Members of the Nuclear Factor 1 Transcription Factor Family Regulate Rat 3
-Hydroxysteroid/Dihydrodiol Dehydrogenase (3
-HSD/DD AKR1C9) Gene Expression: A Member of the Aldo-keto Reductase Superfamily
Chien-Fu Hung1 and
Trevor M. Penning
Department of Pharmacology University of Pennsylvania School of
Medicine Philadelphia, Pennsylvania 19104-6084
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ABSTRACT
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Rat 3
-hydroxysteroid/dihydrodiol
dehydrogenase (3
-HSD/DD; AKR1C9), a member of the aldo-keto
reductase (AKR) superfamily, inactivates nearly all steroid hormones by
converting 5
- and 5ß-dihydrosteroids to their respective
3
,5
- and 3
,5ß-tetrahydrosteroids and protects against
circulating steroid hormone excess. It is highly expressed in rat liver
comprising 0.51.0% of the soluble protein. Previously, we identified
a powerful distal enhancer resident at about -4.0 kb to -2.0 kb in
the 5'-flanking region of the 3
-HSD/DD gene. We now
report the functional dissection of this enhancer. Transfection of
nested deletions of the 5'-end of the gene promoter linked to
chloramphenicol acetyltransferase (CAT) into HepG2 cells located the
enhancer activity between (-4673 to -4179 bp). Further internal and
5'-end deletion mutants revealed that a 73-bp fragment (from -4351 to
-4279 bp) contained a major enhancer element. This fragment spanned
two imperfect direct repeats GTGGAAAAACCCAGGAA and
GTGGA-AAAAACCCAGGAA and contained three direct repeats
of GGAAAAA. This fragment also contained three potential half-nuclear
factor 1 (NF1) sites (TGGA-NNNNNGCCA) and a putative
CCA-AT-enhancer binding protein (C/EBP) binding site. The 73-bp
fragment enhanced CAT activity from the basal 3
-HSD/DD
gene promoter. Recombinant C/EBP
and C/EBPß did not bind to this
fragment. Electrophoretic mobility shift assays showed that HepG2 and
rat liver nuclear extracts bound to this 73-bp fragment. The 73-bp
protein complex was competed out by a NF1 oligonucleotide and was
supershifted by an NF1 antibody. When the 73-bp fragment was fused to
an
1-globin promoter-CAT construct and cotransfected with CCAAT
transcription factor 1 (CTF1)/NF1 into Drosophila Schneider
SL2 insect cells (which lack NF1-like proteins)
trans-activation of CAT activity was observed. These
results indicate that members of the NF1 transcription factor family
regulate high constitutive expression of the rat
3
-HSD/DD gene that is responsible for steroid hormone
inactivation. The potential role of NF1 in regulating other AKR genes
that have protective roles is discussed.
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INTRODUCTION
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The aldo-keto reductase (AKR) gene superfamily consists of highly
related, soluble, monomeric (37-kDa) NAD(P)H oxidoreductases that
convert ketones to alcohols. Their endogenous substrates include
steroid hormones and PGs while their exogenous substrates include drugs
and carcinogens (1, 2). Genes of this superfamily encode for proteins
approximately 320 amino acids in length which adopt an
(
/ß)8-barrel motif and bind NAD(P)(H) in an unusual
extended conformation (3, 4). Loop structures at the back of the barrel
dictate their ligand specificity. Cluster analysis shows that the first
family (AKR1)2 of the superfamily includes
aldehyde reductases (AKR1A), aldose reductases (AKR1B), and the
hydroxysteroid dehydrogenases (HSDs) that comprise the AKR1C subfamily.
The majority of mammalian 3
- and 20
-HSDs belong to the AKR1C
subfamily. Of these, human type 2 3
-HSD (AKR1C3) has been implicated
in the elimination of 5
-dihydrotestosterone from the prostate (5)
while rat ovarian 20
-HSD (AKR1C8), which inactivates progesterone,
has been implicated in the termination of luteal phase pregnancy
(6).
Rat liver 3
-HSD/DD3 (AKR1C9) is
one of the best studied AKRs. It catalyzes the second step in the
metabolism of nearly all circulating steroid hormones, e.g.
androgens, progestins, and glucocorticoids, by converting their 5
-
and 5ß-reduced dihydrosteroids to their corresponding 3
,5
- and
3
,5ß-tetrahydro-steroids (7, 8, 9, 10). Interestingly,
5ß-reductase (AKR1D), which precedes 3
-HSD in steroid hormone
metabolism, is also a member of the AKR1C subfamily, and a single-point
mutation introduces 5ß-reductase activity into 3
-HSD (11),
suggesting that this pathway of steroid hormone metabolism may have
arisen by AKR gene duplication. AKR1C9 is also essential for
the biosynthesis of bile acids and their transport from the sinusoidal
to the canicular pole of the hepatocyte (12, 13, 14).
HSDs in the AKR1C subfamily also have associated dihydrodiol
dehydrogenase (DD) activity, which activates polycyclic aromatic
hydrocarbon (PAH) trans-dihydrodiols by forming reactive and
redox-active o-quinones. These events may contribute to
the carcinogenic potential of the parent PAH. For example, 3
-HSD/DD
converts trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (a
potent proximate carcinogen) to benzo[a]pyrene-7,8-dione with
concomitant production of reactive oxygen species (15, 16). Human
homologs exist for AKR1C9 in which the diverse functions of the rat
enzyme have been distributed across several gene products. Thus, type 1
3
-HSD (DD4 and chlordecone reductase; AKR1C4) is a hepatic specific
3
-HSD, type 2 3
-HSD (DDX and AKR1C3) is expressed in liver and
androgen target tissues, and type 3 3
-HSD (DD2 which is bile
acid-binding protein) shows a similar distribution to the type 2
isoform (17, 18).
AKR1C9 is highly constitutively expressed in rat liver, comprising
0.51.0% of the soluble protein but it is also regulated. In female
rat liver, a 2-fold increase in mRNA and protein expression is
observed, and this female pattern of expression can be established by
administering estrogens to male rats (19, 20, 21). It is unknown whether
this is a direct effect of estrogens or whether this is due to the
regulation of GH secretion by gonadal steroids (20, 21). Additionally,
dexamethasone increases steady-state levels of 3
-HSD/DD mRNA in rat
hepatocytes (22). These findings suggest that the AKR1C9
gene may be autoregulated by circulating steroid hormones.
Progress has been made in understanding the genomic structure of
AKR genes. The human aldose reductase (AKR1B)
gene has been cloned and consists of nine exons and eight introns (23).
This genomic organization is conserved in both the rat and human
3
-HSDs (17, 24, 25). However, less is known about the
transcriptional regulation of the AKR genes. Our earlier
studies have shown that the AKR1C9 gene promoter contains a
basal promoter between (-199 and +55 bp), a weak proximal enhancer
between (-498 to -199 bp), a powerful silencer (-755 to -498 bp)
that binds Oct, and a strong distal enhancer (-4.0 to -2.0 kb) that
controls constitutive expression. The promoter also contains multiple
steroid response elements (SREs), which may comprise a steroid response
unit (26). We demonstrated that in rat hepatocytes the gene was
transcriptionally activated by glucocorticoids via an occupied
glucocorticoid receptor that bound to the proximal glucocorticoid
response element (27). This suggests that the SREs in the gene promoter
are likely to respond to high circulating levels of steroid hormones,
and subsequent gene transcription may protect against steroid hormone
excess. However, the identity of transcription factors that control the
high constitutive expression of AKR genes that serve
protective roles, e.g. AKR1C9 and aldose
reductase, which is osmoprotective (28, 29, 30, 31), is largely unknown.
The aim of this study was to identify the enhancer(s) and transcription
factor(s) responsible for the high constitutive expression of the
AKR1C9 gene. We report the location of a powerful distal
enhancer -4.5 kb upstream from the transcription start site. We have
identified a 73-bp fragment within this region containing a triple
repeat that drives reporter gene activity. Gel mobility shift and
supershift analysis showed that nuclear factor 1 (NF1) transcription
factors bind to this region. When the 73-bp enhancer is fused to an
1-globin promoter-chloramphenicol acetyltransferase (CAT) construct,
it can be trans-activated by cotransfection of NF1 in a
null-environment (SL-2 insect cells). These results suggest that NF1
transcriptionally activates AKR1C9 gene expression and hence
regulates steroid hormone inactivation in rat liver. NF1 transcription
factors have not been previously implicated in AKR gene
regulation. The possibility that NF1 may trans-activate
other AKR genes that play protective roles is discussed.
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RESULTS
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Sequence of the Upstream 5'-Flanking Region of the Rat
3
-HSD/DD (AKR1C9) Gene
We previously reported the sequence of 1593 bp of the 5'-flanking
region upstream from transcription start site of the AKR1C9
gene (26). In that study we located a powerful distal enhancer between
-4673 to -1906 bp. We now report the gene sequence to -4673 bp from
the transcription start site. During the sequence determination,
several errors were identified in the region between -1593 to -1533,
which are now corrected. The entire sequence from -4673 to -1533 is
reported and has been deposited5
(Fig. 1
).

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Figure 1. Sequence of the Distal Promoter of the Rat
3 -HSD/DD (AKR1C9) Gene
The sequence starts at the 5'-end; the first underline
indicates the position of the 73-bp enhancer including its three NF1
binding sites. The second and third underline indicate
the sequence homologous to the promoter sequence of the
MCPII gene. The fourth underline
indicates the position of the GAGA repeat. Numbering is relative to the
sequence of the proximal promoter of the rat 3 -HSD/DD
gene (26 ).
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Identification of cis-Acting Regulatory Elements in the
Distal Promoter of the 3
-HSD/DD (AKR1C9) Gene
To further localize the enhancer within the upstream region, a
p(-4673/+61)CAT construct was deleted stepwise, and the resulting
constructs were analyzed for CAT activity in HepG2 cells (Fig. 2
). Deletion from -4673 to -4197 caused a loss of
64% of CAT activity. Further deletion to -3908 resulted in an another
loss of 20% CAT activity. Subsequent deletion resulted in modest
decreases of CAT activity that ranged from 311% of the activity
observed in the parent construct. These results indicated that the
strongest enhancer element was located between -4673 to -4197 bp. To
test whether this region alone can activate the AKR1C9 gene
promoter, this region was PCR amplified and ligated into a phsdCAT
construct that contained only the basal promoter (-238 to +61) of the
AKR1C9 gene linked to pBLCAT3, to yield the
p(-4673/-4183)hsdCAT construct. The CAT activity of this construct
was 34% that of p(-4673/+61)CAT (Fig. 3
, construct
b). This enhancer region contained 490 bp of upstream sequence and was
designated EN490.

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Figure 2. Analysis of the Distal 3 -HSD/DD
(AKR1C9) Gene Promoter by Transient Expression of 5'-Deletion
CAT Constructs into HepG2 Cells
A series of 5'-nested deletions of the AKR1C9 gene
promoter linked to the CAT reporter gene were generated. The CAT
constructs were transfected into the human hepablastoma cell line,
HepG2, and CAT activity was determined by the incorporation of the
1,3-butyryl groups into [14C]chloramphenicol. Relative
CAT activity is given as a percentage. Transfection with pBLCAT3
represents a negative control since this construct contains no promoter
and no enhancer. Experiments were performed in triplicate.
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Figure 3. Analysis of Portions of the Distal 3 -HSD/DD
(AKR1C9) Gene Promoter by Transient Expression of CAT Reporter
Gene Constructs Containing Internal Deletions
The top panel is the CAT activity of each construct
indicated. Rectangles indicate putative C/EBP binding
sites; circles indicate NF1 binding sites. An
oval indicates a putative HNF1 binding site. Relative
CAT activity is given as a percentage of construct a. Experiments were
performed in triplicate.
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Computer analysis4 showed that there were
four imperfect CCAAT enhancer-binding protein (C/EBP) binding sites
(-4667/-4660, -4536/-4527, -4523/-4514, and -4288/-4279), one
putative hepatocyte nuclear factor 1 (HNF1) binding site
(-4265/-4249), and three putative NF1 binding sites (-4351/-4288)
contained within this region. The three NF1 binding sites were
concentrated in a 64-bp core fragment:
GTGGAAAAACCCAGGAAGCACAGTGGAAAAACA-TGTTTATGCCCTCAGGAAAAAACCCAGGAAC.
A comparison of the sequences of three putative NF1 binding sites in
the 64-bp fragment with the NF1 consensus sequence is shown in Fig. 4B
. NF1 is known to bind to a palindromic
sequence TGGC/A(N)5GCCA (32, 33), and it also binds to one
half of the palindrome with a much lower affinity (34, 35). Based on
this sequence information, several deletion constructs were made by PCR
to identify minimum sequences required to activate the
AKR1C9 gene promoter.

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Figure 4. Oligonucleotides Containing Portions of the Distal
Enhancer Used for Generating Reporter Gene Constructs and for Gel
Mobility Shift Assays
A, NF1 binding sites, E1, E2, and E3, are indicated by
underlines. A putative C/EBP binding site is indicated
in bold. Restriction sites (HindIII)
introduced are indicated in lowercase. B, A comparison
of the sequences of E1, E2, and E3 with the NF1 consensus sequence. The
underline indicates sequence identity to the NF1
consensus sequence.
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Deletion of 189 bp from the 5'-end of the EN490 to remove the three
putative distal C/EBP sites (Fig. 3
, -4484/-4183 construct c) gave a
CAT activity that was 67% of that driven by the whole promoter in
p(-4673/+61)CAT (construct a). These data suggested that the three
putative distal C/EBP binding sites were not involved in the activation
of the AKR1C9 gene promoter. They also indicate that the
dominant enhancer is located in the 301-bp fragment in the
-4484/-4183 construct. Deletion of 62 bp from the 3'-end of the EN490
to remove the putative HNF1 site (Fig. 3
, -4484/-4245, construct d)
did not significantly decrease CAT activity, suggesting that this site
was also not functional in the activation of the AKR1C9 gene
promoter. Deletion of 274 bp (Fig. 3
, -4399/-4183, construct e) from
the 5'-end of the EN490 did not significantly reduce the CAT activity
further (Fig. 3
, compare -4484/-4245, construct d, with construct e).
Removal of 274 bp and 62 bp from the 5'- and 3'-ends of EN490,
respectively (Fig. 3
, -4399/-4245, construct f) gave a CAT activity
that was 39% of that driven by the whole promoter in p(-4678/+61)CAT.
A construct containing two putative NF1 binding sites (Fig. 3
, -4478/-4320 construct g) maintained only 17% CAT activity of
p(-4673/+61)CAT but increased activity of phsdCAT by 3-fold. A
construct containing the third putative NF1 binding site, the proximal
C/EBP binding site, and the HNF1 site (Fig. 4
, -4319/-4183, construct
h) only maintained 6% CAT activity of p(-4673/+61)CAT and was
completely devoid of enhancer activity. When the CAT activity of a
construct containing the three putative NF1 sites and proximal C/EBP
binding site (Fig. 4
, -4399/-4245, construct d or f) is compared with
either construct g (-4478/-4320) or construct h (-4319/-4183), it
is apparent that these binding sites may function as the
AKR1C9 gene enhancer. These binding sites are located within
a 73-bp fragment.
To test the function of this 73-bp enhancer, the 73-bp E1234
(three putative NF1 binding sites and the proximal C/EBP binding site),
E123 (three putative NF1 binding sites), E234 (two putative NF1 binding
sites and the proximal C/EBP binding site), E12 (two putative NF1
binding sites), and E1 (one putative NF1 binding site) (Fig. 4A
) were
ligated into phsdCAT to yield p(E1234)hsdCAT, p(E123)hsdCAT,
p(E234)hsdCAT, p(E12)hsdCAT, or p(E1)hsdCAT, respectively. The CAT
activity of p(E1234)hsdCAT was 3840% of the entire promoter
p(-4673/+61)CAT (Fig. 5
) and was
identical to that observed in -4399/-4245 construct f (Fig. 3
). These
data indicated that the enhancer element in -4399/-4245 (154 bp) was
also located in E1234 (73 bp). The CAT activity of p(E1234)hsdCAT was
13-fold higher than that of phsdCAT. The CAT activity of p(E123)hsdCAT,
which lacked the proximal C/EBP binding site, was 9-fold higher than
that of phsdCAT. p(E234)hsdCAT, which lacked the first putative NF1
binding site, lost almost all of its enhancer activity and had
only 0.65-fold activity of phsdCAT. The CAT activity of p(E12)hsdCAT,
which contained the first two putative NF1 binding sites, had 3-fold
higher CAT activity than phsdCAT. The CAT activity of p(E1)hsdCAT,
which contained only the first putative NF1 binding site, was the same
as phsdCAT (Fig. 5
). These results indicated that E1 was the most
crucial element of the enhancer in E1234, but it did not act alone and
required the presence of E2 and E3 to activate the
AKR1C9 gene promoter. They also indicated that the presence
of the putative proximal C/EBP binding site may be important, because
of the difference in CAT activity between p(E1234)hsdCAT and
p(E123)hsdCAT. The same enhancer elements were also subcloned into
thymidine kinase promoter-CAT constructs and shown to increase
transcription of the reporter gene from the heterologous promoter.
Importantly, E1 was found to be the dominant element required for
promoter activity (data not shown).

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Figure 5. Analysis of the Enhancer Activity in the 73-bp
Fragment Linked to the Basal 3 -HSD/DD (AKR1C9) Gene
Promoter
The top panel is the CAT activity of each construct
indicated. The bottom panel is the sequence of enhancers
tested in each construct. Underlined sequences represent
the first (E1), second (E2), and third (E3) NF1 binding sites.
Bolded sequence is a putative C/EBP binding site (E4).
Relative CAT activity is given as a percentage of p(-4673/+61)CAT.
Experiments were performed in triplicate.
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Identification of the trans-Acting Factors That Bind to
the 73-bp Enhancer
Electrophoretic mobility shift assays were conducted by
using HepG2 nuclear extracts and radiolabeled oligonucleotide probes
containing E1234, E123, E234, E12, or E1 to assess DNA-protein
interactions at these sites. Similar DNA-protein complexes were formed
with each of these oligonucleotide probes (Fig. 6
). When rat liver nuclear extracts were
substituted, these oligonucleotide probes formed DNA-protein complexes
that had the same mobility (data not shown). The formation of these
complexes was abolished by the presence of a 250-fold molar excess of
unlabeled oligonucleotide probe identical in sequence with the labeled
probe (Fig. 6
). In subsequent electrophoretic mobility shift assays, a
10- to 50-fold excess of unlabeled oligonucleotide was sufficient to
abolish the complexes, indicating high specificity of the DNA-protein
interactions. Since the 73-bp fragment E1234 contains three putative
NF1 binding sites and one putative C/EBP binding site, we next tested
whether a C/EBP oligonucleotide could inhibit the formation of the
E1234-protein complexes. These complexes were not inhibited by a
250-fold molar excess of C/EBP oligonucleotide or NFkB oligonucleotide
(used as a negative control), whereas these complexes were completely
competed out in a concentration-dependent manner by unlabeled E1234
oligonucleotide (50- to 250-fold excess) (Fig. 7
). To further confirm that C/EBP
transcription factors do not bind to E1234, we demonstrated that
in vitro transcribed and translated recombinant C/EBP
or
C/EBPß do not bind to E1234. As a positive control, recombinant
C/EBP
or C/EBPß bound to a C/EBP oligonucleotide, and this binding
complex was supershifted by C/EBP
and C/EBPß antibodies,
respectively (Fig. 8
). By contrast, the
DNA-protein complexes formed with E1234 were not supershifted with the
C/EBP
and C/EBPß antibodies (data not shown). Thus, the factors
that bind to E1234 are not members of the C/EBP transcription factor
family.

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Figure 6. HepG2 Nuclear Extracts Bind to Enhancer Elements in
the Rat 3 -HSD/DD (AKR1C9) Gene Distal Promoter
Sequences of the double-stranded oligonucleotides used in the gel shift
analysis are listed in Fig. 4 . HepG2 nuclear extracts (10 µg) were
incubated with [32P]-end-labeled double-stranded
oligonucleotide in the absence (-) or presence (+) of a 250-fold molar
excess of the unlabeled homologous double-stranded oligonucleotide. The
arrows indicate the DNA-protein complexes formed.
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Figure 7. C/EBP and NFkB Oligonucleotides Fail to Compete
with E1234 for HepG2 Nuclear Extracts
E1234 probe was incubated with HepG2 nuclear extracts to form a complex
(lane 1). The complex was competed with a 10-, 50-, and 250-fold molar
excess of unlabeled E1234 (lanes 24), unlabeled C/EBP (lanes 57),
or unlabeled NFkB (lanes 810) double-stranded oligonucleotides.
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Figure 8. E1234 Does Not Bind to Recombinant C/EBP or
C/EBPß
Recombinant C/EBP and C/EBPß were expressed via in
vitro transcription and translation using a rabbit reticulocyte
lysate system. Lane 1, E1234 ( -32P-labeled probe); lane
2, E1234 probe and rabbit reticulocyte lysate; lane 3, E1234 probe and
recombinant C/EBP ; lane 4, E1234 probe and recombinant C/EBPß;
lane 5, C/EBP oligonucleotide and rabbit reticulocyte lysate; lane 6,
C/EBP oligonucleotide and recombinant C/EBP ; lane 7, C/EBP
oligonucleotide and recombinant C/EBPß; lane 8, C/EBP
oligonucleotide, recombinant C/EBP and C/EBP antibody; lane 9,
C/EBP oligonucleotide, recombinant C/EBPß and C/EBPß antibody.
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We next tested whether NF1 transcription factors bind to E1234 by
electrophoretic mobility shift analysis. When E1234 was used as a
probe, the E1234-protein complex was abolished by a 50-fold excess of
NF1 consensus oligonucleotide. When an NF1 consensus oligonucleotide
was used as a probe, the NF1-protein complex was inhibited by a 50-fold
excess of E1234 (Fig. 9
). Furthermore,
the E1234-protein complex was supershifted by the NF1 antibody (Fig. 10
); no supershift was observed when
preimmune serum was substituted. Importantly, identical results were
obtained with nuclear extracts from either HepG2 cells or rat liver.
DNase I footprinting showed that HepG2 cell and rat liver nuclear
extracts protected the E1 fragment consistent with the important role
of this element in driving CAT reporter gene activity (data not shown).
Our data indicated that a member of the NF1 transcription factor family
binds to the E1234 enhancer and that E1 may be the most relevant
site.

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Figure 9. The DNA-protein Complex Formed with the 73-bp
Fragment Is Competed by NF1 Oligonucleotides and Vice Versa
The E1234 probe (lanes 15) was incubated with HepG2 nuclear extracts
to form a complex (lane 1). The complex was competed with a 50- and
250-fold molar excess of unlabeled E1234 (lanes 23) and NF1 (lanes
45), respectively. NF1 probe (lanes 610) was incubated with HepG2
nuclear extracts to form a complex (lane 6). The complex was competed
with a 50- and 250-fold molar excess of unlabeled NF1 (lanes 7 and 8)
and E1234 (lanes 9 and 10), respectively. The NF1 oligonucleotide had
the sequence 5'-TTTTGGATTGAAGCCAATATGATAA-3'.
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Figure 10. NF1 Antibody Supershifts the 73-bp
Fragment-Protein Complex
The E1234 probe was incubated with HepG2 (lanes 1 and 2) and rat (lanes
3 and 4) nuclear extracts, respectively. NF1 antibody was added to the
reactions in lanes 2 and 4 (see Materials and Methods
for details).
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NF1 trans-Activates the 73-bp Enhancer in
Drosophila Schneider SL-2 Cells
The NF1 transcription family is often ubiquitously expressed and
contains many splice variants that could affect the signal observed in
cotransfection paradigms using CAT reporter gene constructs in HepG2
cells. We noted that SL2 insect cells are devoid of NF1 transcription
factors, which suggested that these cells could be used in
cotransfection experiments (36). As a negative control we cotransfected
either pADH (Drosophila Adh promoter) or pADH-CTF1 [in
which expression of CTF1 (CCAAT transcription factor)/NF1 is driven by
the Adh promoter] with p-55
CAT (pBLCAT3 containing the
1-globin
promoter and devoid of NF1 sites) into SL2 cells and noted no
increase in CAT activity (Fig. 11
). As a positive control we
co-transfected either pADH or pADH-CTF1 with p-87(3XAd)
CAT
(which contains four NF1 sites upstream from the
1-globin promoter) and obtained a 7- to 10- fold
increase in CAT activity when pADH-CTF1 and p-87(3XAd)
CAT were
cotransfected. In the experimental system in which either pADH or
pADH-CTF1 was cotransfected with p(E1234)-55
CAT (in which the
1-globin promoter contains E1234 substituted for the four NF1 sites)
a 2- to 3-fold increase was obtained when pADH-CTF1 and
p(E1234)-55
CAT were cotransfected. These data suggest that E1234 is
trans-activated by NF1 transcription factors.
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DISCUSSION
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AKR genes play pivotal roles in steroid hormone metabolism and
endocrinology yet their gene structures and details on how they are
regulated are only just emerging. Genes of the AKR1C subfamily are
involved in the hepatic clearance of steroid hormones and include
5ß-reductase and 3
-HSD. The rat 3
-HSD/DD
(AKR1C9) gene, which is the focus of this study, contains
multiple SREs in its promoter. These are likely to respond to high
circulating steroid hormones, and subsequent gene transcription will
protect against steroid hormone excess. A human homolog, AKR1C1, shares
the dihydrodiol dehydrogenase function of the rat AKR1C9
gene and metabolizes PAH trans-dihydrodiols to yield
reactive and redox active o-quinones. The AKR1C1
gene is induced by reactive oxygen species possibly through an
antioxidant response element to protect against oxidative stress (37).
By contrast, genes of the AKR1B subfamily or aldose reductases catalyze
the first step in the polyol pathway and convert glucose to sorbitol
(28). In diabetes mellitus, high blood glucose is converted to the
hyperosmotic sugar, sorbitol. The resultant increase in water uptake
contributes to the complications arising from diabetes, e.g.
diabetic retinopathy, neuropathy, and nephropathy (28). The rat,
rabbit, and human AKR1B gene promoters have been shown to
contain osmotic response elements that respond to hyperosmolarity and
induce gene transcription to protect against osmotic stress (28, 29, 31). The transcription factors responsible for this response are
unknown. What emerges is that AKR genes serve protective roles; they
are often constitutively expressed yet the identity of transcription
factors that control their expression is unclear. Our studies show that
NF1 contributes to the high constitutive expression of the
AKR1C9 gene in rat liver (0.51.0% of the soluble hepatic
protein).
Sequence analysis of the distal promoter (-4673 to -1533 bp) of the
AKR1C9 gene led to the identification of three novel
elements. First, the promoter contained GAGA repeats (-2401 to
-2271), which are implicated in controlling basal and GH-dependent
gene transcription (38, 39). When these GAGA repeats were deleted from
the 3
-HSD/DD gene promoter (from -2685 to -1906),
transcription of the reporter gene CAT was decreased 5.8-fold (Fig. 2
).
These results suggest that GAGA repeats may contribute to constitutive
expression of the 3
-HSD/DD gene. This gene also displays
sexual dimorphic expression, and GAGA repeats are found in the
promoters of other steroid-metabolizing enzymes that show a similar
pattern of expression. These genes include cytochrome P450
16
-hydroxylase CYP2C11 [expressed in males (40)]
and cytochrome P450 15ß-hydroxylase CYP2C12 [expressed in
females, (41)]. Feminization of rat hepatic steroid hormone metabolism
(i.e. elevated 15ß-hydroxylase and 5
-reductase
activities and decreased 16
-hydroxylase and 5ß-reductase
activities) has been attributed to the gonadal control of GH release
from the anterior pituitary (41, 42, 43, 44). The 3
-HSD/DD gene,
however, is not regulated solely by GH. Thus, hypophysectomy will
reduce 3
-HSD/DD mRNA, protein, and enzyme activity 8-fold, but
normal levels are not restored by the daily administration of GH (21).
It is known that GH is required to maintain hepatic estrogen receptor
expression, and it is likely that GH primes the liver so that the
AKR1C9 gene can be induced by estrogens (45). Although, the
existence of GAGA repeats in all these genes is intriguing, it is
unlikely to be the element that is responsible for their sexual
dimorphic expression because it is resident in genes expressed in both
sexes.
Second, the distal promoter contains sequences in the complementary
strand from -4134 to -3734 and from -3157 to -2838 that are 94%
and 86% identical in sequence to that found between -1578 to -1204
of the MCPII gene, which was identified as a mast
cell-specific enhancer element (46). The 3
-HSD/DD gene
promoter also contains perfect mast cell-specific transcription factor
binding sites defined as GATA binding sites, at -3933/-3928 and
-2965/-2960. This suggests that AKR1C genes may be
expressed upon mast cell activation. Activated human mast cells are
well known to synthesize 9
,11ß-PGF2
(47). AKR1C9
can function as a 9-, 11-, and 15-hydroxy-PG dehydrogenase (48). It
also shares significant sequence homology with PGF2
synthase (AKR1C 7) (49, 50). Further, human type 3 3
-HSD (AKR1C2)
can efficiently reduce PGD2 to yield
9
,11ß-PGF2
(51). It will be of importance to
determine whether expression of AKR1C1 genes is correlated
to mast cell activation to protect against allergens.
Third, we located a triple repeat within a 73-bp fragment which is the
cis-element responsible for high constitutive expression of
the AKR1C9 gene. The enhancer element contained three NF1
binding sites and one imperfect C/EBP binding site (Figs. 3
and 4
).
This enhancer was able to increase the activity of the basal hsd
promoter by 13-fold (Fig. 5
). Electrophoretic mobility shift analysis
demonstrated that the 73-bp enhancer bound NF1 transcription factors
but not C/EBP transcription factors. In cotransfection experiments in
SL2 insect cells, CTF1/NF1 was able to trans-activate the
E1234 enhancer.
When considering how NF1 mediates hepatic specific expression of the
AKR1C9 gene, it is important to consider that NF1 is encoded
by at least four genes (NF1A, NF1B, NF1C, and NF1X) that share high
sequence homology with CTF (34, 52, 53, 54, 55). Alternative splicing of the
NF1 genes adds further diversity to this family (32, 55). NF1 can bind
to DNA as a homo- or heterodimer (34, 56). These factors bind with high
affinity to the palindromic sequence
TGGC/A(N)5GCCA (32, 33) and with
lower affinity to the half-site TGGC/A (34, 35). The NF1 proteins are
highly conserved in their N-terminal DNA-binding domain but diverge in
their C-terminal transactivation domains (34). Although NF1 is known as
a ubiquitous transcription factor, some forms of NF1 are restricted to
certain tissues, and recent analysis of NF1 expression in human and
murine cell lines showed that there are cell line-specific differences
in both the isoforms and the amount of NF1 binding activity observed
(57). NF1 sites are frequently found adjacent to or overlap with other
transcription factor-binding sites, e.g. AP-1 family
members, steroid hormone receptors, and HNF3
(58, 59, 60). It has been
suggested that the interaction of NF1 with other transcription factors
may ultimately determine cell- or tissue-specific gene expression. NF1
will form a composite element by binding in close apposition with
HNF3
to modulate liver-specific expression of the serum albumin gene
(60, 61); NF1 and HNF1 also regulate the liver-specific expression of
the
-fetoprotein gene (62), whereas estrogens and progestins
regulate the amount of NF1 that binds to and trans-activates
the vitellogenin gene (63). Thus, liver-specific expression of genes
regulated by NF1 appears to be governed by its interaction with other
factors. The identity of the NF1 family member that binds to the 73-bp
enhancer of the AKR1C9 gene and the identity of cooperative
transcription factors that govern the liver-specific expression of this
gene remain to be determined.
Identification of NF1 as trans-activators of the
AKR1C9 gene promoter suggests a model for the regulation of
steroid hormone metabolism in rat liver. High constitutive expression
of the AKR1C9 gene leads to the conversion of 5
- and
5ß-dihydrosteroids to 3
/5
- and 3
/5ß-tetrahydrosteroids and
is enhanced by NF1 and repressed by Oct-1 (26). Increases in steroid
hormones in the circulation are sensed by steroid receptors that
increase AKR1C9 gene transcription in an autoregulatory
manner. Although this is the case for glucocorticoids (27), more
complex paradigms may exist for regulation by gonadal steroids (Fig. 12
). Knowing that 5ß-reductase
(AKR1D) precedes 3
-HSD in steroid hormone metabolism, it will be of
interest to determine what transcription factors regulate the
expression of the AKR1D gene especially since it is the
rate-limiting enzyme in this metabolic pathway.
The AKR1C9 gene is also involved in carcinogen metabolism.
NF1 has not been previously implicated in the regulation of genes
involved in PAH activation. The CYP1A1 gene, which codes for
the predominant cytochrome P450 involved in the activation of
PAH-trans-dihydrodiols (proximate carcinogens) to
PAH-anti-diol epoxides (ultimate carcinogens), is regulated
by the occupied Ah receptor binding to a xenobiotic response element
(64, 65, 66). In contrast, NF1 binds to the AKR1C9 gene promoter
to enhance activation of PAH-trans-dihydrodiols to
PAH-o-quinones with the concomitant production of reactive
oxygen species. Since these two pathways of PAH activation are
differentially regulated, which pathway predominates may depend upon
the availability of these trans-acting factors and their
cooperative partners.
NF1 is one of the few transcription factors that have been implicated
in the regulation of AKR genes. We have been struck by the sequence
similarity between the NF1 sites in the AKR1C9 gene promoter
and the osmotic response elements found in the promoters of the rat,
rabbit, and human aldose reductase promoters (see Table 1
). Whether NF1 sites are involved in the
constitutive or regulated expression of other protective AKR genes
remains to be determined. If NF-1 is involved in mediating stress
responses, it is likely to be regulated by stress-activated
kinases.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The human hepatoma cell line, HepG2 (ATCCHB8065), was maintained
in continuous culture in Earles MEM supplemented with 10%
heat-inactivated FBS, 1% L-glutamine, 100 U/ml penicillin,
and 10 µg/ml streptomycin (growth medium) at 37 C in a humidified
atmosphere with 5% CO2. Cells were passaged every 7 days
with a 1:4 dilution. Drosophila Schneider SL-2 cells (a gift
from Dr. James B. Jaynes at Thomas Jefferson University) were grown in
Schneiders insect medium supplemented with 10% FBS at 25 C.
Generation of pCAT Constructs
Six CAT reporter gene constructs containing variable lengths of
the 5'-flanking region of the AKR1C9 gene were generated by
5' deletion of the pBLCAT3 construct containing 6.6 kb upstream of the
rat AKR1C9 gene as described previously (26). Briefly, the
pBLCAT3 plasmid containing the -6.6-kb insert was linearized with
PstI and SphI and subjected to nested deletion
with exonuclease III and S1 nuclease. The deletion mutants were
recircularized by the addition of the Klenow fragment of DNA polymerase
I and T4 DNA ligase and correspond to -4673 to +61 bp; -4197 to +61
bp; -3908 to +61 bp; -3261 to +61 bp; -2685 to +61 bp; and -1906 to
+61 bp where the transcription start site is at +1. The internal
deletion and further 5'-deletion constructs were produced by PCR using
p(-4673/+61)CAT as the template and a combination of oligonucleotides
as primers shown in Table 2
. The
resulting PCR products were cloned into a HindIII site of
phsdCAT, which contains the basal AKR1C9 gene promoter
(-238 to +61) in pBLCAT3 to yield vectors bf in Fig. 3
. Vectors g
and h (Fig. 3
) were created via a slightly different strategy. A 301-bp
fragment, -4484/-4183, was PCR amplified using the primers in Table 2
and blunt-end ligated into the PCR-blunt end vector pCRII
(Invitrogen, San Diego, CA). The -4478/-4320 fragment
was then released by digestion with NcoI and
AfIIII, and the -4319/-4183 fragment was released with
AfIIII alone. Each fragment was then blunt-end ligated into
the HindIII site of phsdCAT. Another strategy was used to
generate p(E1234)hsdCAT, p(E123)hsdCAT, p(E12)hsdCAT, and p(E1)hsdCAT.
Because of the lack of available restriction sites in the basal
promoter construct, phsdCAT (-238 to +61 bp), the basal promoter, was
PCR amplified using primers containing 5' and 3' XbaI amd
BamHI linkers, respectively, and a phsd-CAT construct as
template. The parental pBLCAT3 vector was linearized at the same
restriction sites, and the basal promoter was directionally subcloned
into the vector. The vector was then digested at the HindIII
and XbaI sites in the multiple cloning site upstream from
the hsd promoter, and the enhancer elements in Fig. 4
. were subcloned
into these sites using the compatible HindIII site in the
oligonucleotides encoding those elements.
Constructs for Cotransfection
The constructs for cotransfection in Drosophila
Schneider SL-2 cellspADH, pADH-CTF1, p-55
CAT, and
p-87(3XAd)
-CATpreviously designated as pPADH, pPADH-CTF1,
p
-CAT-55, and p
-CAT-883XAd (36) were gifts from Dr. Nicolas
Mermod (University of Lausanne, Lausanne, Switzerland). pADH is a
Drosophila expression vector containing the proximal and
distal tandem promoters of the Drosophila alcohol
dehydrogenase (Adh) gene. pADH-CTF1 is an expression construct in which
CTF1/NF1 expression is driven by the Adh promoter. p-55
CAT is a
pBLCAT3 vector in which CAT activity is driven by -55 to +36 bp of the
1-globin promoter. p-87(3XAd)
CAT is a pBLCAT3 vector in which CAT
activity is driven by -87 to +36 bp of the
1-globin promoter
containing a CCAAT box (CTF/NF1 site) and three adenovirus NF1 sites.
To construct p(E1234)-55
CAT, p-87(3XAd)
CAT was digested by
EagI and SalI to remove all four NF1 sites and
ligated to a E1234 fragment. The EagI site is located at
position -55 of the
1-globin promoter. The fidelity of the
constructs was confirmed by dideoxy sequencing.
Transient Transfection
HepG2 cells were seeded at a concentration of 1.5 x
106 cells per 60-mm tissue culture plate for 24 h.
Before transfection (4 h), the medium was removed and replaced with a
fresh growth medium. For each pCAT construct, DNA-CaCl2
solutions were prepared by mixing 37 µl 2 M
CaCl2, 10 µg of pCAT constructs or CAT control plasmids
(pBLCAT3 basic promoter and enhancerless, Promega Corp.,
Madison, WI), plus 2 µg of pSV-ß-galactosidase containing plasmid
(Promega Corp.) in a final volume of 300 µl. DNA was
precipitated by mixing with an equal volume of 2 x HEPES-buffered
saline [50 mM HEPES (pH 7.1), 280 mM NaCl, and
1.5 mM Na2HPO4)] with constant
agitation. The precipitants were incubated at room temperature for 30
min before their addition to the cell culture medium. After 3648 h,
the cells were washed twice with PBS (pH 7.1) and harvested using lysis
buffer (Promega Corp.) for CAT and ß-galactosidase
enzyme assays. SL2 cells were cotransfected by calcium phosphate
coprecipitation as described above. Three micrograms of CTF1/NF1 were
cotransfected with 1 µg of reporter construct and 0.5 µg of
hsp82LacZ (ß-galatosidase reporter, a gift from Dr. James B.
Jaynes).
For each ß-galactosidase enzyme assay, a cell lysate (100 µl) was
incubated in 60 mM Na2HPO4, 40
mM NaH2PO4, 1 mM
MgCl2, and 50 mM ß-mercaptoethanol,
containing 0.67 mg/ml
o-nitrophenyl-ß-D-galactopyranoside as a
substrate in a total volume of 300 µl at 37 C for 2 h. The
reaction was terminated by adding 500 µl of 1 M sodium
carbonate, and the absorbance of the o-nitrophenol anion was
read at 420 nm. This end point assay was validated by showing that the
absorbance was in the linear range with respect to time and lysate
protein. For CAT activity, the volumes of the cell lysates were
adjusted to contain the same amount of ß-galactosidase activity to
normalize for transfection efficiency. Lysates (50100 µl) were then
incubated in a final volume of 125 µl containing 25 mM
Tris-HCl (pH 7.5), 40 µM (1.0 µCi) chloramphenicol
(chloramphenicol D-threo-[dichloroacetyl[-1,
2-14C]) (57 mCi/mmol, Dupont NEN,
Boston, MA), and 50 µM butyryl-CoA. The reactions were
initiated by the addition of coenzyme and incubated for 8 h at 37
C. The reactions were terminated by extraction with 500 µl ethyl
acetate, the extracts were dried, resuspended in 20 µl ethyl acetate,
and applied to TLC plates (Silica Gel IB2; J.T. Baker, Inc.,
Phillipsburg, NJ), which were developed in
CHCl3/MetOH, 97:3 (vol/vol). The results were quantitated
by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis of the
butylated and nonbutylated forms of [14C]chloramphenicol
and are reported as relative values in arbitrary units. The results for
each construct represent the mean ± SE from three
separate experiments. All values were normalized for ß-galactosidase
activity cotransfected as pSV-ß-galactosidase.
Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared from HepG2 cells as described
previously (26). Radiolabeled oligonucleotides were
[
32P]-labeled at 5'-end using T4 polynucleotide kinase
and [
32P]ATP (30 Ci/mmol, Dupont NEN).
Nuclear extracts (10 µg) were incubated with 2 fmol of radiolabeled
oligonucleotides (50,000 cpm) for 20 min at room temperature in the
presence of 0.5 µg sheared salmon sperm DNA and 5 µg poly(dI·dC)
(Pharmacia Biotech, Piscataway, NJ) in a binding reaction
of 20 µl containing 40 mM KCl, 25 mM HEPES
(pH 7.9), 1 mM EDTA, 0.5 mM DTT, 5
mM, and 5% glycerol. The mixtures were electrophoresed on
a 4% polyacrylamide gel in 0.5 x TBE buffer (45 mM
Tris-borate and 1 mM EDTA). For the competition studies, a
10- to 250-fold molar excess of unlabeled oligonucleotide were added to
the incubation. For the supershift assays, 1 µl of antibodies for NF1
(a polyclonal antiserum that recognizes CTF-2 and a C-terminal peptide
of CTF-1 was obtained from Dr. Naoko Tanese at New York University) or
two C/EBP isoforms,
and ß (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to the binding reactions and
incubated for another 20 min at room temperature before
electrophoresis. Eukaryotic expression constructs for C/EBP
and
C/EBPß (in the pSG5 vector) were gifts from Dr. Mitchell Lazar
(Department of Medicine, University of Pennsylvania, Philadelphia, PA).
Recombinant proteins were expressed using a rabbit reticulocyte lysate
system (Promega Corp.) according to the manufacturers
protocol. Gel shift analyses were conducted using 1 µl of the
translation mixture in place of the nuclear extracts.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Trevor M. Penning, Department of Pharmacology, 130c John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084.
This work was supported by Grant CA-55711 awarded by the National
Cancer Institute (T.M.P.).
1 Current address: Department of Pathology, Johns Hopkins University
School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205. 
2 3
-HSD/DD, 3
-hydroxysteroid:
NAD(P)+ oxidoreductase (EC1.1.1.213). A
face-specific/dihydrodiol dehydrogenase,
trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC
1.3.1.20) now designated as AKR1C92. 
3 The nomenclature of the AKR superfamily was
recommended by the 8th International Symposium on Enzymology &
Molecular Biology of Carbonyl Metabolism held in Deadwood, South
Dakota, on June 29-July 3, 1996 (2 ). 
4 The TESS search engine (www.cbil.upenn.edu) was
used to search the Transfac database. 
5 The sequence reported in this paper has been
submitted to the GenBank/EMBL Data Bank and given the accession number
AF049661. 
Received for publication January 11, 1999.
Revision received June 10, 1999.
Accepted for publication July 12, 1999.
 |
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