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Departments of Obstetrics and Gynecology and Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032
Address all correspondence and requests for reprints to: William E. Rainey, Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032. E-mail: braine{at}mednet.swmed.edu
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Introduction |
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The expression of each isozyme appears to be controlled at the level of gene transcription. The 5'-flanking region of CYP11B2, the gene encoding aldosterone synthase, has been analyzed in detail for the mouse (4, 5, 6, 7), hamster (8, 9), and human genes (10, 11). However, the mechanisms regulating transcription of CYP11B1, the gene encoding 11ß-hydroxylase, are less completely understood. Although the bovine CYP11B (bCYP11B) gene has been studied in some detail (12, 13, 14, 15, 16), cattle have only one active bCYP11B gene that is expressed in both the glomerulosa and fasciculata of the adrenal (17). Therefore, the regulatory elements in bovine bCYP11B 5'-flanking DNA may differ from those that control the zone-specific expression of the multiple CYP11B genes observed in rodents and humans.
One difficulty in studying the CYP11B genes has been the availability of an appropriate in vitro model system. Previous studies have used the mouse Y-1 adrenal cell line for a preliminary analysis of hCYP11B1 5'-flanking DNA (18, 19). More recently, we and others have characterized a human adrenocortical carcinoma cell line (H295R) that produces both aldosterone and cortisol (20, 21). These cells also express both hCYP11B1 and hCYP11B2 transcripts, which are under the differential control of cAMP and angiotensin II signaling pathways, respectively (22, 23, 24). The current study was undertaken to analyze the 5'-flanking DNA of the hCYP11B1 gene and to define the cis-regulatory elements and transacting factors that are necessary for cAMP induction of transcription. The results suggest that ACTH induction of hCYP11B1 transcription requires primarily two cis-regulatory elements, one (termed Ad1) resembling a consensus ccAMP response element (CRE), and the other (termed Ad4) that binds steroidogenic factor-1 (SF-1).
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Materials and Methods |
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Preparation of mutant constructs
Deoxyribonuclease I footprint analysis of the bCYP11B
5'-flanking DNA demonstrated several regions that bind nuclear proteins
(12). These protected regions were termed Ad-1 to Ad-6.
Similar sequences were observed in the hCYP11B1 gene;
therefore, this terminology will be used to discuss the regulatory
elements. For the Ad1/CRE mutants, the sequence 5'-TGACGTGA-3'
(-71/-64) was changed to 5'-gGtaccGA-3', which included a
KpnI site (underlined). To do so, PCR was
performed on pB1–1102 using a set of primers, 1 and 2 (Table 1
). The Ad4 sequence 5'-ATCCAAGGCTCT-3'
(-244/-233) was changed to 5'-ATCgAAttCTCT-3', and the
Ad5 sequence 5'-CCTGACCTCT-3' (-121/-112) was changed to
5'-CCTGAatTCT-3', each of which included an
EcoRI site (underlined). The pB1–1102 construct
was used as the template for PCR reactions using primers 3 and 4 for
the Ad4 mutant and primers 5 and 6 for the Ad5 mutation (Table 1).
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Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from H295R cells by the method of
Schreiber et al. (26). Double stranded
oligonucleotides (25 pmol) were labeled with 30 µCi
[
-32P]ATP and 10 U T4 polynucleotide kinase
at 37 C for 30 min. Nuclear extract and each radiolabeled probe (40,000
dpm) were incubated at room temperature for 20 min in 20 µl reaction
mixture [20 mM HEPES (pH 8.0), 80
mM KCl, 1 mM EDTA, 10%
glycerol, 1 mM dithiothreitol, 0.5 mg/ml BSA, and
0.025 mg/ml poly(dI-dC) as nonspecific competitor]. When antibodies
were included in the reaction, nuclear extract and antibody were
preincubated on ice for 20 min before the addition of probe and
reaction mixture. For competition analysis, reaction mixtures
containing various amounts of nonradiolabeled oligonucleotide were
added simultaneously with probe. The resulting DNA/protein complexes
were separated from free probe by electrophoresis using a 4% high
ionic strength native polyacrylamide gel with 1 x Tris-glycine
running buffer (2). The gel was dried and visualized after
autoradiography at -70 C for 24 h. The following double stranded
DNA probes used were Ad4 (-250/-227; sequence 7 in Table 1
),
Ad4-mutant (sequence 8 in Table 1), Ad1 (-79/-56; sequence 9 in Table 1), and Ad1-mutant (sequence 10 in Table 1). Rabbit polyclonal
anti-SF-1 (Ad4BP) was provided by Dr. Ken-ichirou Morohashi, Kyushu
University (Fukuoka, Japan). Human SF-1, CRE-binding protein
(CREB), activating transcription factor-1 (ATF-1), and ATF-2 were
prepared using a Promega Corp. in vitro
transcription/translation system.
Protein immunoblotting analysis
Cultured H295R cells were used to prepare nuclear extract as
previously described (26). Two groups of H295R cells were
used to isolate nuclear extracts that were used for the Western
analysis. One group of H295R cells was representative of the cells used
for the transfection EMSA and studies (Fig. 3, H295 NE-1). The second
population of H295R cells was maintained in culture for extended
periods (>1 yr). Adult adrenal glands were obtained and homogenized in
lysis buffer (27). HeLa cell nuclear extract, which was
used as a positive control, was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). In addition, CREB, ATF-1,
and ATF-2 were prepared using a Promega Corp. in
vitro transcription/translation system. PAGE was carried out on
the samples using a precast Novex gel electrophoresis
system with 4–12% bis-Tris NuPage gels (Novex, San
Diego, CA). Proteins were electrophoretically transferred onto
polyvinylidene difluoride membranes by wet transfer for 1 h at 25
V. After transfer the membranes were incubated with ATF-2 (1:1500
dilution), CREB (1:2000 dilution), or ATF-1 (1:800 dilution) antibodies
overnight at 4 C. The ATF-2 and CREB antibodies were obtained from
New England Biolabs, Inc. (Beverly, MA). The ATF-1
antibody was obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). In addition to the manufacturer’s characterization,
antibodies were tested for specificity using purified or in
vitro translated CREB, ATF-1, or ATF-2. Membranes were incubated
with horseradish peroxidase-conjugated secondary antibodies, and
immunoreactive bands were visualized using enhanced chemiluminescence
Western blotting detection reagents from Amersham Pharmacia Biotech (Piscataway, NJ).
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illustrates the reporter activity of
constructs under basal conditions or after treatment with
(Bu)2cAMP (1 mM) for 6
h. Basal luciferase activities of the constructs containing 5'-flanking
sequences from -1102 to -214 bp were similar. Further deletion to
-65 bp decreased luciferase activity to that observed for the pGL3
Basic empty vector, indicating that DNA between -214 and -65 is
essential for basal gene expression. (Bu)2cAMP
treatment of H295R cells transfected with pB1–1102 construct resulted
in a 5-fold increase in luciferase activity. Deletion to -214 bp did
not significantly alter (Bu)2cAMP stimulation of
luciferase activity. Deletion to -65, however, abolished
(Bu)2cAMP induction of reporter gene expression,
indicating that DNA between -214 and -65 contains the minimally
essential regions for both basal and cAMP-regulated transcription of
hCYP11B1.
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).
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). Formation of these complexes was
abolished by the addition of nonradiolabeled competitor. Because the
Ad1 element is almost identical to a consensus CRE, transcription
factors know to interact with this sequence were examined using EMSA.
In vitro prepared CREB, ATF-1, and ATF-2 formed complexes
with the Ad1 element, giving rise to complexes that migrated
differently. ATF-2, but not CREB or ATF-1, formed a radiolabeled
complex that comigrated with a complex observed using H295R cell
nuclear extract (complex 1). Antibody directed against ATF-2
supershifted complex 1, suggesting that it represents a complex
containing ATF-2. Antibodies directed against CREB or ATF-1 did not
significantly modify any of the three major complexes. Oligonucleotides
that included a mutated Ad1 sequence (gGtaaGA) did not form complexes
with in vitro prepared CREB, ATF-1, or ATF-2 (Fig. 2).
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). Nuclear extracts from H295R and HeLa
cells as well as homogenates of human adrenal cortex expressed readily
detectable levels of ATF-2 and varying amounts of CREB and ATF-1. CREB
protein was detectable in HeLa nuclear extract, early passage H295R
nuclear extract (H295 NE-1), as well as adrenal lysate. ATF-1
expression appeared higher in the H295R adrenal cell than adrenal
lysate. Interestingly, CREB expression appeared to be lost in H295R
cells cultured for long periods (Fig. 3, H295 NE-2).
Role of the Ad4 and Ad5 elements in hCYP11B1 transcription
Interestingly, as shown in Fig. 1, deletion of the Ad4 site
(-242/-234) from hCYP11B1 in pB1–214 did not affect basal
or cAMP induction of reporter gene expression. Because cotransfection
of expression vectors containing SF-1 enhanced reporter gene activity
in constructs using 5'-flanking DNA from the bovine CYP11A
(15), human CYP19 (28), mouse CYP21
(29), bovine CYP17 (30), and
bCYP11B (31) genes, we carried out similar
experiments in which H295R cells were transfected with the
hCYP11B1 serial deletion constructs and human SF-1
expression vector (Fig. 4). Reporter gene
expression was similar using constructs containing -1102 to -330 bp
of hCYP11B1 5'-flanking DNA. However, in cells cotransfected
with SF-1 expression plasmid, deletion to -214 bp decreased luciferase
activity to 53% of that seen in cells transfected with the -330 bp
construct (pB1–330). This observation suggests that the Ad4 site is
needed for maximal induction of transcription by SF-1.
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). Mutation of the Ad4 site had little
effect on basal activity, but decreased the stimulatory effect of SF-1
coexpression to 27% of the stimulation seen using the native pB1–1102
construct. In contrast, mutation of the Ad5 did not significantly
affect luciferase activity. Constructs with mutations of both elements
had basal and SF-1-stimulated levels of reporter activity near that
observed with pGL3 Basic empty vector (not shown).
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). When cells were transfected with
pB1–1102 only and treated with (Bu)2cAMP,
activity increased 5-fold over basal levels. Additionally,
cotransfection with SF-1 in the absence of
(Bu)2cAMP caused a 6-fold increase in reporter
activity above basal levels. When cells were cotransfected with
pB1–1102 and the SF-1 expression plasmid and treated with
(Bu)2cAMP, there was a synergistic effect,
leading to a 40-fold increase above basal levels. Treatment with
(Bu)2cAMP also enhanced the effects of SF-1 on
the three mutant constructs tested. However, only the
(Bu)2cAMP-treated cells cotransfected with the
Ad5 mutant and SF-1 displayed levels of activity similar to that of the
native pB1–1102 construct.
To determine whether the Ad4 element could bind SF-1, a synthetic
oligonucleotide probe encompassing this sequence was prepared and used
in EMSA (Fig. 6). In the presence of
H295R nuclear extracts, two major DNA/protein complexes were formed,
only one of which could be completely displaced by nonradiolabeled
oligonucleotide. In vitro prepared human SF-1 caused
formation of a complex that migrated in parallel to that observed using
H295R nuclear extract. In addition, antibody directed against SF-1
abolished the complex observed using H295R nuclear extract (data not
shown). Oligonucleotides that included a mutated Ad4 element did not
form complexes with SF-1 (Fig. 6).
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). Representative constructs (pB1–924,
pB1–330, pB1–214, and pB1–65 bp) from hCYP11B1
5'-flanking DNA were transfected with empty pcDNA3 expression vector or
with the SF-1-containing expression vector. The results were very
similar to those obtained using H295R cells. The basal level of
reporter expression was not effected by deletion to -330 bp. Deletion
to -214 increased basal activity, whereas further deletion to -65 bp
decreased basal expression. Coexpression with SF-1 caused a 2.6-fold
increase in reporter gene activity. This effect was significantly
decreased by deletion to -214 bp and abolished when the pB1–65
construct was tested.
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However, there has been considerable work directed toward defining the mechanisms that regulate transcription of the bCYP11B gene (12, 14, 16, 31, 34). Cattle, unlike humans and rodents, perform 11ß-hydroxylation, 18-hydroxylation, and 18-oxidation though the action of a single 11ß-hydroxylase isozyme (CYP11B) (35) that is expressed in both the glomerulosa and fasciculata. Omura and colleagues using DNase I footprint analysis defined protected regions in the bCYP11B proximal DNA termed adrenal (Ad) 1–6 (12). Analyses of the role of each of the putative cis-elements demonstrated a need for the Ad1 element in basal and cAMP-stimulated expression of reporter activity. The Ad1 element closely resembled a consensus CRE and bound similar proteins to the consensus CRE using nuclear extracts from steroidogenic and nonsteroidogenic cells. However, the nature of these proteins was not determined. Mutation of the Ad1 element blocked cAMP induction, thus furthering the idea that the Ad1 site is most likely the key element in ACTH-regulated induction of transcription of bCYP11B. Our observations on the hCYP11B1 gene are very similar to those observed for the bovine gene in that mutation of the human CRE/Ad1 is critical for both basal and cAMP-dependent transcription.
All CYP11B genes studied to date are positively regulated by the cAMP signaling pathway. It was thought that both CYP11B genes would make use of the Ad1/CRE site for regulation; however, there is a loss of sequence identity in the mouse and rat CYP11B1 gene (5'-AGTGACaTtATCA-3') (36) compared with the human Ad1 sequence (5'-CATGACGTGATCC-3'). The divergence in the rat CYP11B1 sequence may explain why Ishimura and colleagues observed that an AP-1 sequence in the rat CYP11B1 Ad1/CRE flanking DNA was important in cAMP induction of transcription of this gene (37, 38). In support of this premise, the researchers went on to demonstrate that nuclei in the rat adrenal fasciculata expressed much higher levels of AP-1-binding proteins (Jun and Fos) than were observed in the glomerulosa. This contrasts with the hCYP11B1 gene, in which sequence similarity at the Ad1 site to a consensus CRE has been maintained while there is base substitution (compared with the rat AP-1) decreasing the sequence similarly to a consensus AP-1 site. The rationale for specific differences for the differential regulation of CYP11B1 is not clear. However, the CYP11B genes appear to have had considerable evolutionary divergence, particularly when one considers that bovine, sheep, and pig have only one CYP11B gene. Further, study of the rodent CYP11B1 genes will be needed to confirm that the CRE (Ad1) variations in sequence lead to transcriptional differences.
To better define the trans-acting factors that interact with
the Ad1/CRE we used in vitro prepared CRE-binding proteins
and H295R nuclear extract. CREB, ATF-1, and ATF-2 were able to form
complexes with the Ad1/CRE element. However, when compared with nuclear
proteins from H295R cells only the ATF-2/CRE complex migrated in a
similar manner (complex 1; Fig. 2). The ability of an ATF-2 antibody to
supershift complex 1 further supports the idea that ATF-2, within these
cells, binds the hCYP11B1 Ad1/CRE site. The nature of
complex 2 remains to be determined. Although it did not migrate with
in vitro prepared ATF-1 or CREB, the possibility of
heterodimers of these proteins with other factors cannot be ruled out
at this time. To determine whether members of the CREB/ATF family of
proteins are expressed in H295R cells and adrenal cortex, we used
Western analysis. H295R nuclear extracts and adrenal lysates expressed
ATF-2, supporting the EMSA observations. Adrenal lysate also expressed
detectable levels of CREB and ATF-2 with very low levels of ATF-1. A
recent study of H295R cell expression of members of the CREB/ATF family
found no expression of CREB (39). Our data suggest that
certain strains (but not all) of the H295R cells have lost CREB
expression. The mechanism causing the loss of CREB expression is not
clear. However, Groussin and colleagues (39) demonstrate
an increase in the level CREM expression that they suggest compensates
for the loss of CREB. The expression of ATF-2 in adrenal cells and
tissue has not previously been described. Activation of ATF-2 can occur
through a number of signaling pathways used by certain hormones and
growth factors, including alterations in intracellular calcium. Further
experiments will be needed to determine which members of the
CRE-binding protein family can enhance the transcription of the
hCYP11B1 gene.
In addition to the Ad1/CRE, hCYP11B1 gene transcription
relied on the Ad4 element previously characterized in
bCYP11B. The trans-acting protein that binds the
Ad4 sequence was termed Ad4-binding protein (Ad4BP) and was
subsequently cloned from a bovine adrenal complementary DNA (cDNA)
library (31). Ad4BP is equivalent to SF-1 and is expressed
in other steroidogenic cells (29, 40). This protein is
considered a key transcription factor in the regulation of a number of
steroid hydroxylases as well as the steroidogenic acute regulatory
(StAR) protein and 3ß-hydroxysteroid dehydrogenase genes
(41, 42, 43). Herein, we demonstrated that the Ad4 site in the
hCYP11B1 gene was important for transcription; however, its
role was only apparent when cells (either Y-1 or H295R) were
cotransfected with an SF-1 expression vector. The rationale for a need
to overexpress SF-1 to induce hCYP11B1 reporter activity via
the Ad4 element is not clear, as by EMSA (Fig. 6) and immunoblot
analysis (data not shown) the H295R and Y-1 cells express SF-1.
Nevertheless, overexpression of SF-1 has also been shown to greatly
increase reporter gene expression for constructs prepared from the
CYP17, 3ßHSD, CYP11A, and CYP19 genes (15, 28, 29, 30, 31). The
data thus support the requirement for SF-1 for maximal basal and
cAMP-stimulated transcription of hCYP11B1. This need for
SF-1 sharply differs from the effects of SF-1 on hCYP11B2
(aldosterone synthase) transcription (11).
CYP11B2 transcription is not increased by SF-1 coexpression
and mutation of the Ad4 element does not affect reporter gene
expression. In the case of hCYP11B2, we have previously
shown an important role for the Ad5 site in basal and stimulated
expression of reporter genes. Thus, these two isozymes have diverged in
the key cis-elements regulating gene transcription.
In summary, the regulation of the CYP11B genes appears to have evolved species-specific cis-regulatory elements to control transcription. In humans, hCYP11B1 and hCYP11B2 rely on the proximal Ad1/CRE site for transcription. This may differ in mouse and rat CYP11B1, where the Ad1 site has lost identity with the consensus CRE. Second, the requirement for SF-1 appears to have diverged between hCYP11B1 and hCYP11B2. For hCYP11B1 the Ad4 cis-element and expression of SF-1 are both required for maximal transcription. However, this is not the case for hCYP11B2, where the Ad4 element does not appear necessary for transcription (11). Such differences between the CYP11B1 and CYP11B2 genes may play an important role in the differential expression of these isozymes within the adrenocortical zones.
Received December 28, 1999.
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-hydroxylase cytochrome P-450 expression in ovine adrenocortical
cells. Endocrinology 127:1910–1915
in the human adrenocortical cancer cell line
H295R. J Clin Endocrinol Metab 85:245–354
5-4
isomerase promoter by the transcription factor steroidogenic
factor-1/adrenal 4-binding protein and phorbol ester. J Biol Chem 272:7960–7967This article has been cited by other articles:
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