Angiotensin II and Potassium Regulate Human CYP11B2 Transcription through Common cis-Elements
Colin D. Clyne,
Yin Zhang,
Liliya Slutsker,
J. Michael Mathis,
Perrin C. White and
William E. Rainey
Department of Obstetrics & Gynecology (C.D.C., Y.Z., J.M.M.,
W.E.R.) and Department of Pediatrics (L.S., P.C.W.) University
of Texas Southwestern Medical Center Dallas, Texas 75235-9032
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ABSTRACT
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Aldosterone synthase is a mitochondrial enzyme
that catalyzes the conversion of 11-deoxycorticosterone to the potent
mineralocorticoid aldosterone. The gene encoding aldosterone synthase,
CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex.
Although the major physiological regulators of aldosterone production
are angiotensin II (ANG II) and potassium (K+),
the mechanisms by which these compounds regulate CYP11B2 transcription
are unknown. Therefore we analyzed the human CYP11B2 5'-flanking region
using a transient transfection expression system in the H295R human
adrenocortical cell line. ANG II and K+
increased expression of a luciferase reporter construct containing 2015
bp of human CYP11B2 5'-flanking DNA. This response was mimicked by
treatment with the calcium channel activator BAYK8644, whereas
activation of the protein kinase C pathway with
12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene
activity was also increased after activation of cAMP-dependent pathways
by (Bu)2cAMP. Deletion, mutation, and
deoxyribonuclease I footprinting analyses of the CYP11B2 5'-flanking
region identified two distinct elements at positions -71/-64
(TGACGTGA) and -129/-114 (CTCCAGCCTTGACCTT) that were both required
for full basal reporter gene activity and for maximal induction by
either cAMP or calcium-signaling pathways. The -71/-64 element, which
resembles a consensus cAMP response element (CRE), bound CRE-binding
proteins from H295R cell nuclear extracts as determined by
electrophoretic mobility shift analysis. Analysis of the -129/-114
element using electrophoretic mobility shift analysis demonstrated
binding of the orphan nuclear receptors steroidogenic factor 1 and
chicken ovalbumin upstream promoter transcription factor. These
data demonstrate that ANG II, K+, and
cAMP-signaling pathways utilize the same SF-1 and CRE-like
cis-elements to regulate human CYP11B2 expression.
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INTRODUCTION
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The renin-angiotensin system (RAS) is a major regulator of
intravascular volume and blood pressure. Dysregulation of the RAS is
associated with several forms of human hypertension (1, 2). To
completely understand the pathogenesis of such diseases associated with
hypertension, it is necessary to fully delineate the mechanisms
regulating each of the components within this system. A key effector of
the RAS is aldosterone, the primary human mineralocorticoid, which acts
on the distal nephron to regulate sodium resorption, potassium
excretion, and intravascular volume (3). Aldosterone is produced
exclusively in the adrenal zona glomerulosa, and its secretion is
regulated primarily by serum levels of angiotensin II (ANG II) and
potassium (K+) (4, 5). Temporally, the regulation of
aldosterone production can be divided into two phases: an acute phase
that occurs within minutes and reflects cholesterol transfer to
mitochondrial side-chain cleavage enzyme and a chronic phase that
requires several hours and reflects increased expression of aldosterone
synthase (CYP11B2) (6). CYP11B2 is normally expressed only in the
adrenal glomerulosa in contrast to the isozyme 11ß-hydroxylase
(CYP11B1), which is expressed in the adrenal fasciculata (7, 8).
CYP11B2 catalyzes the three successive reactions that lead to the
conversion of deoxycorticosterone to aldosterone (9, 10, 11, 12, 13). Dysregulated
expression of CYP11B2, as seen in glucocorticoid-suppressible
hyperaldosteronism, can lead to elevated circulating aldosterone levels
and hypertension (14). A detailed analysis of the normal mechanisms
regulating human CYP11B2 expression would thus be of significant
interest.
In the only published attempt to analyze the transcriptional regulatory
region of the human CYP11B2 gene, chloramphenicol acetyltransferase
(CAT) reporter constructs were transfected into mouse Y1 adrenocortical
tumor cells, but levels of basal and stimulated expression were too low
for the identification of transcriptional regulatory elements (15). In
contrast, considerable progress has been made in defining the
mechanisms by which cAMP regulates transcription of rat (16) and mouse
(17, 18, 19) CYP11B2, as well as the one isozyme expressed in the bovine
adrenal, CYP11B (20, 21, 22). Cyclic AMP is the second messenger for ACTH,
the major hormonal regulator of glucocorticoid biosynthesis and
expression of CYP11B1 in the adrenal fasciculata. Common elements in
the 5'-flanking region mediating basal and cAMP-induced expression were
identified in each of these genes, including a cAMP-response element
(CRE) and an element binding an orphan nuclear receptor, steroidogenic
factor-1 (SF-1).
The physiological relevance of ACTH in CYP11B2 transcription and
mineralocorticoid production is unclear, however, because chronic
treatment with ACTH decreases both plasma aldosterone levels (23, 24)
and adrenal CYP11B2 expression (25). Thus, the regulated expression of
CYP11B2 cannot be explained through cAMP-dependent mechanisms alone.
Indeed, the principle physiological regulators of CYP11B2 expression,
ANG II and K+, do not increase cAMP levels in adrenal
glomerulosa cells but instead increase the intracellular concentration
of calcium ([Ca2+]i) and activate protein kinase C (5, 26). To date, there have been no studies regarding the effects of ANG
II, K+, or the calcium-signaling pathway on
CYP11B2 transcription.
One difficulty in studying CYP11B2 transcription has been the lack of
an in vitro adrenocortical model system that retains the
abilities to produce aldosterone and respond to ANG II and
K+. We recently described the human adrenocortical H295R
cell line as a model for studying CYP11B2 regulation (27, 28, 29, 30). These
cells respond to ANG II and K+ by increasing both
aldosterone production and CYP11B2 expression. The current study was
undertaken to analyze the 5'-flanking DNA of the human CYP11B2 gene and
to define the cis-regulating elements and
trans-acting factors that are necessary for ANG II and
K+ induction of CYP11B2 transcription. The results indicate
that maximal induction of CYP11B2 transcription by ANG II or
K+ requires two key cis-elements, one of which
binds cAMP-response element (CRE) binding proteins, and the other SF-1
and a second orphan nuclear receptor, chicken ovalbumin upstream
promoter transcription factor (COUP-TF). These two elements are also
required for maximal cAMP-induced expression, suggesting that the
Ca2+ and cAMP-signaling systems use the same
cis-elements to regulate CYP11B2 transcription.
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RESULTS
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Multiple Signaling Pathways Regulate CYP11B2 Reporter Gene
Expression in H295R Cells
H295R cells were transiently transfected with a CYP11B2-luciferase
reporter construct containing 2015 bp of 5'-flanking sequence
(pB22015) and treated with experimental agents for 8 h. As shown
in Fig. 1
, both ANG II and K+ increased
transcription of this reporter construct in a concentration-dependent
manner, with significant induction occurring at 0.1 nM ANG
II and 12 mM K+, respectively. These results
demonstrate that the first 2015 bp of 5'-flanking region contains
sufficient sequence information to direct reporter gene expression in
response to physiological regulators of human CYP11B2.
Previous studies have established that although the primary effect of
K+ on adrenal glomerulosa cells is to increase
[Ca2+]i, treatment with ANG II increases both
[Ca2+]i and protein kinase C activity (5, 26). In
contrast, ACTH increases cAMP levels through activation of adenylyl
cyclase. To assess the relative contributions of these signaling
pathways to the induction of reporter gene expression, H295R cells were
treated with BAYK8644 (an L-type Ca2+ channel agonist),
12-o-tetradecanoylphorbol-3-acetate (TPA; an activator of protein
kinase C) or (Bu)2cAMP (an activator of protein kinase A).
Both BAYK8644 and (Bu)2cAMP increased reporter gene
expression in a concentration-dependent manner, whereas TPA was
completely ineffective (Fig. 1
). To confirm that the observed effects
of BAYK8644 and (Bu)2cAMP were indeed mediated by
Ca2+ and cAMP-dependent pathways, respectively, similar
experiments were performed using the Ca2+ ionophore
ionomycin (1 µM) and the adenylyl cyclase activator
forskolin (10 µM). Both of these agents also increased
expression of the reporter construct (data not shown). Thus, cAMP- and
Ca2+-dependent pathways increase CYP11B2 reporter gene
expression in H295R cells, whereas protein kinase C-dependent pathways
do not appear to play a role in this response. The time course of
reporter gene induction was rapid. As shown in Fig. 2
, luciferase activity increased dramatically within 60 min of agonist
treatment to reach a maximum at 6 h. The induction of reporter
gene expression, however, decreased in response to each agonist after
6 h.
Deletion Analysis of the Human CYP11B2 Promoter
To identify the cis-regulatory elements that mediate
transcriptional activation by ANG II and K+, a series of
deletion constructs containing progressively shorter fragments of human
CYP11B2 5'-flanking DNA (extending from position +2 to -2015, -1521,
-864, -413, -221, and -65 bp, respectively) was prepared. These
constructs were transiently transfected into H295R cells. Figure 3
shows luciferase activity of these constructs under
basal conditions or after treatment with different agonists for 6
h. Basal luciferase activity of each construct containing 5'-flanking
sequences from -2015 to -221 bp was similar. Deletion to -65 bp,
however, decreased luciferase activity by 85%. This result indicates
that DNA sequences located between -65 and -221 bp are essential for
basal gene expression.

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Figure 3. Deletion Analysis of the 5'-Flanking Region of the
Human CYP11B2 Gene
A series of CYP11B2 5'-deletion mutants was transiently transfected
into H295R cells. Panel A shows the positions of putative transcription
factor-binding sites, as identified by sequence comparison, and the
relative lengths of the deletion constructs. B, Luciferase activities
of lysates from control cells or cells treated with ANG II (10
nM), KCl (20 mM), and (Bu)2cAMP (1
mM). Results are expressed as a percentage of the basal
activity of the longest construct (pB22015) and represent the mean ±
SEM of determinations from three to five independent
experiments, each performed in triplicate. *, P <
0.05 vs pB22015.
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ANG II and K+ treatment resulted in 5.5- and 4.5-fold
increases in luciferase activity, respectively (Fig. 3
). In the series
of 5'-deletion constructs, induction of luciferase activity in response
to either agonist was maintained in cells transfected with plasmids
containing up to -221 bp of 5'-flanking sequence. Deletion to -65 bp,
however, completely abolished reporter gene induction in response to
ANG II and K+. The effect of (Bu)2cAMP on
luciferase activity in H295R cells transfected with each deletion
construct was determined in a similar manner. Luciferase activity was
stimulated 13-fold by (Bu)2cAMP in cells transfected with
pB22015. Reporter activity induced by (Bu)2cAMP decreased
by 5060% upon deletion to -864 bp, and was completely abolished
after deletion to -65 bp. These results indicate that the region of
human CYP11B2 between -221 and -65 bp contains positive regulatory
sequences critical for basal, cAMP, and Ca2+-induced
transcription.
DNase I Footprinting Analysis of the Proximal CYP11B2 5'-Flanking
Region
To identify protein-binding sites within this proximal region,
DNase I footprinting analysis was performed using H295R cell nuclear
extracts. As shown in Fig. 4
, two regions of protection
were identified at position -129/-114 (CTCCAGCCTTGACCTT) and at
position -81/-63 (AGTTCTCCCATGACGTGAT). Sequence analysis indicates
that the -129/-114 region contains a nuclear receptor half-site that
resembles the consensus binding site for SF-1 (TGACCT), whereas the
-81/-63 region contains an element (TGACGTGA) sharing seven of eight
bp homology with the consensus CRE.

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Figure 4. DNase I Footprinting Analysis of the Proximal
CYP11B2 Promoter
A 231-bp probe (50,000 cpm) corresponding to CYP11B2 position -229/+2
was digested with DNase I in the absence (control) or presence of H295R
cell nuclear extract (NE, 20 µg) and subjected to denaturing gel
electrophoresis. The protected regions are indicated on the
right of the figure.
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The CYP11B2 -129/-114 Element Binds SF-1 and COUP-TF and Is
Required for Basal and Agonist-Induced Reporter Activity
To begin characterization of nuclear proteins that bind the
-129/-114 bp element, a synthetic oligonucleotide probe encompassing
this sequence was prepared and used in electrophoretic mobility shift
assays (EMSA). In the presence of H295R cell nuclear extracts, three
specific protein-DNA complexes were formed (Fig. 5A
).
Formation of each of these complexes was abolished by the addition of a
10- to 100-fold molar excess of nonradiolabeled wild type competitor.
In the presence of antibody directed against SF-1 protein, complex 3
was displaced (Fig. 5B
). Because COUP-TF is known to bind to some SF-1
elements, we determined whether complexes 1 and 2 might represent
COUP-TF protein binding. As shown in Fig. 5B
, the addition of an
antibody directed against COUP-TF caused these upper two bands to be
displaced.

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Figure 5. Binding of H295R Nuclear Proteins to the
-129/-114 Element
H295R nuclear extracts (NE, 5 µg) were incubated with radiolabeled
probe encompassing the -128/-114 element (20,000 cpm) in the presence
of (A) nonradiolabeled probe (wt) or (B) antibodies directed against
SF-1 (SF-1 Ab) or COUP TF (COUP Ab). DNA/protein complexes (labeled C1,
C2, and C3) were separated from free probe (FP) by gel electrophoresis
and visualized by autoradiography.
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To determine whether this element is necessary for reporter gene
expression, constructs were prepared containing a deletion of the
-129/-114 element. A reporter construct (pB2131), which begins
immediately upstream of the protected region, exhibited basal and
agonist-stimulated activities similar to those of the longer constructs
described above. Deletion of the -129/-114 element resulted in a
reduction of basal activity by approximately 80% compared with the
wild type reporter plasmid (Fig. 6
). In H295R cells
transfected with pB2131, luciferase activity was increased 4.9-,
4.2-, or 7.0-fold above basal after treatment with ANG II,
K+, or (Bu)2cAMP, respectively. Deletion of the
-129/-114 element (pB2106) reduced reporter gene responses to ANG
II (3.0-fold), to K+ (2.8-fold), and to
(Bu)2cAMP (3.7-fold). These results indicate that the
-129/-114 region of human CYP11B2 is essential both for full basal,
as well as maximal Ca2+ and cAMP-induced transcription.

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Figure 6. Deletion Analysis of the 129/-114 Element
CYP11B2 5'-deletion constructs beginning immediately upstream
(pB2131) or downstream (pB2106) of the -129 element were
transiently transfected into H295R cells. After recovery, cells were
treated with: ANG II (10 nM), KCl (20 mM), and
(Bu)2cAMP (1 mM). Luciferase activities are
expressed as a percentage of the basal activity of pB22015 and
represent the mean ± SEM of determinations from four
independent experiments, each performed in triplicate.
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The CYP11B2 -71/-64 Element Binds CREB and Is Necessary for
Calcium Induction
To begin characterization of nuclear proteins that bind the
-71/-64 bp element, a synthetic oligonucleotide probe encompassing
this sequence was prepared and used in EMSA. In the presence of H295R
cell nuclear extracts, at least three specific protein-DNA complexes
were formed (Fig. 7A
). Formation of each of the three
major complexes was abolished by the addition of a nonradiolabeled wild
type competitor but not by a competitor containing a mutation in the
CRE-like sequence (TtctagaA). A fourth faster migrating complex was
observed that was also competed, to some extent, by the wild type
oligonucleotide. However, this complex likely represents nonspecific
interactions since its formation was also inhibited in the presence of
the mutated CRE sequence. With the addition of monoclonal antibodies
that recognized either CRE-binding (CREB) protein or which
cross-reacted with CREB, CRE modulator, or ATF proteins, binding to the
upper complexes 1 and 2 was abolished while complex 3 was not affected
(Fig. 7B
).

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Figure 7. Binding of H295R Cell Nuclear Proteins to the
-71/-64 Element
Panel A, H295R nuclear extracts (NE, 5 µg) were incubated with
radiolabeled probe encompassing the -71/-64 element (20,000 cpm) in
the presence or absence of either nonradiolabeled probe (wt CRE) or
mutated nonradiolabeled probe (m CRE) at the concentrations indicated.
DNA/protein complexes (labeled C1, C2, and C3) were separated from free
probe (FP) by gel electrophoresis and visualized by autoradiography.
Panel B, H295R nuclear extracts were incubated on ice for 20 min in the
presence or absence of antisera directed against either CREM/CREB/ATF,
or CREB specifically.
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To determine whether this element was necessary for reporter gene
activity, constructs were prepared containing the same mutation used in
EMSA. Treatment of H295R cells transfected with the wild type -71/-64
element (pB2354) increased luciferase activity in response to ANG II
(4.9-fold), K+ (3.6-fold), and (Bu)2cAMP
(5.7-fold). Mutation of the -71/-64 element (TGACGTGA to TtctagaA)
reduced basal activity by approximately 50% and drastically reduced
the transcriptional responses to ANG II (1.7-fold), K+
(1.3-fold), and (Bu)2cAMP (1.8-fold) (Fig. 8
). These data suggest that the CRE is required not only
for cAMP but also for Ca2+ induction of reporter gene
activity. However, this element is not in itself sufficient because a
reporter construct that retains the -71/-64 element but lacks the
upstream -129/-114 element exhibited a decreased basal activity and
markedly reduced agonist induction (pB2-106; Fig. 6
). Taken together,
these data suggest that the CRE (-71/-64) and SF-1 (-129/-114)
sequences interact to promote full transcriptional activity in response
to each signaling pathway.

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Figure 8. Analysis of Putative CRE Element within the CYP11B2
Promoter
H295R cells were transiently transfected with a wild type CYP11B2
construct (pB2354) or one containing a mutation in the core CRE-like
sequence (pB2354 mCRE). After recovery, cells were treated with: ANG
II (10 nM), KCl (20 mM), and
(Bu)2cAMP (1 mM). Luciferase activities are
expressed as a percentage of the basal activity of pB22015 and
represent the mean ± SEM of determinations from three to
seven independent experiments, each performed in triplicate.
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DISCUSSION
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Signaling Pathways Regulating CYP11B2
The regulation of CYP11B2 expression is the primary step in
determining the capacity of the adrenal gland to produce aldosterone.
Although ANG II and K+ are the main physiological
regulators of CYP11B2 expression, there have been no studies to define
the molecular mechanisms by which these agonists control CYP11B2
transcription. In addition, the cis-regulatory elements
involved in transcriptional regulation of the human CYP11B2 gene have
not been determined. Herein, we identified two 5'-flanking
cis-elements within the human CYP11B2 gene necessary not
only for ANG II and K+-induced expression, but also for
transcription induced by cAMP.
In adrenal glomerulosa cells, ANG II increases [Ca2+]i
and protein kinase C activity by activating phospholipase C, whereas
K+ increases [Ca2+]i through activation of
voltage-sensitive Ca2+ channels (5, 26). Our data
demonstrate that increases in [Ca2+]i and cAMP can
independently increase CYP11B2 transcription. However, the protein
kinase C pathway does not appear to play a role in regulating human
CYP11B2 transcription. We have recently shown that CYP11B2 mRNA levels
are increased after treatment with K+ or ANG II in a
concentration- and time-dependent manner (27, 28, 29, 30). Because the effects
of these agonists on CYP11B2 reporter gene expression and mRNA levels
are similar, it is likely that changes in CYP11B2 mRNA levels
correspond to changes in transcription of the gene.
CYP11B2 genes of the rat and mouse, when studied as chimeric reporter
constructs in Y-1 adrenocortical cells, are regulated by cAMP (16, 17).
Our data demonstrate the ability of cAMP to similarly increase
transcription of the human gene. However, the role of ACTH and the
cAMP-signaling pathway in long-term regulation of CYP11B2 expression
have been questioned. First, although chronic administration of ACTH to
humans produces a sustained increase in plasma cortisol levels,
aldosterone increases only transiently, then falls to below baseline
within 48 h (24). Second, targeted ablation of pituitary
corticotropes, a maneuver that decreases plasma ACTH to undetectable
levels, does not affect adrenal CYP11B2 expression (31). Thus, in
vivo CYP11B2 expression is not positively regulated by circulating
ACTH. Studies have shown that ACTH treatment increases CYP11B1 but not
CYP11B2 mRNA in rat and human adrenal glomerulosa cells in primary
culture (12, 25). Using the H295R adrenal cell line we have shown a
preferential induction of CYP11B1 by cAMP over that observed for
CYP11B2 (30). Although the physiological role of ACTH and cAMP in
regulation of CYP11B2 expression is unclear, CRE-like motifs are
present in the 5'-flanking region of CYP11B2 in all species thus far
examined. Indeed, using the H295R and the Y-1 adrenal cell we have
demonstrated that the cAMP-signaling pathway can effectively induce
reporter gene expression driven by the CYP11B2 5'-flanking region (32).
The mechanisms by which elevated ACTH levels decrease expression of
CYP11B2 and aldosterone production in vivo will require
further study.
Elements Required for CYP11B2 Expression
Agonist induction of the human CYP11B2 reporter constructs was
dependent on a CRE-like sequence located at position -71. This
proximal CRE is completely conserved between the CYP11B1 and CYP11B2
genes in human, rat, mouse, hamster, and the CYP11B gene of the cow
(9, 10, 11, 12, 13, 21). Previous studies have shown that mutation of this element
within the mouse gene leads to loss of cAMP induction (17). We find
that mutation of this sequence in the human CYP11B2 flanking DNA caused
a loss of induction not only by cAMP, but also by ANG II and
K+, suggesting that this element is necessary for both
Ca2+- and cAMP-induced transcription. It is unclear at
present whether this CRE directly mediates Ca2+-induced
transcription or instead plays a permissive role for other
Ca2+-sensitive elements. There is, however, increasing
evidence that the calcium-signaling pathway can directly utilize CREs
to increase transcription (33, 34). One potential mechanism for this
cross-talk involves the calcium/calmodulin-dependent protein kinases
(CaMK). In vitro, CaMK I, II, and IV can phosphorylate CREB
(35, 36, 37). CaMK are expressed in adrenocortical cells and appear to be
involved in the acute stimulation of aldosterone production (38, 39, 40, 41).
Therefore, the CaMK are likely candidates linking intracellular
Ca2+ signals to CYP11B2 transcription.
The proximal CRE was not sufficient to support human CYP11B2
expression, suggesting that other sequences are required to enhance
transcription. Previous studies have shown that cAMP induction of the
mouse cyp11b2 gene also requires the presence of an element
(AAGGTCTT) that binds SF-1, also referred to as Ad4BP (42, 43).
Mutation of the mouse SF-1-binding site markedly impaired transcription
(18), consistent with the established role of SF-1 in directing the
tissue-specific expression of steroidogenic enzymes (44). Based on
sequence alignments of the CYP11B genes of several mammalian species,
the critical SF-1 site in both mouse CYP11B2 and cow CYP11B corresponds
in human CYP11B2 to a conserved SF-1-like sequence at position
-351/-343 (AAGGCTCC). However, the results of the deletion studies
shown in Fig. 3
do not support a role for the -351/-343 element in
transcription of the human gene, even though this sequence strongly
binds SF-1 from H295R nuclear extracts in EMSA (data not shown)
In contrast to the mouse and cow CYP11B genes, human CYP11B2
transcription required the presence of an element located at
-129/-114 (CTCCAGCCTTGACCT). This element shares 12 of 15 nucleotides
with a region previously identified in the bovine CYP11B gene
(CTCCAACCCTGACCC) termed Ad5 (Adrenal-5, Ref.20). Although Ad5 was
originally identified as an element binding bovine adrenal nuclear
proteins, deletion of this element did not affect the ability of bovine
CYP11B reporter constructs to be induced by cAMP when transfected in
Y-1 cells (21). However, deletion of the Ad5 element from the human
CYP11B2 5'-flanking region drastically impaired basal levels of
transcription as well as preventing maximal induction by cAMP and
Ca2+-signaling pathways. Using EMSA, we demonstrated
binding of SF-1 from H295R cell nuclear extracts to this element.
Sequence analysis revealed an SF-1-like sequence on the noncoding
strand of the element. The Ad5 element is not completely conserved in
the CYP11B2 genes of various species (Fig. 9
). In mouse
cyp11b2 there are 4-bp substitutions that disrupt the SF-1
site, possibly explaining why the Ad5 region neither binds nuclear
proteins (18) nor enhances transcription of the mouse gene
(17, 18, 19).

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Figure 9. Alignment of the Ad5 DNA Sequences of Human, Rat,
and Mouse CYP11B2 and Bovine CYP11B Genes
The nucleotide sequences of the Ad5 regions of the human, rat, and
mouse CYP11B2 and bovine CYP11B are shown. The nucleotide sequences are
numbered from the major transcriptional start site. Sequence
differences with the human Ad5 element are in lowercase
bold.
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Two additional Ad5 protein complexes, which were recognized by COUP-TF
antibody, were observed when H295R cell nuclear proteins were used in
EMSA. Binding of SF-1 and COUP-TF to a common site has been described
in the mouse 21-hydroxylase (CYP21) and bovine 17
-hydroxylase
(CYP17) promoters (45, 46). Moreover, the binding of COUP-TF appears to
occur in competition with SF-1 for the element in the bovine CYP17
promoter (46). Overexpression of COUP-TF in steroidogenic and
nonsteroidogenic cells suggest a role for this orphan receptor in
repression of transcription through its competition with SF-1 (46). It
will therefore be important to determine whether COUP-TF and SF-1 play
synergistic or antagonistic roles in transcription of the human CYP11B2
gene.
Comparison to Previous Studies
In the only previously published analysis of human CYP11B2
transcription, CAT reporter constructs were transfected into mouse Y-1
cells, but no expression was detected unless almost the entire
5'-flanking region (to -64) was deleted (15). In that case, expression
remained very low compared with the corresponding constructs made with
the human CYP11B1 gene. It is possible that these discrepant results
arise through the use of different cell models. For example, Y-1 cells
do not respond to ANG II or K+, suggesting that the
phenotypic characteristics of the Y-1 cell more closely resemble cells
of the zona fasciculata (47, 48). However the Y-1 adrenal cell has
proven to be useful in the analysis of transcriptional regulation of
the mouse and bovine CYP11B genes. In addition, we have obtained good
basal and cAMP-induced expression in Y-1 cells using human CYP11B2
reporter constructs (32).
In summary, ANG II, K+, and cAMP increase reporter gene
expression driven by the 5'-flanking region of human CYP11B2. Two
cis-elements have been identified, both of which are
necessary for maximal induction of CYP11B2 by either Ca2+-
or cAMP-signaling pathways. The mechanism by which these independent
pathways converge to enhance transcription of CYP11B2 will need to be
defined. In addition, the potential interactions between CRE-binding
proteins and the orphan nuclear receptors SF-1 and COUP-TF will need to
be investigated.
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MATERIALS AND METHODS
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Plasmids
A transient expression system using the luciferase reporter gene
was used to characterize the CYP11B2 promoter. The 5'-end of the
CYP11B2 gene was amplified from a bacteriophage
clone carrying
CYP11B2 (13) and a 2017-bp fragment extending from position +2
[relative to the transcription start site, (13)] to the
EcoRI site at -2015 bp was cloned into the promoterless
pGL2-Basic (Promega, Madison, WI) luciferase reporter plasmid to create
pB22015. Several 5'-deletion constructs were prepared using available
restriction endonuclease sites (SmaI, position -1521;
XbaI, position -864; and PstI, position -413).
Smaller deletion constructs were prepared by PCR, introducing
appropriate restriction sites (5', KpnI; 3',
XhoI) or desired mutations. PCR fidelity was confirmed by
sequencing (Sequenase II: USB, Cleveland, OH) and the PCR products
cloned into KpnI/XhoI-digested pGL2. These
deletion fragments corresponded to the following positions: -354,
-221, -131, -106, and -65 bp. The promoterless vector (pGL2-Basic)
and vector containing the SV40 early promoter (pGL2-Control, Promega)
were used as controls.
Cell Culture and Transient Transfection
H295R adrenocortical cells were cultured as previously described
(27, 28), using 2.0% Ultroser G (BioSepra SA, Villeneuve la Garenne
Cedex, France) instead of Ultroser SF. Transient transfection was
carried out using Lipofectamine reagent (GIBCO/BRL, Gaithersburg, MD)
following the manufacturers instructions. Cells were seeded onto
12-well plates to 3040% confluency and used 48 h later.
Transfection was carried out for 6 h at 37 C in a final volume of
0.5 ml DMEM/Hams F12 medium (DMEM/F12, 1:1) (GIBCO/BRL) containing
5.0 µg Lipofectamine and 190 fmol plasmid DNA. After transfection,
cells were incubated for 14 h to allow recovery and expression of
foreign DNA. Cells were then incubated with 2.0 ml low serum medium
(DMEM/F12 containing 0.1% Ultroser G) for a further 24 h before
being rinsed and treated with test substances for the times indicated.
Cells were then rinsed twice with PBS and lysed. Luciferase activity of
the cell lysates was measured using the Luciferase Assay System
(Promega). Luciferase activities were expressed as a percentage of the
basal activity observed for the longest construct (pB22015), which
allowed data from multiple experiments to be pooled for analysis. In
addition, at least two separate plasmid DNA preparations were used for
each reporter construct. Statistical significance of transformed data
was determined using Mann-Whitney U test with a value of
P < 0.05 considered significant.
Electrophoretic Mobility Shift Assay
Nuclear extracts from cultured H295R cells were prepared by the
method of Dignam et al. (49). Double-stranded
oligonucleotides were end-labeled using [
-32P]dCTP and
Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and
incubated (20,000 cpm) with 4 µg nuclear extract and 2.0 µg
poly(deoxyinosinic-deoxycytidylic)acid (as nonspecific competitor) in a
final volume of 20 µl for 20 min at 25 C. Where antibodies were
included in the reaction, nuclear extract and antibody were
preincubated on ice for 20 min before addition of probe. The following
antibodies were used: mouse monoclonal anti-CREB, mouse monoclonal
anti-CREB/CREM/ATF (both provided by Dr. James P. Hoeffler, Invitrogen
Corp., San Diego, CA), rabbit polycolonal anti-COUP-TF (provided by Dr.
Ming-Jer Tsai, Baylor College of Medicine, Houston Texas), and rabbit
polyclonal anti-SF-1 (provided by Dr. Ken-ichirou Morohashi, Kyushu
University, Fukuoka, Japan). The COUP-TF antibody does not discriminate
between COUP-TF I and COUP-TF II (50) and appears to bind at least two
proteins on Western analysis (51). For competition analysis, reaction
mixtures contained various amounts of nonradiolabeled oligonucleotide
added simultaneously with probe. The resulting DNA/protein complexes
were separated from free probe by electrophoresis using a 5.4%
polyacrylamide gel and 0.5 x TBE (final concentrations 44.5
mM Tris, 44.5 mM boric acid, 1 mM
EDTA, pH 8.0) as running buffer for 2 h at 200 V. Gels were dried
and radioactive complexes visualized after autoradiography at -70C for
24 h. Each figure is representative of a minimum of four
independent analyses.
Dnase 1 Footprinting Assay
A 231-bp fragment of CYP11B2 5'-flanking DNA (-229/+2) was
amplified by PCR and labeled using MMLV reverse transcriptase and
[
32P]-dCTP. Assays were performed using the HotFoot
Footprinting kit (Stratagene, La Jolla, CA), following the
manufacturers instructions, using 50,000 cpm probe and 20 µg
nuclear extract. Naked probe was digested with 0.2 U DNase I and, in
the presence of H295R cell nuclear extracts, 3.0 U DNase I (2 min at 25
C). Digested fragments were separated by denaturing electrophoresis
using an 8% polyacrylamide 7 M urea gel and 1 x TBE
(89 mM Tris, 89 mM boric acid, 2 mM
EDTA, pH 8.0) as running buffer. The positions of the protected regions
were confirmed in three independent footprinting analyses.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Ken-ichirou Morohashi (Kyushu
University, Fukuoka, Japan) for providing the SF-1 antiserum, Dr. James
Hoeffler (Invitrogen Corp., San Diego, CA) for kindly providing the
CREB/CREM/ATF and CREB antibodies, and Dr. Ming-Jer Tsai (Baylor
College of Medicine, Houston, TX) for providing the COUP-TF
antiserum.
 |
FOOTNOTES
|
---|
Address requests for reprints to: William E. Rainey, Ph.D., Department of Obstetrics & Gynecology, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9032.
This work was supported by awards from the American Heart Association
(95010570) and the NIH (DK-43140) (to W.E.R.), from the American
Heart Association (Texas Affiliate 94G-086) (to J.M.M.), and from the
NIH (DK37867 & DK42169) (to P.C.W.).
Received for publication November 14, 1996.
Revision received January 21, 1997.
Accepted for publication January 28, 1997.
 |
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