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INTRODUCTION |
Aldosterone synthase is the product of the CYP11B2 gene
and is responsible for the final step of aldosterone synthesis in most
mammalian adrenal glands (1). This key regulatory enzyme is under the
control of many factors including angiotensin II (AII),1 adrenocorticotropin
(ACTH), and changes in blood Na+ and K+
concentrations (1). The hamster adrenal CYP11B2 gene has
been cloned and its promoter activity studied (2, 3). In transfected human adrenocortical carcinoma NCI-H295 cells, the first 161 base pairs
of the 5'-flanking region of the hamster CYP11B2 were shown to be sufficient for maximal promoter activity and for the stimulatory effect of AII, KCl, and Bt2cAMP (4). In comparison, the
first 129 base pairs of the 5'-flanking region of the human
CYP11B2 gene were also shown to be responsive to the
stimulatory action of AII, KCl and dibutyryl cyclic adenosine
3':5'-monophosphate (Bt2cAMP) (5).
The involvement of protein kinase C (PKC) in the expression of adrenal
steroidogenic enzymes has been studied using activators and inhibitors
of PKC. For example, 12-O-tetradecanoylphorbol-13-acetate (TPA) has been reported to increase the mRNA levels of
3-
-hydroxysteroid dehydrogenase (6) and cytochrome P450
21-hydroxylase in the NCI-H295 cell line (7, 8). In addition, phorbol
esters have been shown to decrease the mRNA levels of cytochrome
P450 17
-hydroxylase (7) and cytochrome P450 side chain cleavage (7)
and to abolish the enhancing effect of BAYK 8644 on cytochrome P450
11
-hydroxylase (P450C11) and cytochrome P450 aldosterone synthase
(P450aldo) (9). Steroidogenesis is also affected by inhibition of PKC. For instance, staurosporine has been reported to stimulate
steroidogenesis in Y-1 cells (3, 10), to elevate the levels of mRNA
of cytochrome P450 side chain cleavage and P450C11 (10), and to
stimulate the activity of the hamster CYP11B2 promoter in
transfected Y-1 cells (3). We have reported that GF109203X, a specific
PKC inhibitor, has stimulated the output of aldosterone and the
activity of the hamster CYP11B2 promoter in
NCI-H295-transfected cells (4). In addition, staurosporine has been
reported to reverse the inhibitory effect of TPA on aldosterone
production in rat adrenal glomerulosa cells and to enhance the
stimulatory effects of AII and K+, but not of ACTH, in
these cells (11).
PKC comprises several isoforms of serine/threonine kinases that can be
divided in three families (12-14). Members of the conventional family
(PKC
, -
1, -
2, and -
) are calcium-dependent and
are activated by phosphatidylserine and diacylglycerol. Members of the
novel family (PKC
, -
, -
, and -
) do not require calcium for
their activity but are activated by diacylglycerol, whereas members of
the atypical family (PKC
and -
/
) do not respond to calcium or diacylglycerol.
Current knowledge supports the interpretation that PKC is involved in
the regulation of steroidogenesis at least at two separate enzymatic
steps of aldosterone synthesis, namely the transformation of
cholesterol to pregnenolone (10, 15) and the conversion of
deoxycorticosterone to aldosterone (3, 4). However, the role of
individual PKC isozymes in the regulation of aldosterone synthesis
remains unknown. We have addressed this question by investigating the
role of PKC isoforms on the regulation of the hamster
CYP11B2 gene promoter activity. We show here that
cotransfection of NCI-H295 cells with constitutively active (CA) or
dominant negative (DN) mutants of PKC isoforms, along with the
CYP11B2 gene promoter, resulted in a differential regulation
of promoter activity of conventional (
) and atypical (
) PKC.
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EXPERIMENTAL PROCEDURES |
Tissues--
Adult male Syrian Golden hamsters (120 ± 10 g) and 2-month-old Long Evans male rats were purchased from
Charles River Inc. (St. Constant, Quebec, Canada) and kept on Purina
rat chow and tap water ad libitum. Animals were sacrificed
in accordance with the ethical standards of our local institutional
review committee. The adrenal glands were removed and freed of fatty
tissue. The zona glomerulosa was separated from the zona
fasciculata-reticularis and the medulla by the method of Giroud
et al. (16). Three adult adrenal glands were obtained from
50-, 52-, and 55-year-old renal transplant donors, in accordance with
the Human Subject Review Committee of our institution.
Reagents--
The PKC inhibitors GF109203X and Gö6976 were
purchased from Calbiochem, Cedarlane Laboratories Ltd. (Hornsby,
Ontario, Canada). TPA, AII, and Bt2cAMP were obtained from
Sigma-Aldrich (St. Louis, MO).
Western Blot Analysis of PKC Isoforms--
NCI-H295 cells were
harvested in Laemmli's sample buffer (17), and tissues were
homogenized in the same buffer. Samples were passed through a 26-gauge
needle, heated in boiling water for 10 min, and centrifuged
(12,000 × g) for 10 min. Triton X-100-soluble and
-insoluble fractions of NCI-H295 cells or hamster adrenal zona
glomerulosa were prepared according to the technique described by
Garcia-Paramio et al. (12). Thin human adrenal slices were obtained using a Stadie-Riggs tissue slicer (Thomas Scientific, St.
Laurent, Quebec, Canada). The first slices consisted of the adrenal
capsule and the zona glomerulosa, and the next slices consisted of the
zona fasciculata (18). Contamination of the zona glomerulosa by the
zona fasciculata was estimated to be ~20%, based on P450
17
-hydroxylase expression, as determined by Western blotting
analysis, using the same quantity of proteins from each zone
preparation (data not shown). Proteins were separated by electrophoresis on 7.5% polyacrylamide gel and analyzed by Western blotting as described previously (19, 20) using the following polyclonal antibodies: anti-rabbit PKC
, anti-rabbit PKC
1
(Sigma-Aldrich), anti-rabbit PKC
2, anti-mouse PKC
, anti-rabbit
PKC
, and anti-rabbit PKC
(Sigma-Aldrich), anti-mouse PKC
and
anti-mouse PKC
(Transduction Laboratories, Lexington, KY), and
anti-rabbit PKC
(N-17) (Santa Cruz Biotechnology Inc., Santa Cruz,
CA). Immunoreactive proteins were detected using ECL+ light emitting
reagents (Amersham Pharmacia Biotech UK Ltd, Little Chalfont,
Buckinghamshire, United Kingdom). Autoradiograms were observed by
exposing the blots to Kodak X-Omat RP films.
In Situ Detection of PKC Isoforms--
Adrenal glands were
excised from three different animals. Human adult adrenal glands were
also used. The glands were fixed in buffered neutral formalin solution
for 24 h. The fixed adrenal glands were dehydrated by successive
treatments with graded aqueous ethanol, cleared in toluene, and
embedded in paraffin. Five to seven 5-µm-thick sections were prepared
according to the usual histologic procedures. Sections were freed of
paraffin, hydrated, and treated with NH4Cl in 50 mM PBS (20 min) to block aldehydic groups. After two
washes, tissue sections were incubated for 2 h at room temperature
with the respective anti-PKC
, -
, or -
antibodies (diluted in
PBS containing 1% bovine serum albumin), and then washed twice.
Tissues sections were then incubated for 30 min with a
fluorescein-conjugated goat anti-rabbit IgG (Roche Molecular
Biochemicals, Laval, Quebec, Canada) diluted 1/50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1%
phenylenediamine (21). Control sections were incubated with the second
antibody only. Paraffin-embedded sections were examined with a Reichert
Polyvar 2 microscope equipped for epifluorescence.
RNA Analysis--
Total RNA was isolated from NCI-H295 cells
using RNeasy® (Qiagen Inc., Mississauga, Ontario, Canada)
and treated with DNase I. Two hundred ng total RNA was reversed
transcribed and amplified using a Titan One Tube RT-PCR Kit (Roche
Diagnostics, Laval, Quebec, Canada) following the manufacturer's
protocol. A 392-base pair (bp) fragment corresponding to exons 1 and 2 of P450C11 and P450aldo was amplified using the following primers:
5'-ATGGCACTCAGGGCAAAGGCA-3' (sense) and 5'-CAAGAACACGCCACATTTGTGC-3'
(antisense) (7). The PCR products were then digested with
BglI (P450aldo-specific) and separated on a 2% agarose gel
containing ethidium bromide. GAPDH was used as an internal control; a
239-bp fragment containing a BglI restriction site was
amplified using 5'-CATCCTGGGCTACACTGAGC-3' (sense) and
5'-TCTCTCTTCCTCTTGTGCTC-3' (antisense) primers. The BglI
digestion distinguished P450C11 from P450aldo by cleaving the 392-bp
P450aldo fragment to 307- and 85-bp fragments; the conversion of the
GAPDH 239-bp fragment to 176- and 63-bp fragments demonstrates complete
BglI digestion. The marker pBR322 DNA-MspI digest
was purchased from New England Biolabs Inc. (Beverly, MA).
Generation of PKC Mutants--
CA- and DN-PKC mutants were
engineered by point mutation using a PCR technique (22). In the case of
the CA mutants, a specific alanine residue in the pseudo-substrate
region was exchanged for a glutamic acid residue (PKC
A25E, PKC
A159E, PKC
A148E, PKC
A119E). The pseudo-substrate region refers
to the consensus sequence XRXX(S/T)XRX found in the
phosphorylation sites of prominent PKC substrates. However, in the
pseudo-substrate region of PKC, one alanine residue replaces a serine
or threonine residue normally found in the substrate motif. It is
generally thought that the pseudo-substrate region interacts with the
catalytic (substrate binding) site of PKC and maintains the enzyme in
an inactive state (23). In the case of the DN mutants, a critical
lysine in the 5'-(pyro)-triphosphates of adenosine binding site was
exchanged for an alanine residue (PKC
K368A, PKC
75 437A, PKC
K409A, PKC
K281A) (24, 25). The pcDNA3 vector (Invitrogen,
Carlsbad, CA) was used as the expression vector.
Deletion Plasmids of the CYP11B2 Promoter--
The modified pCAT
basic vector (Promega, Madison, WI) was used to construct
CYP11B2 gene deletion plasmids as we have described previously (3, 4). The modifications consisted of the removal of an
AP-1 consensus sequence and of an AP-1 like sequence. In this study
83-,
102-,
134-,
167-,
328-,
350-,
486-, and
2600-CYP11B2-CAT deletion plasmids were used (4). The
sequence 5'-CAGGGACAGC-3' of the
102-CYP11B2 deletion
plasmid was changed to 5'-tggtacccat-3' to produce the
102 mutant
(mut-102).
Cell Cultures--
NCI-H295 cells were maintained in Dulbecco's
modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 1%
ITS Premix (Becton Dickinson Labware, Bedford, MA), 2% UtroSer SF (Bio
Sepra S.A., Villeneuve la Garenne, France), 200 µg/ml streptomycin, and 200 units/ml penicillin G. The cells were grown at 37 °C under 95% air, 5% CO2.
Transient Transfections and CAT Assays--
Transient
transfections were performed in NCI-H295 cells as described previously
(4). NCI-H295 cells (5 × 105) were seeded into
six-well tissue culture plates and grown until 80-90% confluence. The
cells were transiently transfected for 5 h with plasmid
constructions using the SuperFect transfection reagent (Qiagen),
according to the manufacturer's protocol. After transfection, the
cells were incubated with medium containing 2% UtroSer SF for 16 h to allow recovery and expression of foreign DNA. Media were changed
to low serum media (Dulbecco's modified Eagle's medium/Ham's F-12
containing 0.1% UtroSer SF), and the cells were incubated for 16 h in the presence/absence of AII (10
7
M), KCl (16 mM), Bt2cAMP (1 mM), or TPA (0.16 µM). Cell lysates were
prepared by freeze-thawing, and CAT assays were performed using
[14C]chloramphenicol as described previously (4). In
experiments where CAT activity was too high, the assays were performed
with smaller amounts of cell proteins to keep the rate of acetylation linear. Quantification was done using a Storm 860 laser scanner instrument (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
Immunodetection of PKC--
Western blotting analysis was used to
determine which PKC isoforms were expressed in hamster adrenal glands
and in NCI-H295 cells. Adrenal glands of humans and rats were also
included in our analysis to determine the similarity of PKC expression
among species. Rat brain preparations were used as control (Fig.
1, left lane).
Results showed that the PKC
, -
, and -
isoforms were present in
hamster adrenal inner zones and zona glomerulosa, whereas PKC
1,
-
2, -
, -
, -
, and
were absent. PKC
, -
, -
, and
-
and low levels of PKC
were detected in NCI-H295 cells, whereas
PKC
1, -
2, -
, and -
were absent. PKC
, -
, and -
were revealed in rat zona glomerulosa and inner zones, in human zona glomerulosa and zona fasciculata-reticularis. Very low levels of PKC
and -
were also detected in adult human adrenal preparations. PKC
was present in the human adrenal zona fasciculata and in very low
levels in rat brain. The ubiquitous presence of PKC
, -
, and -
in the adrenal cortex of species studied is indicative of their
physiological importance in this tissue. Indirect immunofluorescence was also used to investigate the expression of PKC isozymes in paraffin
sections of the hamster and the human adrenal cortex. Results showed
(Fig. 2) that PKC
, -
, and -
were
present in hamster and human adrenal zona glomerulosa and, to a lesser
extent, in the zona fasciculata.

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Fig. 1.
Immunoblot of adrenal gland preparations
probed with anti-PKC antibodies. The zona glomerulosa
(ZG) of rat and hamster (Ham) adrenal glands were
separated from inner zones (IZ) containing the medulla.
Adult human adrenal zona glomerulosa and zona fasciculata
(ZF), NCI-H295 cells (C), and rat brain
(Rb) were also used. Tissues were homogenized, and 50 µg
of proteins of each preparation were used for immunodetection with
antibodies directed against PKC , - 1, - 2, - , - , - ,
- , - , and - , respectively.
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Fig. 2.
Indirect immunofluorescence detection of PKC
in micrographs of paraffin sections of hamster and human adrenal
glands. A, the fluorescein-conjugated antibody revealed
PKC in the zona glomerulosa and in some cells of the zona
fasciculata. B, PKC was revealed in zona glomerulosa and
in some cells of the zona fasciculata. C, PKC was
revealed in the zona glomerulosa and in some cells of the zona
fasciculata. Hum, human; Ham, hamster.
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We have analyzed the activation state of PKC in NCI-H295 cells and in
hamster adrenal zona glomerulosa. Activated PKCs associate with
cellular components and their distribution can be revealed in detergent
Triton X-100-insoluble fraction (26). Furthermore, activation of PKC
isoforms makes them susceptible to dephosphorylation and degradation
(27). In NCI-H295 cells, PKC
and -
isoforms were found in the
Triton X-100-soluble (nonactivated) fraction whereas the
and
isoforms were distributed in the Triton X-100-soluble and -insoluble
fractions (Fig. 3). Similarly, in hamster
adrenal zona glomerulosa, PKC
was also found in both the Triton
X-100-soluble and -insoluble fraction. Furthermore, PKC
and -
were detected in the detergent-insoluble fraction. These data suggest
that, under basal conditions, PKC
, -
, and -
are activated in
hamster adrenal zona glomerulosa and that PKC
and -
are also
activated in NCI-H295 cells. In contrast, PKC
and -
were found to
be mostly associated with the Triton X-100-soluble fraction in NCI-H295 cells and therefore presumably nonactivated.

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Fig. 3.
Determination of the activation state of PKCs
in NCI-H295 cells and in hamster adrenal zona glomerulosa.
NCI-H295 (H295) cells and hamster adrenal zona glomerulosa were
fractionated into Triton X-100-soluble (S) and Triton
X-100-insoluble (I) fractions. Equivalent samples of each
fraction were separated by electrophoresis and then subjected to
immunoblot assays with antibodies specific to each PKC. Rat brain
(Rb) was used as control.
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TPA Inhibition of P450aldo mRNA Expression in NCI-H295
Cells--
The presence of phorbol ester/diacylglycerol
(DAG)-sensitive PKC in NCI-H295 cells led us to investigate the effect
of TPA on the P450aldo mRNA expression. This was done by reverse
transcriptase-PCR performed as described under "Experimental
Procedures." Fig. 4A shows
the PCR-amplified products. BglI digestion distinguished P450aldo from P450C11 bands, and the complete BglI digestion
was demonstrated by GAPDH (Fig. 4, B and C). TPA
inhibited basal and stimulated P450aldo mRNA expression by AII,
Bt2cAMP, or KCl. Although TPA inhibited AII- and
KCl-stimulated P450C11 mRNA expression, it did not inhibit its
basal or Bt2cAMP-stimulated expression.

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Fig. 4.
Effect of TPA on the mRNA levels of P450
11 -hydroxylase (C11) and P450
aldosterone synthase (AS) in NCI-H295 cells.
NCI-H295 cells were incubated for 16 h without (control,
Ctr) or with AII (10 7
M), Bt2cAMP (1 mM) or KCl (16 mM), in the absence (-) or presence (+) of TPA (0.16 µM). Two hundred ng of total RNA were reverse transcribed
using specific primers to both P450C11, P450aldo, and to GAPDH, and
PCR-amplified for 30 cycles. A, the PCR products were
separated through a 2% agarose gel containing ethidium bromide.
B and C, the PCR products from two independent
experiments were digested with BglI. M
corresponds to DNA markers of indicated molecular sizes.
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Effect of TPA on the Hamster CYP11B2 Promoter Activity in
Transfected NCI-H295 Cells--
NCI-H295 cells transiently transfected
with the hamster
2600-CYP11B2-CAT deletion plasmid were
exposed to TPA and CAT activity determined as before (4). Results
showed (Fig. 5) that TPA decreased CAT
activity. Importantly, TPA abolished the stimulatory effects of AII,
Bt2cAMP, or KCl, suggesting that activation of phorbol
ester/DAG-sensitive PKC has a negative effect on the regulation of the
CYP11B2 promoter activity. The KCl concentration used for stimulation in the above experiments was 16 mM. Although
less efficient, lower concentrations of KCl (4-7 mM) were
still able to stimulate the promoter activity (data not shown).

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Fig. 5.
Effect of TPA on the hamster
CYP11B2 promoter activity. Transfected NCI-H295
cells with the hamster 2600-CYP11B2-CAT deletion plasmid
were incubated with AII (10 7 M),
TPA (0.16 µM), Bt2cAMP (cAMP) (1 mM), or KCl (16 mM). Incubations in the
presence of either AII and TPA, cAMP and TPA, or KCl and TPA were also
done. Results shown are measurements of three different experiments
(mean ± S.E.) performed in triplicate.
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Effect of Gö6976 on the Hamster CYP11B2 Promoter Activity in
Transfected NCI-H295 Cells--
The inhibitor Gö6976
specifically blocks the action of conventional PKCs (28). This
characteristic was used to discriminate between the action of the
unique conventional PKC
and that of other DAG-dependent
PKCs expressed in hamster adrenal and NCI-H295 cells. At
10
6 M Gö6976 enhanced CAT
activity (percentage of acetylation: mean ± S.E., control
8.43 ± 1.37; Gö6976: 16.51 ± 1.43, n = 3, performed in triplicate). These observations provided further
evidence that PKC
was involved in suppression of the hamster
CYP11B2 promoter activity.
Effect of CA-PKC
and DN-PKC
Mutants on the CYP11B2 Promoter
Activity in Transfected NCI-H295 Cells--
To confirm the suppressive
effect of PKC
on the expression of CYP11B2,
cotransfection experiments were performed using CA-PKC
and DN-PKC
mutants. Results indicated that CYP11B2 promoter activity was inhibited when cotransfected with CA-PKC
(Fig.
6A). The inhibition was also
observed when the cells had been transfected with 0.5 or 1 µg of
PKC
-containing plasmid (Fig. 7). Of
interest, expression of CA-PKC
also inhibited, in a plasmid
concentration-dependent manner, the stimulatory effect of
AII (Fig. 7). In contrast, expression of DN-PKC
enhanced promoter
activity (Fig. 6A).

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Fig. 6.
Effect of CA- and DN-PKC mutants on the basal
CYP11B2 promoter activity. NCI-H295 cells
were cotransfected with the hamster 2600-CYP11B2-CAT
deletion plasmid and pcDNA3 or cotransfected with pcDNA3
harboring different mutated PKC DNAs. Results shown are mean ± S.E. of three to five experiments performed in triplicate
(n = 5 for CA- , DN- , DN- ; n = 4 for CA- , DN- , DN- ; n = 3 for CA- and
CA- ). A: PKC and PKC mutants; B: PKC
and PKC mutants.
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Fig. 7.
CA-PKC decreases the
hamster 2600-CYP11B2-CAT expression in transfected
NCI-H295 cells. NCI-H295 cells were cotransfected with 2 µg of
the hamster 2600-CYP11B2-CAT deletion plasmid and 1 µg
of pcDNA3 per well (0). In other wells, increasing
quantities (0.05-1 µg) of pcDNA3 harboring the mutated CA-PKC
DNA was substituted to equivalent quantities of pcDNA3. Results
shown are measurements of three different samples (mean ± S.E.).
Filled columns, treated angiotensin II
(10 7 M).
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CA-PKC
also abolished the stimulatory effects of KCl and
Bt2cAMP on CAT activity (Fig.
8A), whereas DN-PKC
increased the basal promoter activity (Fig. 6A) and did not
prevent the stimulatory effects of AII, KCl and Bt2cAMP
(Fig. 8A). The fact that the DN mutant enhanced rather than
decreased these effects adds weight to the hypothesis that PKC
suppresses the hamster CYP11B2 promoter activity.

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Fig. 8.
Effects of CA- and DN-PKC mutants on the
CYP11B2 promoter activity in transfected NCI-H295
cells stimulated by AII, Bt2cAMP, or KCl. NCI-H295
cells were cotransfected with the hamster 2600-CYP11B2-CAT
deletion plasmid and pcDNA3 or pcDNA3 harboring CA-PKC or
DN-PKC DNAs (A) or pcDNA3 or pcDNA3 harboring
CA-PKC or DN-PKC DNAs (B). C, pcDNA3 or
pcDNA3 harboring CA-PKC or DN-PKC DNAs. Cells were incubated
for 16 h with AII (10 7 M),
Bt2cAMP (cAMP) (1 mM), or KCl (16 mM). Results are mean ± S.E. of six independent
experiments performed in triplicate for cells cotransfected with
pcDNA3 and three experiments for cells cotransfected with
PKCs.
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Effect of CA-PKC
and DN-PKC
Mutants on the CYP11B2 Promoter
Activity in Transfected NCI-H295 Cells--
The fact that PKC
was
present in NCI-H295 cells and, to a lesser extent in hamster adrenal
zona glomerulosa (Figs. 1 and 2), led us to address whether this PKC
plays a role on the hamster CYP11B2 promoter activity in
cotransfected NCI-H295 cells. We found that CA-PKC
had an inhibitory
effect on CAT activity (Figs. 6A and 8B) similar
to that of the CA-PKC
mutant. In contrast, DN-PKC
had a
stimulatory effect (Figs. 6A and 8B). Moreover, the stimulatory effects of AII, KCl, and Bt2cAMP were
blocked in cells transfected with CA-PKC
but not with DN-PKC
(Fig. 8B). These results indicated that PKC
could also be
involved in the regulation of the CYP11B2 promoter activity
in NCI-H295 cells.
Effect of CA-PKC
and DN-PKC
Mutants on the CYP11B2 Promoter
Activity in Transfected NCI-H295 Cells Stimulated by AII, KCl, and
Bt2cAMP--
The role of atypical PKC
, which is
expressed in the hamster adrenal zona glomerulosa and NCI-H295 cells
(Fig. 1), on the hamster CYP11B2 promoter activity was
investigated in the next series of experiments. In contrast to results
obtained with CA-PKC
, CA-PKC
stimulated the CYP11B2
promoter basal activity (Figs. 6B and 8C). In
addition, cell stimulation with AII or KCl were not abolished in
cotransfection experiments with CA-PKC
; the effect of
Bt2cAMP, however, was abolished. DN-PKC
increased the basal activity of the promoter and the stimulatory effects of AII, KCl,
and Bt2cAMP were observed (Figs. 6B and
8C).
Cellular Distribution of CA-PKC
and DN-PKC
--
The aim of
the next series of experiments was to obtain evidence of the state of
activation of PKC
, CA-PKC
, and DN-PKC
by comparing their
distribution in Triton X-100-soluble and -insoluble fractions in
transfected NCI-H295 cells stimulated by AII or TPA. Fig.
9 showed that wild type PKC
was mainly
present in the soluble fraction whereas faint protein bands were found
in the insoluble fraction. The pattern of PKC
distribution was not
altered when cells were exposed to AII. In contrast, TPA induced an
increase of PKC
in the insoluble fraction. Although speculative, the
slow and fast moving bands observed in the soluble and insoluble
fractions are presumably phosphorylated and dephosphorylated protein,
respectively, which is in agreement with results reported by
Garcia-Paramio et al. (12). Marked differences were observed
with preparations of cells transfected with CA-PKC
. In a situation
where CAT activity was inhibited, the insoluble fraction of these cells
contained large amounts of immunoreactive protein migrating faster than the single band detected in the soluble fraction, suggesting a translocation of PKC
within the cell and presumably
dephosphorylation of the protein. The presence of AII did not change
this profile. A similar profile was observed with TPA except for the
appearance of a small fast migrating band in the soluble fraction. Fig.
9 also shows results obtained in the case of the DN form of PKC
in a
situation where CAT activity was not inhibited. Without treatment, the
main immunoreactive band found was a slow migrating protein in the
soluble fraction. When the cells were treated with AII or TPA, a second
fast migrating band was revealed in the soluble and in the insoluble
fractions in addition to the main upper band. These data suggested
that, upon stimulation, this mutant PKC was able to translocate and was
presumably dephosphorylated.

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Fig. 9.
Detergent analysis of the distribution of
wild type (Wt) and PKC
mutants in stimulated transfected NCI-H295 cells. NCI-H295
cells transfected with wild type PKC , CA-PKC , or DN-PKC were
fractionated into Triton X-100-insoluble (I) and -soluble
(S) fractions. Cells were incubated without (Ctr)
or with TPA (0.16 µM) or AII
(10 7 M). Equivalent amounts of
proteins from each fraction were separated by electrophoresis. PKC
was analyzed by Western blotting.
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Effect of TPA on the Activity of Different Deletion CYP11B2
Plasmids--
Eight different deletion plasmids were used (listed
under "Experimental Procedures") to locate the promoter region
responsible for TPA inhibition of the hamster promoter
CYP11B2 gene. The basal, AII-, KCl-, and
Bt2cAMP-stimulated activity profiles of the different deletion plasmids were similar to those reported previously by us (4).
TPA slightly inhibited the enhancing effects of AII, KCl, and
Bt2cAMP on the activity of the
83 deletion plasmid. However, the enhancing effects of AII, KCl, and Bt2cAMP on
the activity of the
102 and longer CYP11B2 deletion
plasmids was abolished by TPA (Fig.
10).

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Fig. 10.
TPA inhibition of the hamster
CYP11B2 promoter activity stimulated by AII,
Bt2cAMP, and KCl. NCI-H295 cells were transfected with
eight different (from 83- to 2600)-CYP11B2-CAT deletion
plasmids. Transfected cells were incubated with AII
(10 7 M), KCl (16 mM),
or Bt2cAMP (cAMP) (1 mM) alone or
combined with TPA (0.16 µM). Data are measurements of
three different samples (mean ± S.E.).
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Effect of CA-PKC
, -
, -
, and -
on the Activity of
Different Deletion CYP11B2 Plasmids--
CA-PKC
, -
, and -
were able to prevent the enhancing effects of AII, KCl, and
Bt2cAMP on the activity of the
102-CYP11B2 deletion plasmid (Fig. 11A)
in cotransfection experiments, as observed in the case of TPA-treated
cells. In contrast, the enhancing effect of CA-PKC
on the basal
promoter activity was observed in cotransfection experiments using
102-CYP11B2 (Fig. 11B). In addition, promoter stimulation with AII or KCl was not abolished in cotransfection experiments with CA-PKC
, whereas the effect of Bt2cAMP
was prevented.

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Fig. 11.
Inhibition of the 102-CYP11B2
promoter activity by the constitutively active PKC mutants.
NCI-H295 cells were cotransfected with the hamster
102-CYP11B2-CAT deletion plasmid and pcDNA3 or
pcDNA3 harboring CA-PKC , CA-PKC , CA-PKC , or CA-PKC
DNAs. Cells were incubated for 16 h with or without AII
(10 7 M), KCl (16 mM),
or Bt2cAMP (cAMP) (1 mM). Results
shown are (mean ± S.E.) from one experiment (A) and
two experiments (B) performed in triplicate. A:
DAG-dependent PKC- , , and ; B: atypical
PKC .
|
|
Mutation of the Ad2 cis-Element--
Bases
95 to
86 of the Ad2
cis-element (3) in the hamster
102-CYP11B2-CAT
deletion plasmid, were all mutated (mut-102). As shown in Fig.
12, this mutation resulted in the
decrease of basal and stimulated promoter activities. The effect of
Bt2cAMP and AII on mut-102 were partially inhibited by TPA
while that of KCl was completely inhibited. TPA completely abolished
the stimulatory effect of the three regulators on wild type
102
deletion plasmid (Fig. 12).

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Fig. 12.
Effect of mutating the
Ad2-cis-element on the CYP11B2
promoter activity. NCI-H295 cells were transfected with the
hamster 102-CYP11B2-CAT ( 102) or the
102-mutated CYP11B2-CAT deletion plasmid
(mut-102); bases 95 to 86 comprised in the Ad2
cis-element were all mutated. Cells were incubated for
16 h without (control, Ctr) or with AII
(10 7 M), Bt2cAMP
(cAMP) (1 mM), or KCl (16 mM) in the
absence or presence of TPA (0.16 µM). Data shown are
measurements of three different samples (mean ± S.E.).
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DISCUSSION |
Previous studies have shown that PKC are involved in the control
of several adrenal steroidogenic genes (4-10). Evidence was derived
from experiments using PKC activators, phorbol esters, and various PKC
inhibitors. The present work was designed to determine which PKCs were
involved in the control of the hamster CYP11B2 gene
expression. We report that DAG-dependent PKC can suppress whereas the atypical PKC
can enhance the basal expression of this
gene in transfected NCI-H295 cells.
High levels of expression of PKC
, low levels of PKC
, and very low
levels of PKC
were detected (Western blotting) in the hamster
adrenal zona glomerulosa. Indirect immunofluorescence analysis
confirmed the presence of PKC
, -
, and -
in the hamster and
human adrenal zona glomerulosa and in lower levels in the zona
fasciculata. In comparison, these isoforms were found in the zona
glomerulosa of rat and in NCI-H295 cells. Our results are in agreement
with previous reports indicating that the rat adrenal zona glomerulosa
expressed PKC
and -
(28). PKC isoforms
,
, and
were
also commonly expressed in the human adrenocortical NCI-H295 cell line
(Fig. 1) and in the mouse adrenal cell line Y-1 (15). The expression of
PKC
, -
, and -
in the zona glomerulosa of all adrenal tissues
studied is indicative of their specificity and functional importance in
this zone. Moreover, the fact that under basal conditions PKC
, -
,
and -
were partially activated (as determined by their presence in
the Triton X-100-insoluble fraction) in hamster adrenal zona
glomerulosa was also indicative of their functional importance in these
adrenocortical cells.
We have shown for the first time that the PKC activator TPA inhibited
basal, AII-, Bt2cAMP-, and KCl-stimulated P450aldo mRNA level in NCI-H295 cells, thus validating this model for studying hamster CYP11B2 promoter activity. We also observed that TPA
did not inhibit basal and cAMP-stimulated P450C11 mRNA levels,
suggesting a differential control of mRNA expression of the
CYP11B1 and CYP11B2 genes by phorbol
ester/DAG-sensitive PKC in NCI-H295 cells.
In the present study, we have concentrated our efforts to elucidate the
role of the three PKC isoforms
,
, and
present in the hamster
adrenal zona glomerulosa, on the expression of the hamster
CYP11B2 promoter activity. We have found that the PKC
activator TPA inhibited the enhanced expression of the hamster CYP11B2 by AII, Bt2cAMP, or KCl, suggesting that
phorbol ester/DAG-dependent PKC were involved in inhibition
of the promoter activity.
We have used a combined approach to discriminate between the role of
PKC
and -
, present in hamster zona glomerulosa and in NCI-H295
cells, and of PKC
also present in NCI-H295 cells. The specific
inhibitory action of Gö6976 on the classical PKC isoforms (28)
resulted in stimulation of promoter activity. These observations led us
to conclude that PKC
was responsible for inhibition of promoter
activity since this PKC isoform is the only member of conventional PKC
found in the zona glomerulosa and in NCI-H295 cells.
Additional evidence for the inhibitory action of phorbol
ester/DAG-dependent PKC on the expression of the hamster
CYP11B2 promoter activity was obtained in cotransfection
experiments with CA-PKC
and DN-PKC
mutants. The data clearly
showed not only an inhibition of promoter activity by the
constitutively active mutants but also a blockade of the stimulatory
effects of AII, KCl, or Bt2cAMP. As expected, inactive
DN-PKC
isoform, showed an absence of inhibitory effect. These
results were consistent with the interpretation that the inhibitory
action of endogenous PKC
could be reversed by overexpression of the
DN mutant form of PKC
. A dose dependence of the effects of the
CA-PKC
expression vector on the promoter activity showed that
inhibition was maximal at a concentration of 1 µg of transfected DNA.
Dose-response studies were not done in the case of other PKCs since, at
this concentration of transfected DNA, CA-PKC
and CA-PKC
efficiently blocked the CYP11B2 promoter activity. It
remains to be seen whether, in the case of these two mutant PKCs, a
lower concentration of transfected DNA would be as effective.
The fact that PKC
was not detected in hamster adrenal glands rules
out any interference between the PKC
and -
isoforms in regulating
CYP11B2 promoter activity in hamster adrenal zona glomerulosa. It cannot be excluded however, that PKC
could play a
role in NCI-H295cells. To test this possibility we have overexpressed the CA-PKC
and DN-PKC
mutants in NCI-H295 cells. We found that the CA but not the DN mutant had an inhibitory effect on CAT activity similar to that of CA-PKC
. Moreover, the stimulatory effects of AII,
KCl, and Bt2cAMP were all blocked when CA-PKC
was
overexpressed (data not shown). It cannot be excluded that endogenous
PKC
could have interfere and/or participate in the control of the
hamster CYP11B2 expression in transfected NCI-H295 cells.
Although PKC
is present in very low levels in hamster adrenal zona
glomerulosa and in NCI-H295 cells, its role on CYP112B2 expression remains to be elucidated. It is clear, however, that, when
highly expressed, CA-PKC
but not DN-PKC
was able to inhibit CYP11B2 promoter activity and block the stimulatory effects
of AII, KCl, and Bt2cAMP (Fig. 8B). It is
therefore possible that, in hamster adrenal zona glomerulosa and
NCI-H295 cells, endogenous PKC
is involved in CYP11B2
gene expression. Garcia-Paramio et al. (12) have reported a
lack of specificity of DN mutants of PKC
and -
. Both mutants were
able to attenuate the effect of CA-PKC
on the TPA responsive
element-luciferase activity and to inhibit the normal accumulation of a
fully phosphorylated PKC
in cotransfected COS-1 cells. Although low
levels of PKC
were found in NCI-H295 cells and in hamster adrenal
glands, the degree of cross-talk between PKC
and -
could not be
evaluated until the turnover of PKC
was assessed.
In contrast to the results on the action of DAG-dependent
PKC, data obtained in the case of cotransfection experiments using PKC
mutants indicated that this PKC isoform did not play any negative regulatory role on the basal expression of hamster adrenal CYP11B2. CA-PKC
, however, did not prevent the enhancing
effects of AII and KCl on promoter activity but inhibited the effect of Bt2cAMP, suggesting additional differential regulation
between DAG-dependent PKC and atypical PKC
.
In agreement with our results, Reyland et al. (15) have
reported that steroidogenesis in Y-1 cells inversely correlates with
total PKC activity and with the expression of PKC
but not with
PKC
or -
. Induction of PKC
expression in these cells resulted in decreased cytochrome P450 side chain cleavage activity and reduced
transcription of a cytochrome P450 side-chain cleavage promoter-luciferase construct. However, the same authors found no
effects on cAMP induction of steroidogenesis, indicating that these
pathways function independently to regulate steroidogenesis. In
contrast, our study showed that the overexpression of the
constitutively active PKC
inhibited the enhancing effect of
Bt2cAMP on the CYP11B2 promoter activity. These
observations suggest that genes involved in the regulation of the first
step of steroidogenesis, the transformation of cholesterol to
pregnenolone, and in the last step of aldosterone synthesis, the
transformation of deoxycorticosterone to aldosterone, may be
differently regulated by cAMP.
The fact that TPA and CA-PKC
, -
, and -
blocked the enhancing
effect of AII on the CYP11B2 promoter activity indicated
that AII did not act through the activation of
DAG-dependent PKC. This finding is in agreement with the
conclusion of Nakano et al. (30), who reported a full
aldosterone response to AII despite marked cellular PKC depletion in
rat adrenal zona glomerulosa cells after prolonged preincubation with
TPA. Our results are also in agreement with those of Hajnóczky
et al. (11), who showed that, regardless of whether PKC is
activated by phorbol esters or physiological stimuli such as AII or
K+, it exerts an inhibitory action on steroid production in
rat adrenal zona glomerulosa cells. Therefore, the mode of action of
stimuli enhancing CYP11B2 expression in adrenal zona
glomerulosa remains to be fully understood. However, in bovine adrenal
zona glomerulosa cells, it has been recently reported that AII
stimulated Raf-1 kinase activity through Ras, independently of PKC
activation and elevation of intracellular calcium (31), thus opening a new avenue of investigation for the action of this hormone.
AII binding to AII receptor type 1 stimulates inositol phosphate and
DAG formation, activates PKC, and enhances aldosterone secretion in
adrenal zona glomerulosa cells. At first sight, it seems paradoxical
that AII stimulates aldosterone biosynthesis while activating
DAG-dependent PKC that inhibit CYP11B2
expression and therefore P450aldo synthesis. As an explanation to this
apparent discrepancy, we suggest that AII exerts a positive action on
CYP11B2 expression by other signaling pathway(s) yet to be
discovered, and a negative action through the PKC signaling pathway.
One has to speculate that the negative regulatory action would be
delayed in comparison to the positive action so producing a feedback
control to restore normal levels of CYP11B2 expression and
to end the enhancing AII action.
Results of Western blotting experiments showed that activated PKC
translocated to the Triton X-100-insoluble cell fraction, where it was
presumably dephosphorylated. This observation was in agreement with
previous reports indicating that PKC
is constitutively phosphorylated, the protein being fully active albeit in a latent effector-dependent state (26). PKC
is dephosphorylated
and inactivated in a desensitization process following activation and
is then degraded (27). Our results showed that mutants CA-PKC
and
DN-PKC
could be dephosphorylated following stimulation with TPA,
suggesting that phosphorylation and dephosphorylation could occur for
both mutants.
Different deletion plasmids of the CYP11B2 promoter were
used in an attempt to identify the region for TPA-dependent
PKC inhibition. TPA only partially prevented the enhancing effects of
AII, KCl, and Bt2cAMP on the activity of the
83-CYP11B2-CAT deletion plasmid (containing a CRE
cis-element (Ref. 4)) and completely blocked their actions
on the activity of the
102-CYP11B2-CAT and on that of
longer deletion plasmids. The inhibition by TPA on the
102 deletion
plasmid was mimicked by cotransfection with CA-PKC
, -
, or -
mutants. In contrast, the CA-PKC
mutant had a stimulatory effect on
the basal
102-CYP11B2-CAT deletion plasmid activity. The
hamster 102-CYP11B2-CAT deletion plasmid contains a
cis-element (Ad2 (Ref. 4)) conserved among
CYP11B2 genes of rat, mouse, and human species (3). Mutation
of bases
86 to
95 of the Ad2 cis-element in the
102-CYP11B2-CAT deletion plasmid dramatically decreased
basal and AII-, Bt2cAMP-, and KCl-stimulated promoter activity (Fig. 12). Similar results were obtained when the same mutation was done in the
486-CYP11B2-CAT deletion plasmid
(data not shown), thus emphasizing the importance of this
cis-element in the control of the gene expression. Although
TPA attenuated the stimulatory effect of AII and Bt2cAMP to
a lesser extent than in wild type, we cannot draw a firm conclusion on
the role of this cis-element in TPA action due to the very
low promoter activity of the mut-102.
In conclusion, our findings suggest that the hamster CYP11B2
gene is under differential control by conventional (
) and atypical (
) PKC.