNF4 Binding Is Reduced following RA
Exposure--
Because transfection analysis localized the RA effect to
a 125-bp promoter region, gel shift studies were set up to survey this
region for altered transcription factor binding. Two HNF1 recognition
sites were located within 131 bp of the transcription start site (13,
18, 19, 22), and HNF1 has been shown to be an important activator of
AFP gene expression (13). To accommodate the two HNF1 sites, the survey
was enlarged to encompass the region to
Other assays examined the remainder of the promoter region for
additional effects (Fig. 5c). This region contained known
C/EBP and NF1-binding sites. Oligonucleotides were also designed to survey regions without known binding factors, in case RA-mediated effects were predominant to cryptic binding sites that had not been
described previously. In contrast to the HNF1 and HNF4 binding, NF-1
and C/EBP binding were increased by RA treatment. Observation of these
effects was helpful, because it demonstrated that the RA effects were
specific for HNF1 and HNF4 and did not reflect a general effect on the
Hep3B nuclei or other effects that might have altered the entire
nuclear extract. Gel shift with oligonucleotides A-D indicated some
additional weak binding factors that had not been characterized, but
none of these showed significant change with RA. Together, these data
indicated that RA selectively decreased binding of both HNF1 and its
regulator, HNF4, and the magnitude of the decreases was comparable with
the reduced gene expression observed in the transfection assays.
Moreover, the localization of the RA effect to the HNF1 and HNF4 mRNA Expression Is Reduced by RA
Treatment--
Northern blot analysis of mRNA levels was carried
out to follow up the observation of reduced nuclear levels of HNF1 Reversal of RA-mediated Down-regulation--
The studies so far
imply that RA acts to inhibit HNF1 Down-regulation of AFP gene expression can occur by a variety of
mechanisms. Previous studies have implicated AP-1 transcription factors
in glucocorticoid-mediated down-regulation (43), c-Jun in
12-O-tetradecanoylphorbol-13-acetate-mediated
down-regulation (44), and COUP-TF as a general negative regulator (45,
46). In this report we have demonstrated a distinctive effect in which RA-mediated down-regulation is dependent on the inhibition of transcription factors HNF1 and HNF4. This effect suggests a
relationship between retinoids and the two main transcription factors
that control the hepatocyte phenotype.
RA both activates and represses AFP expression in different settings.
In cases where RA activates the AFP gene, the mechanism appears to
involve direct activation by binding of RXR A variety of signaling pathways has been implicated in the
down-regulation of the AFP gene, but all can be distinguished from RA-mediated repression. Similar to the effect of RA, glucocorticoid can
either turn AFP gene expression on and off (reviewed in Ref. 48)
through binding sites in the upstream promoter region. Postnatal repression may involve the action of a DNA silencer element in a region
upstream of the minimal promoter between A number of mechanisms can be envisioned for the negative regulation of
the AFP gene by RA. One possibility is that RA reduces the expression
of HNF4, which in turn reduces expression of HNF1 How might RA regulate the expression of HNF1 and HNF4? The specific
mechanism for RA inhibition of each gene remains to be investigated.
One possibility is that RA treatment might reduce expression or
activity of particular RARs or RXRs. We have found that RXR We thank Dr. G. Crabtree for the HNF1 *
This work was supported by National Institutes of Health
Grants CA53596 and CA68440 and American Cancer Society Grant NP-955.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.
¶
Present address: Dept. of Cancer Biology, National Cancer
Institute, University of Cairo, Cairo, Egypt.
The abbreviations used are:
RA, retinoic acid; AFP, 2
T. R. Magee, Y. Cai, M. E. El-Houseini, J. Locker, and Y.-J. Y. Wan, unpublished results.
Retinoic Acid Mediates Down-regulation of the 
-Fetoprotein
Gene through Decreased Expression of Hepatocyte Nuclear Factors*
,,
Department of Pathology, Harbor-UCLA Medical
Center, Torrance, California 90509 and the § Department
of Pathology, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Fetoprotein (AFP), a protein highly induced
during fetal liver development, is down-regulated by retinoids in the
human hepatoma cell line Hep3B, in contrast to up-regulation observed in other cell types. Previously, we have documented that such up-regulation involves direct effects through cis-retinoid
X receptor-binding sites in the AFP enhancer. In this report, we show a
distinctive effect of all-trans-retinoic acid (RA) in Hep3B
cells. RA caused a marked decrease in AFP transcripts. Deletion
analysis of the upstream regulatory region of the AFP gene revealed
that cis-acting sites required for down-regulation resided
near the promoter. Gel mobility shift assays for factors binding to key
elements in the AFP promoter region demonstrated that hepatocyte
nuclear factor (HNF) 1 binding was diminished in nuclear extracts from RA-treated cells. In addition, HNF4, which is not known to bind to the
AFP promoter but does regulate HNF1, was also diminished. The levels of
HNF1 and HNF4 mRNA were also decreased following RA treatment. AFP
promoter-chloramphenicol acetyltransferase transient transfection
assays demonstrated that the level of HNF1 had a direct impact on basal
transcription as well as RA-mediated down-regulation of the AFP gene,
and that co-transfection of HNF1 and HNF4, but not transfection of
either factor alone, reversed the RA-mediated inhibition. Taken
together these data point to an interaction among the RA, HNF1, and
HNF4 signals, which is reflected in decreased expression of AFP.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2 (1-3), RAR2 (4), RAR
2 (5), rat
growth hormone (6), and cellular retinol-binding protein type II (7),
as well as the -fetoprotein (AFP) gene (8, 9). RA can also repress
certain gene transcripts. In F9 cells, the 2.3-kb RXR gene is
completely repressed by RA (10). The rat stromelysin gene, encoding an
enzyme involved in extracellular matrix remodeling, is also
down-regulated by RA, possibly through the AP-1 signaling pathway
(11).
and ,
albumin, and aldolase B (Ref. 21 and references therein). HNF1 can
associate with the highly related HNF1 (vHNF1) homeodomain protein
to form homo- or heterodimers (21). HNF1 dimers bind at two sites in
the rat AFP promoter (
131 to 116 bp; 66 to 51 bp) (13, 18, 19).
Basal level expression of AFP is strongly dependent on HNF1, since
selective mutation of both HNF1-binding sites abolished expression of
AFP promoter-CAT constructs, with or without enhancers (22).
2.4 kb (8). This activation is present in
several AFP-expressing cell lines and has been characterized in detail
in McA-RH8994 cells (8). However, we observed an unusual paradoxical
response in Hep3B cells in that AFP expression was strongly inhibited
by RA. The mechanism of this repression is likely to involve important
features of RA-mediated gene control that are obscured by the
AFP-activation observed in other cell lines. The studies in this paper
demonstrate a distinctive RA-induced repression of the AFP gene and
further show that repression is mediated by elements of the proximal
promoter that interact with HNF1, a factor which is in turn controlled
by HNF4.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
X, pNsiX,
p1483BN, p3195XN, and p1334 (14); AFP-gene plasmids with promoter
deletions 191, 178, 155, 134, and 125 (31); HNF4 reporter
plasmid pAPF-HIVCAT and HNF4 expression plasmid pLEN4S (23); and HNF1 expression plasmid pONHNF1 (32). Among the AFP constructs, pSpeX, which is deleted to 60, contains only half of the HNF1-2 site and
the basal promoter and serves as a negative control. The RXR-specific agent LG100153 was a generous gift from Ligand Pharmaceutical Co.
(28), HNF4 (23),
AFP (34), rRNA, and -actin cDNA (35) restriction fragments were
labeled by random priming and hybridized to membranes in 7% (w/v) SDS,
0.5 M sodium phosphate, pH 6.5, 1 mM EDTA, and
1 mg/ml bovine serum albumin at 68 °C overnight. The membranes were
washed twice in 1% SDS, 50 mM NaCl, and 1 mM EDTA at 68 °C for 15 min each and autoradiographed using
intensifying screens.
135 to 112, 5'-GTGCTGTTAATTATTGGCAAATTG-3'; AFP HNF1-2, 70 to
47, 5'-CTGAAGGTTACTAGTTAACAGACA-3'); the HNF1 consensus binding site
(5'-GTTAATAATTAAC-3') (36); the HNF4 consensus binding site
(5'-CCTAGGCAAGGTTCATATTTG-3') (23); the NF-1 and C/EBP-binding sites in
the rat AFP promoter (AFP NF-1, 126 to 105, 5'-TTGGCAAATTGCCTAA-3';
AFP C/EBP, 118 to 97, 5'-CAAATTGCCTAACTTCAACATA-3'); and the NF-1
and C/EBP consensus binding sites (NF-1, 5'-ATTTTGGCTTGAAGCCAATATG-3';
C/EPB, 5'-ATCTTACTCAACAT-3') (24, 37) were end-labeled using the Klenow
fragment of DNA polymerase I and [32P]dCTP. Additionally,
oligonucleotides spanning the AFP promoter but with no identified
binding sites were similarly labeled. These consisted of
oligonucleotide A (104 to 77; 5'-TCAACATAAGGAAAAAAAGTAACATGTT-3'), oligonucleotide B (84 to 61, 5'-AACATGTTTGCCCACTGAAGGTTAT-3'), oligonucleotide C (50 to 22, 5'-GACATCACTTAAAAAGGATATAAAAGAACT-3'), and oligonucleotide D (21 to +5, 5'-TTCAGCGCTACTGCTCACAGTATCCG-3'). All oligonucleotides contain HindIII and
XbaI restriction sites on the 5' and 3' ends, respectively.
Labeled probes (105 cpm) were incubated with Hep3B nuclear
extracts (5 µg of protein unless otherwise noted in the figure
legends) in a 20-µl reaction consisting of 2 µg of
poly(dI-dC)·poly(dI-dC), 10 mM Hepes, pH 7.9, 50 mM KCl, 5% glycerol, 2% Ficoll, 0.05% Nonidet P-40, and 1 µl of rabbit serum. Reactions were incubated for 10 min on ice, radiolabeled probe was added, and reactions were continued for another
10 min on ice. For supershift experiments, 1 µl of 1:3 diluted HNF4
antibody (provided by F. M. Sladek) was added after probe incubation,
and the mixture was further incubated for 10 min on ice. Reactions were
run on a 5% nondenaturing polyacrylamide gel in 0.5 × TBE (1 × TBE: 89 mM Tris base, 89 mM boric acid, and 50 mM EDTA at pH 8.0) buffer. For competition experiments, unlabeled oligonucleotides were added to the reactions at a 30-fold molar excess over labeled oligonucleotides.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
7 to 105 M) (Fig.
1a). This caused clear reduction of AFP mRNA in Hep3B cells. In contrast, 9-cis-RA (c-RA) increased AFP mRNA
in McA-RH8994 cells. A representative study (Fig. 1b) is
presented to illustrate this difference. In Hep3B cells, RA and c-RA
caused comparable inhibition of AFP mRNA. Both agents are ligands
for RARs, whereas only c-RA is a ligand for RXRs. To further clarify
receptor specificity, we also used LG100153, an RXR-specific ligand.
This agent also inhibited, but the effect was weaker than with RA and
c-RA (Fig. 1, a and c). The I0.5
values were calculated as 1 × 107 M for
c-RA and 2 × 10-7 M for t-RA, a
difference that was not significant within the accuracy of our
measurements. In contrast, the I0.5 for LG100153 was
3.3 × 106 M. Though this agent is
expected to have comparable potency to c-RA for RXR activation, the
data show that a degree of inhibition can be mediated solely by
liganded RXR. Together, these observations indicate that activation of
RXRs alone cannot mediate the observed phenomena. However, RXRs
mediated some inhibition, although the inhibition might be a dual
effect of RARs and RXRs. For both RA and c-RA, inhibition was clear at
the physiological dose of 107 M.
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Fig. 1.
The effect of retinoids and cycloheximide on
AFP mRNA. a, Hep3B cells were cultured in the
absence (Control) or presence of 10 7,
106, and 105 M
all-trans-RA (t-RA), 9-cis-RA
(c-RA), or LG100153 for 48 h. b, McA-RH8994
cells were cultured in the absence (Control) or presence of
106 M of c-RA. c, the mRNA
determinations were scanned and the measurements plotted as a function
of molar dosage of t-RA, c-RA, or LG100153. d, Hep3B cells
were cultured in the absence (Control) or presence of RA for
48 h, with or without 10 µg/ml cycloheximide. For all the
panels, RNA was extracted, and Northern blot hybridization was
performed for AFP and control rRNA or -actin mRNA.
6 or 105 M showed
down-regulation of mRNA to the same extent as for treatment with RA
alone (Fig. 1d). This result indicated that no new protein synthesis was required for RA to mediate the repression of AFP gene
expression. Even with cycloheximide, we saw no detrimental effects on
the cells such as necrosis or apoptosis, as determined by light
microscopic evaluation of trypan blue exclusion (data not shown). Since
these data demonstrated that RA (105 M)
showed strong inhibition without cytotoxicity, this agent and dosage
were chosen for further studies.
5 M) can block AFP gene expression in
Hep3B cells by inhibiting cell proliferation when cells are treated for
long periods of time (30 days) in serum-free medium (38). To exclude a
similar effect, we measured cell growth over a period of 72 h. As
seen in Fig. 2c, there was little change in cell
proliferation over this time, indicating that the RA-mediated decrease
in AFP mRNA did not coincide with decreased cell growth.
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Fig. 2.
Time course of RA effects on AFP mRNA and
cell growth. a, equal number of Hep3B cells (5 × 106) were plated and cultured in the absence (C)
or presence of 10 5 M RA for various periods
of time ranging from 4 to 48 h. RNA was extracted from the cells
and Northern blot hybridization was performed. b, the
mRNA determinations were scanned and the measurements plotted
versus time in culture. Values are relative to the mRNA
level at initial plating. c, Hep3B cells (5 × 106) were plated and cultured in the absence (C)
or presence of 105 M RA for various periods
of time ranging up to 72 h. Cells were detached from plates using
trypsin and counted by light microscopy. Each data point consists of
the average of three independent determinations.
BN (Enhancer 1), p3195XN (Enhancer
2), and p1334 (Enhancer 3) contained a single enhancer combined with
the full-length promoter region. pNsiX contained only the promoter
and associated transcription controls, whereas pSpeX, deleted to
60, contained only the basal promoter and served as a negative
control (Fig. 3c). All three enhancers together, or each
single enhancer, showed comparable down-regulation as the plasmid
construct containing only the AFP promoter (2.5-fold). These data
therefore localized the negative regulation to a region near the
promoter.
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Fig. 3.
Transfection to localize the general region
of RA-repression. a, the 5'-upstream region of the rat
AFP gene is shown with enhancer and promoter regions detailed. A series
of AFP gene expression plasmids, derived from the parent plasmid
pAFP7300 (14), p1483 BN, p3195XN, p1334 contained single enhancer
regions combined with the promoter, whereas pNsiX contained only the
full promoter region, and pSpeX, a minimal promoter. b
and c, cell transfections were carried out as described
under "Experimental Procedures." Transfectants were treated with or
without 105 M RA for 2.5 days. Results, the
average of three samples (n = 3), were plotted as total
activity (counts/min (CPM) of acetylated chloramphenical).
The error bars are S.D. values.
191, 178, 155, 134, or 125 bp from the transcription start
site (31). As seen in Fig. 4b, progressive deletion caused a
reduction of overall CAT expression, but all constructs continued to
show the same magnitude of RA-mediated down-regulation. CAT expression
from all of these plasmids showed comparable reduction, ranging from 2.2- to 2.7-fold. Further deletion of the promoter region abolished the
basal activity. Thus, the RA-mediated repression required only 125 bp
of the promoter region.
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Fig. 4.
Resolution of RA mediate repression to the
proximal promoter region. a, a series of AFP promoter
deletions were created from the parent plasmid pAFP7300 (31). Plasmids
191, 178, 155, 134, and 125 were sequential deletions of the
AFP promoter, which retained all three enhancers. b, cell
transfections and treatment with 105 M RA
were as described in the legend to Fig. 3. Results, the average of
three samples (n = 3), were plotted as total activity
(counts/min (CPM) of acetylated chloramphenical). The
error bars are S.D. values.
135 (Fig.
5a). We also studied HNF4
binding. Although the region contained no known binding sites for this
factor, HNF4 is the primary transcriptional regulator of HNF1 and may
also be down-regulated (26). Oligonucleotides representing the two AFP
HNF1-binding sites were 32P-labeled and incubated with
nuclear extracts from cells treated with or without RA (Fig.
5b). These were compared with the HNF1 consensus binding
site in both direct binding and competition assays, demonstrating that
all three oligonucleotides bound in a fashion specific for HNF1. The
binding pattern consisted of two specific bands, marked by
arrows in the figure. The upper band represented HNF1
homodimers and the lower HNF1: heterodimers (42).2 Specific and
nonhomologous competition demonstrated that additional bands in the
patterns were nonspecific. Both AFP promoter HNF1 sites were weaker
than the consensus site, though the more upstream site was the stronger
of the two. Notably, after RA treatment, binding of HNF1 to all three
oligonucleotides was reduced by more than half compared with the
controls. HNF4 binding to an HNF4 consensus binding site was similarly
reduced in nuclear extracts following RA exposure (Fig. 5b,
right panel). For this factor, the specificity of the assay
was verified by supershifting of HNF4 protein with an HNF4-specific
antibody (Fig. 5b).
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Fig. 5.
RA-induced changes in transcription factor
binding. a, the AFP proximal promoter region is
detailed. Oligonucleotides used for gel shift analysis are indicated
under the promoter map. b, promoter sites AFP HNF1-1, AFP
HNF1-2, an HNF1 consensus binding site, and an HNF4 consensus binding
site, were 32P-labeled and used as probes when incubated
with Hep3B nuclear extracts. In addition to specific competitors, an
AP1 oligonucleotide was used as a nonspecific competitor. 1 µl of
anti-HNF4 antibody was included for supershift assays. Extracts were
prepared from cells cultured in the absence (C) or presence
(RA) of 10 5 M RA for 3 days. In
the three panels to the left, arrows
indicate the positions of HNF1-specific bands, whereas in the
right panel, the arrows mark the specific HNF4
band or its supershift band. c, gel shifts were conducted in
the same manner as above using oligonucleotides to survey the entire
promoter region. For competition experiments, unlabeled competitors
were added at a 30× molar ratio. In addition to specific competitors,
a C/EBP oligonucleotide was used as a nonspecific competitor for
oligonucleotides A-D. For all panels, arrows indicate
undefined gel shift bands that disappear only on self-competition,
indicating a specific binding interaction.
125-bp region of
the promoter could be explained by the effects on HNF1, even though
there was an additional binding site outside this region, since earlier
studies indicated that both sites make significant contributions to AFP
gene expression (13, 22). Thus the shorter promoter was weaker, but
still regulated through its remaining HNF1-binding site.
and HNF4. Fig. 6 demonstrates that both
HNF1 and HNF4 mRNA expression decreased 2-3-fold following
treatment of Hep3B cells with RA. These observations verify the
transcription factor binding study by also indicating that the RA
effect on AFP expression is mediated by transcriptional down-regulation
of HNF1 and HNF4.
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Fig. 6.
Effect of RA on HNF1 and HNF4
mRNA. Hep3B cells were cultured in the absence (C)
or presence of 105 M RA for 3 days. Northern
blot hybridization was performed to examine the expression of the
HNF1 (a) and HNF4 (b) genes. -Actin
mRNA expression was used to measure loading.
expression and that reduced
HNF1 expression in turn leads to reduced AFP expression. Moreover,
since HNF4 regulates HNF1, the primary effect could be at the level
of HNF4 expression. Alternatively, RA might act independently on both
HNF4 and HNF1 expression. It is even possible that RA could have an
independent effect on AFP that we failed to localize. To examine these
possibilities and study the direct impact of HNF1 and HNF4 on the
RA-mediated AFP gene regulation, HNF1 and HNF4 expression plasmids
were used in co-transfection experiments to determine whether increased expression of either factor would reverse the RA effect. Plasmid 134
was chosen as the reporter for these experiments, since it had
relatively strong expression through two functional HNF1 sites (Fig.
4a). Overexpression of HNF1 increased AFP promoter-driven CAT expression by 3-fold and also blocked 80% of the
RA-dependent down-regulation (Fig.
7a). Overexpression of HNF4
alone had no effect in preventing RA-mediated down-regulation of the
AFP gene. Even though HNF4 is a strong positive regulator of HNF1
expression, transient expression of HNF4 did not necessarily have an
effect on the endogenous HNF1 gene. Control reporter plasmids
pAPF-HIV-CAT, containing two HNF4-binding sites, demonstrated that the
transfection effectively increased HNF4 expression (Fig.
7b). Nevertheless, when HNF1 and HNF4 were
co-transfected, the effect was greater than transfection with HNF1
alone. AFP expression was stimulated, and RA repression was totally
blocked. Thus, although the mechanisms appear to be complex, the effect
of RA on AFP expression was mainly due to decreased expression of
HNF1, but HNF4 may also play a role.
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Fig. 7.
Restoration of AFP expression by
co-transfection with HNF1 and HNF4 expression plasmids.
a, Hep3B cells were transiently transfected with 134 as an
AFP gene reporter and expression plasmids for HNF1 or HNF4.
b, in control studies, basal promoter reporter pBS-HIV-CAT
or HNF4 reporter pAPF-HIV-CAT was studied with and without
co-transfection with the HNF4 expression plasmid. pAPF-HIV-CAT has two
tandem promoters. All transfections contained 1 µg of reporter
plasmid and 170 ng of expression plasmid DNA per plate. Transfected
cells were treated with or without 106 M RA
for 2.5 days and assayed for CAT activity. Results were the average of
three determinations (n = 3) and plotted as counts/min
(CPM) (counts/min of acetylated chloramphenical). The
error bars represent S.D. values.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
/RXR homodimers to
cis-acting sites in the 5'-flanking region of the AFP gene. We have identified an RXRE at 2406 to 2378 bp active in McA-RH8994 cells that mediates this stimulation (8). Other studies have indicated
additional binding sites (9, 41, 47). Thus, RA has dual effects in
controlling the expression of the AFP gene. As reported previously
(46), a weak RXRE exists within the AFP promoter region at 143 to
131 bp. However, deletion of the AFP promoter to within 125 bp of the
transcriptional start site did not prevent RA-mediated repression (Fig.
4), indicating that this RXRE had little effect on AFP expression with
or without RA treatment. Study of the various enhancer-promoter
combinations also ruled out a role for various upstream RAR- or
RXR-binding sites. Thus, in contrast to stimulation, the RA-mediated
repression of AFP is indirect, through regulation of HNF1 and HNF4
gene expression.
1010 and 250, which also
can act as a repressor for heterologous gene expression (39, 40, 49).
However, deletion of the glucocorticoid-binding sites or the silencer
element had no effect on the actions of RA in Hep3B cells. In
fibroblasts, the AFP gene is repressed by the binding of unknown
transcription factors to the promoter region between 57 and 43 bp
(50), but our findings showed no increased binding of factors to this
region. A variety of other transcription factors and signaling pathways
such as c-Ha-Ras (51), c-Jun (AP1) (44), ATBF1 (A-T binding factor),
which binds only the human upstream enhancer (52), transforming growth
factor- (53), and hepatocyte growth factor (54) have also been
associated with some degree of AFP gene repression. A possible role for
RA in these signaling pathways remains to be investigated.
. Hence
down-regulation of AFP would be due to decreased binding activity of
HNF1 to the two AFP HNF1-binding sites. It is not known whether HNF4
has a direct effect on the AFP gene, because no HNF4-binding site has
been identified within the AFP gene. Moreover, reduced expression of
HNF4 by itself was not sufficient for RA-mediated down-regulation,
since overexpression of HNF4 did not prevent down-regulation of an
AFP-CAT reporter plasmid (134). In contrast, overexpression of HNF4
and HNF1 together could completely overcome the RA-mediated
down-regulation. It is not clear why HNF4 worked only when both HNF1
and HNF4 were overexpressed, but this observation does indicate a level
of regulation that is not recapitulated by transfection of either
factor alone. Nevertheless, the data strongly suggest that the
decreased expression of HNF1 is the immediate effector of decreased
AFP expression. Another case where HNF1 activity modulates AFP gene
expression comes from genetic persistence of AFP expression in humans.
Analysis of the AFP promoter sequence in affected individuals
identified a single point mutation, a G to A transition at 119 in the
AFP HNF1-1-binding site (55). This mutation strengthened the site, increased HNF1 binding, and also increased the expression of the AFP
promoter region. A number of other genes are down-regulated when HNF1
expression is decreased in a manner strikingly similar to the AFP gene.
For example, diabetes mellitus decreases HNF1 and albumin gene
expression in the rat (56). Human renal cell carcinoma provides another
example, where glutathione S-transferase mRNA expression
is reduced concomitant with reduction of HNF1 binding to its promoter
(57).
mRNA
expression is selectively reduced following RA treatment of Hep3B cells
and that total transcription factor binding to DR1 and DR5 sites in
RA-treated Hep3B nuclear extracts is also decreased (58). Though
neither the HNF4 nor the HNF1 promoter appears to have retinoic acid
receptor element (DR5) sites, the HNF4-binding site in the HNF1
promoter is also a DR1 site. The role of possible RXR or RAR binding
sites in the HNF1 and HNF4 promoter regions is currently being
investigated. There are several known cases where DR1 sites bind both
HNF4 and RXR dimers. The hepatitis B virus enhancer I, for example,
contains an RXRE-like element (DR1) that can bind HNF4, RXR, or
COUP-TF (59). In F9 cells, RA induces the expression of HNF1 (28,
60), and the HNF1 promoter has an another DR1 site that interacts
with several members of the steroid hormone receptor superfamily
including HNF4, COUP-TF, and RXR (60). Thus, complex interaction of
HNF4 and RXR homo- and heterodimers at DR1 sites, and change in the mixture of RARs and RXRs, are likely to contribute to the RA effects we observed.
ACKNOWLEDGEMENTS
expression plasmid and Dr. F. Sladek for the HNF4 expression plasmid
and HNF4 antibody.
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Pathology, Harbor-UCLA Medical Center, 1000 West Carson St., Torrance, CA 90509. Tel.: 310-222-3876; Fax: 310-782-6649; E-mail:
agarose{at}ucla.edu.
-fetoprotein; RXR, retinoid X receptor; RXRE, retinoid X
receptor-responsive element; RAR, retinoic acid receptor; kb, kilobase pair(s); HNF, hepatocyte nuclear factor; C/EBP, CCAAT/enhancer-binding
protein; bp, base pair(s); t-RA, all-trans-retinoic acid; c-RA, 9-cis-retinoic acid.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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