Atrial Natriuretic Peptide Inhibits Calcium-Induced Steroidogenic Acute Regulatory Protein Gene Transcription in Adrenal Glomerulosa Cells
Nadia Cherradi,
Yves Brandenburger,
Michel F. Rossier,
Michel B. Vallotton,
Douglas M. Stocco and
Alessandro M. Capponi
Division of Endocrinology and Diabetology (N.C., Y.B., M.F.R.,
M.B.V., A.M.C.) Department of Internal Medicine Faculty of
Medicine CH-1211 Geneva 14, Switzerland
Department of
Cell Biology and Biochemistry (D.M.S.) Texas Tech University Health
Sciences Center Lubbock, Texas 79430
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ABSTRACT
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Atrial natriuretic peptide (ANP) is a potent
inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa
cells by physiological agonists activating the calcium messenger
system, such as angiotensin II (Ang II) and potassium ion
(K+). While the role of calcium in mediating
Ang II- and K+-induced aldosterone production
is clearly established, the mechanisms leading to blockade of this
steroidogenic response by ANP remain obscure. We have used bovine
adrenal zona glomerulosa cells in primary culture, in which an
activation of the calcium messenger system was mimicked by a 2-h
exposure to an intracellular high-calcium clamp. The effect of ANP was
studied on the following parameters of the steroidogenic pathway: 1)
pregnenolone and aldosterone production; 2) changes in cytosolic
([Ca2+]c) and
mitochondrial
([Ca2+]m)
Ca2+ concentrations, as assessed with targeted
recombinant aequorin; 3) cholesterol content in outer mitochondrial
membranes (OM), contact sites (CS), and inner membranes (IM); 4)
steroidogenic acute regulatory (StAR) protein import into mitochondria
by Western blot analysis; 5) StAR protein synthesis, as determined by
[35S]methionine incorporation,
immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot
analysis with a StAR cDNA; 7) StAR gene transcription by
nuclear run-on analysis.
While clamping Ca2+ at 950
nM raised pregnenolone output 3.5-fold and
aldosterone output 3-fold, ANP prevented these responses with an
IC50 of 1 nM and a
maximal effect of 90% inhibition at 10 nM. In
contrast, ANP did not affect the
[Ca2+]c or
[Ca2+]m changes
occurring under Ca2+ clamp or Ang II
stimulation in glomerulosa cells. The accumulation of cholesterol
content in CS (139.7 ± 10.7% of control) observed under
high-Ca2+ clamp was prevented by 10
nM ANP (92.4 ± 4% of control).
Similarly, while Ca2+ induced a marked
accumulation of StAR protein in mitochondria of glomerulosa cells to
218 ± 44% (n = 3) of controls, the presence of ANP led to a
blockade of StAR protein mitochondrial import (113.3 ± 15.0%).
This effect was due to a complete suppression of the increased
[35S]methionine incorporation into StAR
protein that occurred under Ca2+ clamp
(94.5 ± 12.8% vs. 167.5 ± 17.3%, n = 3).
Furthermore, while the high-Ca2+ clamp
significantly increased StAR mRNA levels to 188.5 ± 8.4 of
controls (n = 4), ANP completely prevented this response. Nuclear
run-on analysis showed that increases in intracellular
Ca2+ resulted in transcriptional induction of
the StAR gene and that ANP inhibited this process.
These results demonstrate that Ca2+ exerts a
transcriptional control on StAR protein expression and that ANP appears
to elicit its inhibitory effect on aldosterone biosynthesis by acting
as a negative physiological regulator of StAR gene
expression.
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INTRODUCTION
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In the zona glomerulosa cells of the adrenal cortex, the
octapeptide hormone angiotensin II (Ang II) and potassium
(K+) are the most powerful stimuli of aldosterone
production. Mineralocorticoid biosynthesis and secretion are highly
dependent upon the increase in intracellular calcium triggered by these
two agonists through distinct mechanisms (1, 2, 3, 4, 5).
The rate-limiting step in the activation of steroidogenesis is the
delivery of cholesterol from the mitochondrial outer membrane to the
inner membrane, where the cytochrome P450 side-chain cleavage
(P450scc) enzyme is located (6, 7, 8, 9, 10). Earlier studies have
shown that cycloheximide, an inhibitor of protein translation, blocked
pregnenolone production elicited by either cAMP- or
Ca2+-mobilizing hormones (8, 9, 11). Indeed, the appearance
of newly synthesized mitochondrial phosphoproteins, referred to as the
30 kDa-proteins, has been shown to correlate directly with steroid
production in adrenal cells and MA-10 mouse Leydig tumor cells
(12, 13, 14, 15). Currently, the 30-kDa steroidogenic acute regulatory (StAR)
protein is considered to be a key regulator of cholesterol delivery to
the P450scc enzyme (16, 17, 18, 19). The decisive demonstration
came from an inherited disease that leads to a dramatic deficiency in
all steroid hormones, congenital lipoid adrenal hyperplasia: mutations
in the StAR gene have been shown to underlie this disorder
(20, 21).
Atrial natriuretic peptide (ANP), a hormone originally identified in
atrial cardiomyocytes, affects blood pressure through its concerted
actions on various target organs, including vascular smooth muscle,
kidney, and adrenal cortex. In adrenal glomerulosa cells, ANP strongly
impairs aldosterone secretion stimulated by Ang II (4, 22, 23, 24, 25),
K+ (26), or ACTH (27, 28). In vitro and in
vivo studies indicate that ANP-induced inhibition of aldosterone
synthesis is mediated by type A natriuretic peptide receptors endowed
with intrinsic guanylyl cyclase activity (29, 30), and cGMP was
initially thought to be the intracellular messenger mediating all
effects of ANP (29, 31). However, a number of studies reported that the
inhibition of aldosterone synthesis by ANP could not be mimicked with
membrane-permeant, nonhydrolysable analogs of cGMP, such as
8-bromo-cGMP or dibutyryl-cGMP (4, 32, 33). The intracellular sites of
the antisteroidogenic action of ANP remain, therefore, to be
elucidated.
In theory, ANP could impede the generation of the cytosolic
Ca2+ ([Ca2+]c) signal and the
resulting rise in mitochondrial free Ca2+ concentration
([Ca2+]m) that are observed upon challenge
with Ang II and K+ (34). Alternatively, ANP could affect
cholesterol supply to the mitochondria or intramitochondrial
cholesterol transfer or interfere with either the expression or the
action of the StAR protein (35, 36).
In the present study, we have examined whether ANP interferes with any
of the above processes, using the Ca2+-clamp technique to
mimic Ang II or K+ activation. We report that ANP
inhibition is exerted on intramitochondrial cholesterol transfer and
results from an inhibition of the Ca2+-induced synthesis of
StAR protein and therefore of StAR protein accumulation within
mitochondria. We provide evidence that the increase in StAR protein
synthesis elicited by Ca2+ is a consequence of an increase
in the steady state level of StAR mRNA, suggesting that
Ca2+ activates StAR protein gene transcription and that ANP
modulates this activation.
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RESULTS
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ANP Inhibits Ca2+-Induced Pregnenolone and
Aldosterone Biosynthesis
In Ca2+-clamped bovine glomerulosa cells, pregnenolone
production was stimulated in a concentration-dependent manner by
[Ca2+]c as previously reported (37). This
activation was inhibited in a concentration-dependent manner by both
Thr-Ala-Pro-Arg-human (h)ANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) (urodilatin, an analog of ANP
originally isolated from human urine) and hANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) (Fig. 1
). Similar results were obtained for
aldosterone production (data not shown). Both peptides were equipotent
in preventing pregnenolone production in cells submitted to an
intracellular calcium clamp of 600 nM, with an
IC50 of approximately 1 nM and a maximal
inhibition of 90% (Fig. 1B
). They were therefore used indifferently in
subsequent experiments. Since the Ca2+ signal generated by
the high-Ca2+ clamp is, by definition, constant, these
results globally suggested to us that the target for the inhibitory
mechanism of ANP resides downstream of the production of the calcium
signal.

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Figure 1. Effect of ANP on Ca2+-Induced
Pregnenolone Formation in Bovine Adrenal Glomerulosa Cells
A, Glomerulosa cells were stimulated with a cytosolic
Ca2+-clamp for 2 h, in the absence or in the presence
of 10 nM urodilatin, as described in Materials and
Methods. Pregnenolone production is expressed as a percentage
of the production determined in control cells incubated in nominally
Ca2+-free medium ([Ca2+]c < 100
nM). **, Significantly different (P <
0.01, n = 6) from the respective control without urodilatin. B,
Concentration dependence of the inhibitory effect of hANP(4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ) ( )
and urodilatin () on pregnenolone production in
high-Ca2+ (600 nM) clamped cells. Percent
inhibition was calculated by comparing pregnenolone production above
zero-Ca2+ control in the presence of ANP peptides to that
measured in the absence of peptide. Each point is the mean ±
SEM of two to nine separate experiments. Pregnenolone
production: Control, 0.102 ± 0.02 nmol/mg prot/2 h;
high-Ca2+, 0.319 ± 0.135; control + hANP (10
nM), 0.092 ± 0.037; high-Ca2+ + hANP,
0.120 ± 0.054.
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The membrane-permeant analogs of cGMP, 8-Br-cGMP and dibutyryl-cGMP (10
µM), failed to mimick the antisteroidogenic action of
ANP, as shown in Fig. 2
. Ten-fold higher
concentrations (100 µM) of the analogs were equally
uneffective (data not shown).

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Figure 2. Lack of Effect of cGMP Analogs on
Ca2+-Induced Pregnenolone Production
Glomerulosa cells were stimulated with a cytosolic
Ca2+-clamp for 2 h, in the absence or in the presence
of 8-Br-cGMP ( ) or dibutyryl-cGMP () (10 µM), as
described in Materials and Methods. Pregnenolone
production is expressed as a percentage of the production determined in
control cells incubated in nominally Ca2+-free medium
([Ca2+]c < 100 nM). Each point
is the mean value of triplicate samples from two separate
experiments.
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ANP Does Not Affect Ang II-Induced
[Ca2+]c and
[Ca2+]m Responses
The lack of an effect of ANP on Ca2+ responses was
confirmed in bovine glomerulosa cells transfected with targeted
aequorin. In cells that had been pretreated with 100 nM
hANP for 330 min, the [Ca2+]c response to
Ang II in glomerulosa cells transfected with nontargeted-aequorin was
not affected by the presence of hANP, and the
[Ca2+]c values established under
Ca2+ clamp were not altered (not shown). Since the
mitochondrion is a known target for the Ca2+ signal, we
also examined [Ca2+]m changes. When adrenal
glomerulosa cells were challenged with Ang II (10 nM), a
biphasic [Ca2+]m response was observed, as
previously reported (34). In the presence of hANP, this response was
superimposable to that recorded in the absence of hANP (Fig. 3
). When added after Ang II challenge,
hANP did not alter the plateau [Ca2+]m phase.
Clearly, therefore, the generation of the calcium signal was unaffected
by hANP.

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Figure 3. Lack of Effect of ANP on the
[Ca2+]m Response to Ang II in Bovine
Glomerulosa Cells
Cells were transfected with targeted aequorin as described in
Materials and Methods. Aequorin luminescence was
recorded under stimulation with Ang II (10 nM) in control
cells (dotted trace) or in cells that had been
pretreated with hANP (100 nM) (solid trace)
for 30 min. This trace is representative of four similar experiments.
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ANP Prevents Ca2+-Induced Stimulation of
Intramitochondrial Cholesterol Transfer
We have previously shown that the stimulation of ionomycin-treated
bovine adrenal glomerulosa cells with Ca2+ markedly
decreased cholesterol content in outer mitochondrial membranes (OM) and
concomitantly increased cholesterol content in contact sites (CS) and
inner membranes (IM), reflecting a stimulation of intramitochondrial
cholesterol transfer (35, 36). Since ANP almost entirely prevented
Ca2+-supported pregnenolone formation, the first enzymatic
step after cholesterol supply to P450scc, we therefore
examined the effect of ANP on Ca2+-induced
intramitochondrial cholesterol distribution. As we have previously
shown, stimulation of glomerulosa cells with Ca2+ led to a
significant increase of cholesterol content in CS and IM to 139.7
± 10.7% and 131.5 ± 7% of the respective controls
(P < 0.05, n = 3) and to a concomitant
significant decrease of cholesterol content in OM, to 70.4% ± 2.1%
of controls (P < 0.001, n = 3) (Fig. 4
). hANP (10 nM) completely
prevented this Ca2+-induced transfer of cholesterol to CS
and IM (92.4 ± 4% and 103.1 ± 16.1% of the respective
controls, n = 3). Moreover, no significant change was observed in
cholesterol content in OM when glomerulosa cells were simultaneously
treated with Ca2+ and hANP (88.2 ± 6.5% of controls,
n = 3). hANP alone had no effect on cholesterol content in OM, CS,
and IM (105.6 ± 2.9%, 95.5 ± 9.8%, and 104.2 ±
1.4% of the respective controls, n = 3) (Fig. 4
).

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Figure 4. Effect of ANP on Ca2+-Induced
Intramitochondrial Cholesterol Transfer in Bovine Glomerulosa Cells
Cells were stimulated with a cytosolic low- (<100 nM,
control cells) or high- (600 nM) Ca2+ clamp for
2 h, in the absence or in the presence of 10 nM
hANP(4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ) as described in Materials and Methods. After
submitochondrial fractionation, the cholesterol content of OM, CS, and
IM in Ca2+ clamped cells was determined and expressed as a
percentage of that measured in the respective submitochondrial
fractions of control low-Ca2+ clamped cells (mean ±
SEM, n = 3). In a typical experiment, mass unit values
for cholesterol in OM, CS, and IM were, respectively, 10.9, 3.4, and
1.6 µg/mg protein for controls. (* and ***, significantly
different from the respective control with P <
0.05 and P < 0.001, respectively).
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ANP Prevents Ca2+-Induced Accumulation of
StAR Protein within Mitochondria
We showed recently that Ca2+-activated
intramitochondrial cholesterol transfer is accompanied in bovine
glomerulosa cells by an increase in StAR protein within mitochondria, a
finding consistent with a role for StAR protein in cholesterol
transport (36). To determine whether ANP affects the StAR protein
accumulation induced by Ca2+, we performed immunoblot
analysis on mitochondrial proteins from glomerulosa cells that had been
calcium-clamped in the presence or in the absence of hANP. As shown in
Fig. 5
, while Ca2+ induced
the expected marked increase of StAR protein content in mitochondria
(to 218.2 ± 44.4% of controls, n = 3, P <
0.01), hANP (10 nM) completely prevented this
Ca2+-induced increase in StAR (94.5 ± 12.8% of
controls). hANP had no significant effect in itself.

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Figure 5. Effect of ANP on the Ca2+-Induced
Increase in StAR Protein Content in Mitochondria of Bovine Glomerulosa
Cells
A, Mitochondria were isolated from low- or high-Ca2+
clamped cells incubated in the presence or in the absence of hANP (10
nM). Shown is a Western blot from one representative
experiment. For each sample, 10 µg of mitochondrial proteins were
analyzed by SDS-PAGE and immunoblotting as described in
Materials and Methods. B, The immunospecific bands for
StAR protein were quantitated by densitometry in three independent
experiments. Control, low-Ca2+-clamp; Ca2+,
high-Ca2+ clamp (600 nM); hANP,
low-Ca2+ clamp in the presence of hANP (10 nM);
Ca2+ + hANP, high-Ca2+ clamp in the presence of
hANP. Integrated optical density (IOD) values are expressed as a
percentage of that measured in mitochondria from control cells. **,
Significantly different from control with P <
0.01, ++, significantly different from Ca2+ stimulation
with P < 0.01.
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ANP Prevents Ca2+-Induced Stimulation of
StAR Protein Synthesis
The Ca2+-induced accumulation of StAR protein content
in mitochondria could result from increased StAR protein synthesis.
This hypothesis was tested in glomerulosa cells radiolabeled with
[35S]methionine/cysteine during Ca2+
stimulation in the presence or in the absence of hANP. StAR protein was
then immunoprecipitated from total cellular extracts as described in
Materials and Methods. Upon SDS-PAGE analysis and
autoradiography of StAR immunoprecipitates, we observed that, in high
Ca2+-clamped cells, the labeling of StAR protein was
significantly stimulated (167.5 ± 17.3% of controls, n = 3,
P < 0.01) (Fig. 6
),
reflecting increased StAR protein synthesis. The addition of hANP (10
nM) simultaneously with Ca2+ completely
prevented this increase in StAR protein labeling.

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Figure 6. Effect of ANP on the Ca2+-Induced
Increase in StAR Protein Radiolabeling
A, Immunoprecipitation of StAR protein from
[35S]methionine/cysteine-labeled bovine glomerulosa cells
treated as described in the legend of Fig. 5 . StAR protein was
immunoprecipitated from equivalent amounts of radioactivity for each
treatment as described in Materials and Methods, and the
antibody-StAR protein complexes were analyzed by SDS-PAGE and
autoradiography. B, Immunoprecipitated labeled StAR protein was
quantitated by densitometry in three independent experiments.
Integrated optical density (IOD) values are expressed as a percentage
of that measured in control cells. **, Significantly different
from control with P < 0.01; ++, significantly
different from Ca2+ stimulation with P
< 0.01.
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ANP Inhibits Ca2+-Induced Increase in StAR
mRNA Levels
The above results prompted us to examine whether ANP could exert
its antisteroidogenic effect by acting directly on StAR mRNA production
and/or stability. Upon Northern blot analysis of StAR mRNA, we observed
consistently two, sometimes three, transcripts that hybridized with
StAR cDNA. Two major bands migrated at approximately 2.6 and 1.6 kb
(Fig. 7A
), and a third one migrated at
0.9 kb. These transcripts showed coordinate induction. We therefore
quantified only the most abundant 2.6-kb transcript. The quantification
of StAR 2.6 kb mRNA from four independent experiments indicated that
Ca2+ provoked a significant accumulation of this transcript
(189 ± 14.5% of controls, P < 0.001) and that
hANP (10 nM) prevented the Ca2+-induced
increase in StAR mRNA (116 ± 13% of controls) (Fig. 7B
).

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Figure 7. Effect of ANP on the Ca2+-Induced
Increase in Steady State StAR mRNA Levels in Bovine Glomerulosa Cells
A, Northern blot analysis of StAR mRNA in control (C) and
high-Ca2+ clamped cells, in the absence or in the presence
of 10 nM hANP (lanes 13). In lanes 46, the same
experiment was performed in the presence of actinomycin D (5 µg/ml).
Total RNA was isolated as described in Materials and
Methods and 15 µg of each sample were analyzed by agarose gel
electrophoresis. StAR specific transcripts were detected by
hybridization with a mouse full-length StAR cDNA. The blot was then
stripped and rehybridized with a cDNA probe specific of the mouse GAPDH
to normalize for equivalent loading of RNA. B, Quantification of StAR
mRNA levels in four independent experiments (panel A, lanes 13). The
major 2.6-kb transcript was quantitated and normalized to GAPDH mRNA
levels. The corrected values for the IOD are expressed as a percentage
of the IOD of StAR mRNA levels in control cells. ***, Significantly
different from controls with P < 0.001; ++,
significantly different from Ca2+ stimulation with
P < 0.01 (n = 3). C, Quantification of StAR
mRNA in control and high Ca2+-clamped cells incubated in
the presence of actinomycin D, with or without hANP (10 nM)
(panel A, lanes 46) (n = 3).
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The Ca2+-induced accumulation of StAR mRNA may result from
changes in transcription rate and/or in mRNA turnover. To determine
whether Ca2+ or hANP affected StAR mRNA stability,
glomerulosa cells were submitted to a high-calcium clamp (600700
nM) for 2 h. The incubation buffer was then removed
and replaced with fresh buffer containing ionomycin and actinomycin D.
Incubation was then continued for 6 h in the presence or in the
absence of Ca2+ and/or hANP (10 nM). After a 6
h-incubation period in Krebs buffer, StAR mRNA levels decayed to
69.6 ± 6.6% (n = 3) of the zero time value, whereas in the
presence of Ca2+, StAR mRNA decayed to 52 ± 15% of
the zero time value (n = 3). These differences were not
statistically significant. The addition of hANP did not elicit any
significant effect on StAR mRNA stability, as measured 6 h after
the addition of actinomycin D (63.6 ± 4.7% of zero time
value).
These results suggested that the effect of Ca2+ on the
steady state levels of StAR mRNA must reflect transcriptional
activation. To assess this possibility, glomerulosa cells were treated
simultaneously with Ca2+ and actinomycin D (5 µg/ml), in
the absence or in the presence of hANP (10 nM).
Densitometric analysis of Northern blots from three independent
experiments clearly indicated that actinomycin D completely prevented
the Ca2+-induced StAR mRNA accumulation (108 ± 5% of
controls) (Fig. 7C
). In separate experiments, we observed that
actinomycin D inhibited Ca2+-induced pregnenolone
production by 69.8 ± 11.8% (n = 4). Since ANP had no effect
on StAR mRNA stability, it is likely that ANP exerts its inhibitory
effect on Ca2+-induced StAR mRNA accumulation by preventing
StAR gene transcription.
Ca2+ Stimulates ANP-Sensitive Transcription
of the StAR Gene
The above hypothesis was confirmed by the results of nuclear
run-on experiments performed in bovine glomerulosa cells. As shown in
Fig. 8
, after 2 h of a
high-Ca2+ clamp, transcriptional activity of the
StAR gene was 171 ± 19% (P < 0.01,
n = 3) of that measured in control (low-Ca2+ clamp)
cells. Once again, hANP (10 nM) prevented this
Ca2+-induced activation of StAR gene
transcription. Transcription of the ß-actin gene was not
significantly affected by Ca2+ or hANP, and no signal was
detected with the empty vector (Fig. 8A
).

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Figure 8. Nuclear Run-on Transcriptional Analysis of the
StAR Gene
A, Bovine glomerulosa cells were submitted to a low- (control) or a
high-Ca2+ (600 nM) clamp in the presence or in
the absence of hANP (10 nM), as described in
Materials and Methods. The transcription of the
StAR gene was assessed in isolated nuclei incubated with
[32P]UTP to generate radiolabeled RNA transcripts. RNA
was hybridized to membranes on which StAR cDNA cloned in a Bluescript
plasmid, empty Bluescript plasmid (pBSK, negative control), and mouse
ß-actin cDNA cloned in Bluescript (invariant internal control) had
been immobilized. B, Quantification of nuclear run-on analysis from
three independent experiments. Results are expressed as a percentage of
controls.
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DISCUSSION
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ANP exerts one of its major physiological antihypertensive effects
by preventing the stimulation of aldosterone secretion elicited in
adrenal glomerulosa cells by agonists such as Ang II and K+
(4, 26, 38). Although ANP is known to stimulate cGMP formation in
glomerulosa cells (29, 31), the intracellular mechanism of action of
ANP as well as its molecular targets in adrenocortical cells are still
poorly defined, due in part to the failure of membrane-permeant,
nonhydrolysable analogs of cGMP to mimic the biological effect of ANP
(4, 32, 33). This lack of effect of cGMP analogs was also observed in
this work.
The present study was therefore undertaken in an attempt to identify
intracellular targets for the inhibitory action of ANP on aldosterone
biosynthesis. In a previous work on the mechanisms of activation of
aldosterone biosynthesis by Ang II and Ca2+ in bovine
adrenal glomerulosa cells, we have shown that physiological rises in
[Ca2+]c acutely stimulate the rate-limiting
step in this process, namely cholesterol translocation from the
mitochondrial outer membrane to intermembrane contact sites and inner
membrane (35). Concomitantly, Ca2+ induced a specific
increase in StAR protein in the inner mitochondrial membrane,
confirming the well documented role of StAR in intramitochondrial
cholesterol redistribution (36). In addition, both
Ca2+-stimulated intramitochondrial cholesterol transfer and
StAR accumulation in the inner membrane were cycloheximide-sensitive,
indicating the requirement for ongoing protein synthesis in the acute
activation of aldosterone production. Using the ionomycin-mediated
calcium clamp technique as a tool to mimic agonist-induced
physiological rises of [Ca2+]c (37), we have
investigated whether ANP affects any of these
Ca2+-sensitive events.
Two major conclusions can be drawn from the results of the present
work: 1) Ca2+ regulates StAR gene expression at
the transcriptional level and 2) ANP inhibits aldosterone production by
preventing Ca2+-induced StAR mRNA accumulation.
While the role of cAMP in mediating the activation of StAR
gene transcription via steroidogenic factor 1 (SF-1), an orphan nuclear
receptor, has been extensively investigated (39, 40, 41, 42, 43), the potential
participation of Ca2+ in regulating StAR gene
expression has received little attention to date. We first examined the
effect of Ca2+ stimulation on StAR mRNA levels. In
agreement with previous data on bovine StAR gene expression
(43), Northern blot analysis of StAR mRNA showed that two major StAR
mRNA transcripts of 2.6 and 1.6 kb were present in bovine adrenal
cells. Importantly, Ca2+ induced a marked and significant
increase in StAR mRNA levels within 2 h of exposure of glomerulosa
cells to a high Ca2+-clamp. This observation raised the
question of whether Ca2+ could increase StAR mRNA
half-life. Our results show that StAR mRNA decayed to the same extent
in low- and high-Ca2+ clamped cells for up to 6 h
after addition of actinomycin D, thus excluding a stabilizing effect as
the mechanism leading to Ca2+-induced acute accumulation of
StAR mRNA.
Rather, the clear-cut inhibitory effect of actinomycin D on the
Ca2+-induced increase in StAR mRNA steady state levels
suggested a direct relationship between StAR gene
transcription and Ca2+-stimulated pregnenolone production.
However, data from the literature on the requirement of transcription
for the acute steroidogenic response are conflicting. Indeed,
actinomycin D exerted little or no effect on ACTH-induced RNA synthesis
and corticosteroid production in rat adrenal quarters or dispersed
cells (44, 45), while it blocked to a variable extent RNA synthesis and
steroid production in bovine adrenal slices (44), in a mouse testicular
interstitial cell line (46), in rat testicular interstitial cells in
primary culture, and in Leydig cells (47, 48). More recently, Clark
et al. (49) demonstrated that actinomycin D potently
inhibits StAR mRNA accumulation and steroid production in MA-10 mouse
Leydig tumor cells. Our results with bovine glomerulosa cells are
consistent with the studies on testicular cells, MA-10 cells, and, more
importantly, bovine adrenal slices and strongly speak in favor of a
transcriptional activation exerted by Ca2+.
Increased [Ca2+]c up-regulates eukaryotic
gene and, in particular, immediate-early response gene transcription in
a variety of cell types (50). In rat aortic smooth muscle cells (51),
as well as in bovine adrenal glomerulosa (52) and fasciculata (53)
cells, Ang II was found to induce the expression of the early response
genes c-jun, c-fos and/or Jun B.
Whether these protooncogenes could be involved in the control of
StAR gene transcription remains to be determined. However,
at the present time, no activator protein-1 (AP-1)-responsive elements
have been found in the 1.3 kb of DNA upstream of the transcription
start site (39, 41). Interestingly, Sugawara et al. (39)
reported the presence within the StAR promoter of two Sp1-responsive
elements. This transcription factor has recently been shown to mediate
Ca2+-regulated gene expression in various cell types (50, 54). Alternatively, StAR gene transcription could be
promoted by a Ca2+-dependent relief of a blockade to
elongation in intragenic sites, as has been proposed for
c-fos transcription (50). Whatever the mechanism, our
results clearly demonstrate a dual site of action for the calcium
messenger in the acute activation of mineralocorticoid
biosynthesis: in addition to its previously described
intramitochondrial effects (35, 36, 37, 55), Ca2+ exerts
clear-cut rapid genomic actions that result in de novo
synthesis of StAR protein.
The second important finding of this work relates to the
antisteroidogenic action of ANP. While ANP did not affect the
[Ca2+]c and [Ca2+]m
changes elicited by either Ang II or a Ca2+ clamp, it
markedly reduced the Ca2+-supported increase in
pregnenolone formation, indicating that the inhibition occurred
downstream of the generation of the Ca2+ signal. By
contrast, ANP did not significantly affect pregnenolone synthesis in
control nonstimulated cells. These results are consistent with previous
studies suggesting that ANP may interfere with the early steps of
steroidogenesis (22, 23, 24, 56). Elliott and Goodfriend (23) reported that
the inhibitory effect of ANP on Ang II-elicited aldosterone synthesis
was less effective at high extracellular Ca2+
concentrations, suggesting that ANP may impair a process that is
stimulated by Ca2+. Our data demonstrate for the first time
that ANP decreases the availability of endogenous cholesterol to
the P450scc enzyme, by preventing
Ca2+-stimulated intramitochondrial cholesterol transfer.
Earlier studies on the hormonal regulation of cholesterol
mobilization in steroidogenic cells had demonstrated that ongoing
synthesis of StAR protein is required to facilitate cholesterol
translocation to the inner mitochondrial membrane. Our results show
that ANP completely abolished both the accumulation of mature StAR
protein within mitochondria and the increase in
[35S]methionine labeling of StAR protein in high
Ca2+-clamped glomerulosa cells. This finding indicates that
ANP inhibits the Ca2+-induced de novo synthesis
of StAR protein and is consistent with previous data reported by
Elliott et al. (57). Using two-dimensional gel
electrophoresis, these authors have shown that Ang II induces in bovine
adrenal glomerulosa cells the rapid appearance of a family of 28- to
30-kDa proteins, subsequently assumed to be StAR proteins (58), and
that cotreatment with ANP and Ang II markedly reduced the amount of
these proteins in mitochondria.
Interestingly, ANP abolished the Ca2+-elicited increase in
StAR mRNA levels. We therefore examined whether this inhibitory effect
of ANP was due to blockade of StAR gene transcription and/or
to a decrease in StAR mRNA stability. RNA decay experiments performed
in the presence of actinomycin D showed that ANP did not alter StAR
mRNA stability. Hence, it was likely that ANP counteracted the positive
effect of Ca2+ on StAR transcription. Further
evidence for the transcriptional control of the StAR gene by
Ca2+ and ANP was obtained using nuclear run-on assays. To
our knowledge, this is the first report relating increases in
intracellular Ca2+ to transcriptional induction of
StAR gene as well as an inhibitory effect of ANP on this
process. The process by which ANP exerts its negative control through
non-cGMP-mediated mechanisms remains to be elucidated. Interestingly,
DAX-1, a member of the nuclear-receptor superfamily of transcription
factors, has been recently shown to repress StAR gene
expression and steroidogenesis (59), and one could speculate that ANP
might induce DAX-1 expression in adrenal glomerulosa cells.
In summary, our results directly show that Ca2+ ion is a
potent physiological regulator of StAR gene expression in
bovine adrenal glomerulosa cells and that ANP impedes this
transcriptional activation, thus exerting its physiological inhibitory
action on aldosterone biosynthesis.
 |
MATERIALS AND METHODS
|
---|
Chemicals
Ionomycin was purchased from Calbiochem (Lucerne, Switzerland).
Human ANP [hANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] and urodilatin
[Thr-Ala-Pro-Arg-hANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] were obtained from Bachem (Bubendorf,
Switzerland). [3H]pregnenolone was purchased from
Amersham (Zurich, Switzerland), [
-32P]dCTP (3000
Ci/mmol) and [
-32P]UTP (800 Ci/mmol) from Hartmann
Analytic (Braunschweig, Germany).
[35S]methionine/cysteine labeling mix was purchased from
ICN Biomedicals GmbH (Eschwege, Germany). Antipregnenolone antiserum
was obtained from Biogenesis Ltd (Poole, UK). Win 19758 was purchased
from Farillon (London, UK). All other chemicals used were purchased
from Sigma (St. Louis, MO) or from Fluka (Buchs, Switzerland).
Bovine Adrenal Zona Glomerulosa Cell Culture and Treatments
Bovine adrenal glands were obtained from a local slaughterhouse.
Zona glomerulosa cells were prepared by enzymatic dispersion with
dispase and purified on Percoll density gradients (60). Primary
cultures of purified glomerulosa cells were established as described in
detail elsewhere (60) and kept in serum-free medium for 1 day before
experiments, which were performed on the third day of culture.
Cells cultured in 10 cm plastic Petri dishes (107 cells per
dish) or in 24-well plates (500,000 cells per well) were washed twice
with a modified Krebs-Ringer (NaCl, 136 mM;
NaHCO3, 5 mM; KH2PO4,
1.2 mM; MgSO4, 1.2 mM; KCl, 1.8
mM; EGTA, 0.2 mM; D-glucose, 5.5
mM; HEPES, 20 mM; pH 7.4) and preincubated at
37 C for 30 min in the same buffer. Ca2+-clamping was
performed at 37 C for 2 h, in the presence of 2 µM
ionomycin, 1 mM total extracellular Ca2+, and
0.2 mM EGTA, to achieve a [Ca2+]c
of 600700 nM (high-Ca2+ clamp), as described
elsewhere (37). Control cells were Ca2+-clamped in
Krebs-Ringer buffer lacking Ca2+, in the presence of
0.2 mM EGTA (low-Ca2+ clamp,
[Ca2+]c < 100 nM).
Thr-Ala-Pro-Arg-hANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), ANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), or membrane-permeant
cGMP analogs were added to the incubation medium during stimulation
with Ca2+ as indicated. In experiments designed to measure
cholesterol content in submitochondrial membranes, aminogluthetimide
(500 µM) was included in the incubation medium to inhibit
cholesterol side-chain cleavage. At the end of the incubation period,
the cells were scraped and sedimented at 200 x g for
15 min before subcellular fractionation or processing for total RNA
isolation as described hereafter.
Steroid Measurement
Aldosterone content in incubation media was measured by direct
RIA, using a commercially available kit (Diagnostic Systems
Laboratories, Webster, TX). For the assessment of pregnenolone
production, WIN 19758 (5 µM) was included in the
incubation medium to prevent conversion of pregnenolone into
progesterone. At the end of the incubation period, pregnenolone was
determined directly in the medium by RIA. Steroid production was
normalized and expressed per milligram cellular protein.
Measurement of
[Ca2+]c and
[Ca2+]m
[Ca2+]m was measured in bovine
glomerulosa cells transfected with a plasmid coding for mitochondrial
matrix-targeted aequorin as previously described in detail (34).
[Ca2+]c was determined in cells transfected
with the same plasmid lacking the targeting mitochondrial presequence
cDNA. Cells were transfected by the phosphate calcium precipitation
method using the CellPhect Transfection Kit (Pharmacia Biotech,
Zürich, Switzerland), according to the specifications of the
manufacturer.
Preparation of Submitochondrial Fractions and Cholesterol
Measurements
Glomerulosa cells were homogenized with a Potter-Elvejhem
homogenizer (1200 rpm, 35 strokes), in a 5 mM Tris-HCl
buffer, pH 7.4, containing 275 mM sucrose. The homogenate
was centrifuged at 200 x g for 15 min to remove large
debris and nuclei. Further centrifugation of the supernatant at
10,000 x g for 10 min yielded the mitochondria. The
mitochondrial pellet was washed twice at 8,000 x g
with the same buffer. Osmotically shocked adrenocortical mitochondria
were fractionated on a sucrose density gradient into OM, CS, and IM as
previously described (35). Protein was quantified using the Bio-Rad
protein microassay (Bio-Rad Laboratories, Richmond, CA) and BSA as a
standard. The cholesterol content of submitochondrial fractions (1520
µg protein for OM, 3040 µg protein for CS, and 80100 µg for
IM) was determined by a coupled cholesterol oxidase-peroxidase assay as
reported elsewhere (35).
SDS-PAGE
SDS-PAGE was performed according to Laemmli (61). Mitochondrial
proteins (10 µg/lane) were solubilized in sample buffer (60
mM Tris-HCl, pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 10%
glycerol, 0.01% bromophenol blue) and loaded onto a 12% SDS-PAGE
minigel (Mini Protean II System, Bio-Rad). Electrophoresis was
performed at 150 V for 1 h.
Blotting Procedure and Immunodetection
SDS-PAGE-resolved proteins were electrophoretically transferred
onto a nitrocellulose membrane (Schleicher & Schuell, Feldbach,
Switzerland) according to Towbin et al. (62). After
transfer, the membrane was incubated in a blocking buffer (PBS buffer
containing 0.4% Tween 20 and 5% nonfat dry milk) for 1 h at room
temperature, and then incubated with an antiserum generated by CovalAb
(Lyon, France) against a peptide fragment (amino acids 8898) of the
30- kDa StAR for 1 h in PBS/Tween 20 buffer (36). The membrane was
thoroughly washed with the same buffer (3 x 10 min), and then
incubated for 1 h with horseradish peroxidase-labeled goat
antirabbit IgG (CovalAb). The nitrocellulose sheet was then washed four
times for 15 min, and the antigen-antibody complex was revealed by
enhanced chemiluminescence, using the Western blotting detection kit
and Hyper-ECL film from Amersham.
Radiolabeling of Glomerulosa Cells and Immunoprecipitation of
StAR
For radiolabeling of cellular proteins, 3 x
106 glomerulosa cells, plated in 6-cm petri dishes, were
calcium-clamped for 2 h at 37 C, in the presence or in the absence
of ANP. The incubation medium (3 ml) contained
[35S]methionine-cysteine (300 µCi). At the end of the
labeling period, cells were washed three times with ice-cold PBS and
lysed in RIPA buffer (10 mM sodium phosphate, pH 7.4,
containing 150 mM NaCl, 1% Triton, 1% sodium
deoxycholate, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml
aprotinin, and 1 µg/ml leupeptin). The lysate was cleared by
centrifugation for 5 min at 12,000 x g at 4 C. The
StAR antiserum was added at a 1:100 dilution to aliquots of the
supernatant (200 µl,
100 µg protein), which were then gently
rocked for 2 h at 4 C, before being incubated for 30 min with
Protein-A Sepharose beads (Pharmacia Biotech AG, Dubendorf,
Switzerland). Immunoprecipitates were pelleted, washed four times with
RIPA buffer and analyzed by SDS-PAGE and autoradiography.
RNA Isolation and Northern Blot Analysis
Glomerulosa cell total RNA was extracted using the RNAgents kit
(Promega, Zurich, Switzerland) according to the instructions of the
manufacturer. This system consistently yields 5080 µg total
RNA/107 cells. For Northern blot analysis, 1520 µg RNA
were size-fractionated on a 1% formaldehyde agarose gel,
vacuum-transferred onto Nytran membranes (Schleicher & Schuell) and
fixed by UV cross-linking. The integrity of the 18 S and 28 S RNA was
checked by ethidium bromide staining of the gel. Hybridization was
performed using the previously cloned 1.5-kb mouse StAR cDNA (16). The
cDNA was labeled with [
32P]dCTP using the Rediprime
random primer labeling kit from Amersham. Northern blots were
prehybridized in Rapid Hybridization Buffer (Amersham) at 65 C for 30
min. The
32P-labeled probe (specific activity: 2 x
106 cpm/ng DNA) was then added and the incubation was
continued for 2 h at 65 C. Blots were washed for 5 min and 15 min
successively at room temperature in 2 x saline sodium citrate
(SSC), 0.1% SDS, and then for 15 min in 1 x SSC, 0.1% SDS. The
final wash was performed at 65 C for 15 min in 1 x SSC, 0.1%
SDS. RNA-cDNA hybrids were visualized on Hyperfilms (Amersham) after a
12- to 24-h exposure period. Blots were stripped and reprobed with
mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Ambion,
Lugano, Switzerland) to assess RNA loading.
Nuclear Run-on Assays
Nuclear run-on was performed according to described protocols
(63). Bovine glomerulosa cells (5 x 107 cells per
treatment) were subjected to a low- or high-calcium clamp in the
presence or in the absence of hANP for 2 h. Transcription
reactions were then carried out on isolated nuclei using 150 µCi
32P-labeled UTP and 1 mM ATP, CTP, and GTP
(Pharmacia Biotech AG, Dübendorf, Switzerland) in reaction buffer
(5 mM Tris-HCl, pH 8, 2.5 mM MgCl2,
150 mM KCl) at 30 C for 30 min. Nuclear RNA was isolated
and hybridized for 36 h at 65 C to Hybond-N+-charged
nylon membranes (Amersham) on which 5 µg of a Bluescript plasmid
containing a 1.5-kb StAR cDNA insert, 5 µg of a Bluescript plasmid
containing a 250-bp mouse ß-actin insert (Ambion), or 5 µg of empty
Bluescript plasmid had been immobilized. Membranes were washed and
exposed to hyperfilms for visualization.
Analysis of Data
Results are expressed as means ± SEM. The mean
values were compared by ANOVA using Fishers test. A value of
P < 0.05 was considered as statistically
significant. Quantification of immunoblots and autoradiograms was
performed using a Molecular Dynamics (Sunnyvale, CA) Computing
Densitometer.
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to Liliane Bockhorn, Walda Dimeck, and
Gisèle Dorenter for their excellent technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Nadia Cherradi, Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail:
nadia.cherradi{at}diogenes.hcuge.ch
This work was supported in part by Swiss National Science Foundation
Grants 31.4217894 (to A.M.C.) and 32.49297.96 (to M.F.R.), by NIH
Grant HD-17841 (to D.M.S.) and by grants from the Ciba-Geigy
Jubiläumsstiftung and the Sandoz Foundation. M.F.R. is a
recipient of a grant from the Professor Max Cloëtta
Foundation.
Received for publication January 16, 1998.
Revision received March 11, 1998.
Accepted for publication March 17, 1998.
 |
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