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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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.
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). 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.
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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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). 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
pB2–2015. Reporter activity induced by (Bu)2cAMP decreased
by 50–60% 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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).
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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). In H295R cells
transfected with pB2–131, 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 (pB2–106) 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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). 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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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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-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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
pB2–2015. 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 manufacturer’s instructions. Cells were seeded onto
12-well plates to 30–40% 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/Ham’s 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 (pB2–2015), 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
manufacturer’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 J Song, I Narita, S Goto, N Saito, K Omori, F Sato, J Ajiro, D Saga, D Kondo, M Sakatsume, et al. Gender specific association of aldosterone synthase gene polymorphism with renal survival in patients with IgA nephropathy J. Med. Genet., May 1, 2003; 40(5): 372 - 376. [Full Text] [PDF]
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J. Gu, Y. Wen, A. Mison, and J. L. Nadler 12-Lipoxygenase Pathway Increases Aldosterone Production, 3',5'-Cyclic Adenosine Monophosphate Response Element-Binding Protein Phosphorylation, and p38 Mitogen-Activated Protein Kinase Activation in H295R Human Adrenocortical Cells Endocrinology, February 1, 2003; 144(2): 534 - 543. [Abstract] [Full Text] [PDF]
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P. O. Lim, T. M. Macdonald, C. Holloway, E. Friel, N. H. Anderson, E. Dow, R. T. Jung, E. Davies, R. Fraser, and J. M. C. Connell Variation at the Aldosterone Synthase (CYP11B2) Locus Contributes to Hypertension in Subjects with a Raised Aldosterone-to-Renin Ratio J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4398 - 4402. [Abstract] [Full Text] [PDF]
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J. C. Condon, V. Pezzi, B. M. Drummond, S. Yin, and W. E. Rainey Calmodulin-Dependent Kinase I Regulates Adrenal Cell Expression of Aldosterone Synthase Endocrinology, September 1, 2002; 143(9): 3651 - 3657. [Abstract] [Full Text] [PDF]
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N. Yamamoto, H. Yasue, Y. Mizuno, M. Yoshimura, H. Fujii, M. Nakayama, E. Harada, S. Nakamura, T. Ito, and H. Ogawa Aldosterone Is Produced From Ventricles in Patients With Essential Hypertension Hypertension, May 1, 2002; 39(5): 958 - 962. [Abstract] [Full Text] [PDF]
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P. O. Lim and T. Rankinen Role of Aldosterone in the Pathogenesis of Hypertension * Response Hypertension, February 1, 2002; 39 (2): e14 - e14. [Full Text] [PDF]
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A. Peri, P. Luciani, B. Conforti, S. Baglioni-Peri, F. Cioppi, C. Crescioli, P. Ferruzzi, S. Gelmini, G. Arnaldi, G. Nesi, et al. Variable Expression of the Transcription Factors cAMP Response Element-Binding Protein and Inducible cAMP Early Repressor in the Normal Adrenal Cortex and in Adrenocortical Adenomas and Carcinomas J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5443 - 5449. [Abstract] [Full Text] [PDF]
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 K. Ranade, K. D. Wu, N. Risch, M. Olivier, D. Pei, C.-F. Hsiao, L.-M. Chuang, L.-T. Ho, E. Jorgenson, R. Pesich, et al. Genetic variation in aldosterone synthase predicts plasma glucose levels PNAS, October 25, 2001; (2001) 221467098. [Abstract] [Full Text] [PDF]
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S. Portrat, P. Mulatero, K. M. Curnow, J.-L. Chaussain, Y. Morel, and L. Pascoe Deletion Hybrid Genes, due to Unequal Crossing Over between CYP11B1 (11{beta}-Hydroxylase) and CYP11B2(Aldosterone Synthase) Cause Steroid 11{beta}-Hydroxylase Deficiency and Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3197 - 3201. [Abstract] [Full Text] [PDF]
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 C. Delles, J. Erdmann, J. Jacobi, K. F. Hilgers, E. Fleck, V. Regitz-Zagrosek, and R. E. Schmieder Aldosterone synthase (CYP11B2) -344 C/T polymorphism is associated with left ventricular structure in human arterial hypertension J. Am. Coll. Cardiol., March 1, 2001; 37(3): 878 - 884. [Abstract] [Full Text] [PDF]
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 Y. Mizuno, M. Yoshimura, H. Yasue, T. Sakamoto, H. Ogawa, K. Kugiyama, E. Harada, M. Nakayama, S. Nakamura, T. Ito, et al. Aldosterone Production Is Activated in Failing Ventricle in Humans Circulation, January 2, 2001; 103(1): 72 - 77. [Abstract] [Full Text] [PDF]
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X.-L. Wang, M. Bassett, Y. Zhang, S. Yin, C. Clyne, P. C. White, and W. E. Rainey Transcriptional Regulation of Human 11{beta}-Hydroxylase (hCYP11B1) Endocrinology, October 1, 2000; 141(10): 3587 - 3594. [Abstract] [Full Text] [PDF]
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L. Groussin, J. F. Massias, X. Bertagna, and J. Bertherat Loss of Expression of the Ubiquitous Transcription Factor cAMP Response Element-Binding Protein (CREB) and Compensatory Overexpression of the Activator CREM{tau} in the Human Adrenocortical Cancer Cell Line H295R J. Clin. Endocrinol. Metab., January 1, 2000; 85(1): 345 - 354. [Abstract] [Full Text]
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B. J. Clark and R. Combs Angiotensin II and Cyclic Adenosine 3',5'-Monophosphate Induce Human Steroidogenic Acute Regulatory Protein Transcription through a Common Steroidogenic Factor-1 Element Endocrinology, October 1, 1999; 140(10): 4390 - 4398. [Abstract] [Full Text]
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F. Paillard, D. Chansel, E. Brand, A. Benetos, F. Thomas, S. Czekalski, R. Ardaillou, and F. Soubrier Genotype-Phenotype Relationships for the Renin-Angiotensin-Aldosterone System in a Normal Population Hypertension, September 1, 1999; 34(3): 423 - 429. [Abstract] [Full Text] [PDF]
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 C. Delcayre and J.-S. Silvestre Aldosterone and the heart: towards a physiological function? Cardiovasc Res, July 1, 1999; 43(1): 7 - 12. [Full Text] [PDF]
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K. Zeitoun, K. Takayama, M. D. Michael, and S. E. Bulun Stimulation of Aromatase P450 Promoter (II) Activity in Endometriosis and Its Inhibition in Endometrium Are Regulated by Competitive Binding of Steroidogenic Factor-1 and Chicken Ovalbumin Upstream Promoter Transcription Factor to the Same cis-Acting Element Mol. Endocrinol., February 1, 1999; 13(2): 239 - 253. [Abstract] [Full Text]
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S. Tamaki, N. Iwai, Y. Tsujita, and M. Kinoshita Genetic Polymorphism of CYP11B2 Gene and Hypertension in Japanese Hypertension, January 1, 1999; 33(1): 266 - 270. [Abstract] [Full Text] [PDF]
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E. Brand, N. Chatelain, P. Mulatero, I. Fery, K. Curnow, X. Jeunemaitre, P. Corvol, L. Pascoe, and F. Soubrier Structural Analysis and Evaluation of the Aldosterone Synthase Gene in Hypertension Hypertension, August 1, 1998; 32(2): 198 - 204. [Abstract] [Full Text] [PDF]
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M. Kupari, A. Hautanen, L. Lankinen, P. Koskinen, J. Virolainen, H. Nikkila, and P. C. White Associations Between Human Aldosterone Synthase (CYP11B2) Gene Polymorphisms and Left Ventricular Size, Mass, and Function Circulation, February 17, 1998; 97(6): 569 - 575. [Abstract] [Full Text] [PDF]
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J.-G. LeHoux, G. Dupuis, and A. Lefebvre Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms J. Biol. Chem., March 9, 2001; 276(11): 8021 - 8028. [Abstract] [Full Text] [PDF]
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K. Ranade, K. D. Wu, N. Risch, M. Olivier, D. Pei, C.-F. Hsiao, L.-M. Chuang, L.-T. Ho, E. Jorgenson, R. Pesich, et al. Genetic variation in aldosterone synthase predicts plasma glucose levels PNAS, November 6, 2001; 98(23): 13219 - 13224. [Abstract] [Full Text] [PDF]
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), BAYK8644 (
), (Bu)2cAMP
(
), or TPA (
) at the concentrations indicated for 8 h before
being lysed and assayed for luciferase activity. Results are expressed
as a percentage of the control (untreated) activity and represent the
mean ± SEM of determinations from three to six independent
experiments, each performed in triplicate.
