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(Received for publication, October 17, 1994) From the
The rat genome contains four P450c11 genes. One of these (CYP11B1) encodes P450c11 The synthesis of 11-deoxycorticosterone (DOC) (
Full-length P450c11B3 and P450c11
Figure 1:
Sequence
of rat adrenal P450c11B3 cDNA. The amino acids encoded by the cDNA are
shown above the cDNA sequence.
Figure 2:
Alignment of P450c11
To assess the validity of
this approach we prepared pure mRNAs for P450c11
Figure 3:
RNase
protection assay of P450c11
Figure 4:
RNase
protection assay of RNA isolated from male and female rat adrenals at
2, 10, 12, and 18 days of age. One microgram of adrenal RNA from rats
2, 10, 12, and 18 days old was combined with 1
To confirm that P450c11B3 mRNA is
abundantly expressed in neonatal rat adrenals, and to define when
P450c11B3 expression begins, we amplified cDNA from day 2 to day 32 rat
adrenals by Reverse Transcriptase/PCR using P450c11B3-specific
oligonucleotide primers (Fig. 5). An amplified DNA product is
evident in adrenals from rats 8-32 days old, but not from animals
2-6 days old, consistent with our RNase protection data in Fig. 4. Sequencing the amplified products from day 12 and day 18
adrenals confirmed that they were P450c11B3 (data not shown). Thus,
P450c11B3 gene expression in the newborn rat adrenal is turned on
between the 6th and 8th day of life.
Figure 5:
Ethidium bromide-stained agarose gel
containing cDNA amplified from adrenal RNA. Adrenal RNA (1 µg) from
rats 2-30 days old was reverse-transcribed into cDNA and
amplified by PCR using P450c11B3-specific primers (top) or
glyceraldehyde-3-phosphate dehydrogenase-specific primers (bottom) as control for cDNA synthesis from days 2-8.
Amplified products were separated on 2% agarose gels and stained with
ethidium bromide. The P450c11B3 fragment is 445 bp and the
glyceraldehyde-3-phosphate dehydrogenase fragment is 254
bp.
P450scc encodes the
mitochondrial cholesterol side chain cleavage enzyme and is involved in
glucocorticoid and mineralocorticoid synthesis. P450scc mRNA also
changes during the early neonatal period. The abundance of P450scc mRNA
is greater at 12 and 18 days than it is at 2 or 10 days. (Fig. 6) and shows no sex-specific differences (not shown).
Although expression of P450scc mRNA increases during early neonatal
life, it does not parallel the temporal expression of any of the three
P450c11 mRNAs. Thus, the ontogenic patterns of expression of the three
forms of P450c11 mRNA are distinct from one another and also distinct
from the ontogenic patterns of P450scc expression.
Figure 6:
RNase
protection of neonatal adrenal P450scc mRNA. One microgram of adrenal
RNA from rats 2, 10, 12, and 18 days was combined with 1
Figure 7:
Dark
field photomicrographs of in situ hybridization of 18 day rat
adrenals. Rat adrenals from day 18 were hybridized with either an
Figure 8:
RNase protection assay of adrenal RNA from
animals treated with ACTH. Day 12 and day 18 rats were given ACTH or
saline injection and were killed 24 h later. One microgram of adrenal
RNA was combined with 1
The regulation of
P450c11B3 by ACTH may be sex-dependent (Fig. 9). When we
analyzed RNA from both male and female rats given a single injection of
ACTH at 12 or 18 days, we found that accumulation of P450c11B3 mRNA
decreased only in adrenals from male rats and not from female rats.
Neither P450c11
Figure 9:
RNase protection assay of adrenal RNA from
male and female rats treated with ACTH. Day 12 and day 18 rats were
given ACTH or saline injection and were killed 24 h later. One
microgram of adrenal RNA was combined with 1
Figure 10:
Autoradiogram of a thin layer
chromatography of
It is unclear why the rat expresses three different P450c11
genes during the neonatal period. All three genes are expressed in a
zone-specific manner and have different but overlapping enzymatic
activities. To date, only two P450c11 genes have been isolated from
human beings(21) . The existence of P450c11B3 will permit the
production of abundant 18-OH DOC and 18-OH corticosterone while
limiting the production of aldosterone. However, roles for these 18-OH
steroids that are distinct from the corticosterone produced by
P450c11 While the bovine genome also contains multiple
P450c11 genes(22, 23) , it is not clear if they are
all functional. Bovine P450c11 The
regions of difference and similarity among P450c11
Our
results demonstrate that P450c11B3 produces more 18-OH
11-deoxycorticosterone (18-OH DOC) than does P450c11 The Dahl salt-sensitive
rats are a widely studied genetic model of salt-sensitive hypertension.
In this strain, supplementary dietary sodium chloride increases blood
pressure, but in the salt-resistant (R) strain, supplementary dietary
sodium chloride has little effect on blood pressure(30) . Two
groups have recently shown that the gene for P450c11 Since we found marked differences in the
regulation of P450c11 P450c11B3 is the first gene encoding a steroidogenic
enzyme not involved in sex steroid production that is regulated in a
sex-specific fashion in the same tissue. This sexually dimorphic
regulation, along with the time course for expression of this gene,
suggests that P450c11B3 may play a role in the ``stress
hyporesponsive period'' (33, 34, 35) .
At birth, the concentration of plasma corticosterone in the rat is the
same as in the adult rat. However, a few days later, the concentration
of corticosterone drops dramatically and stays low for several weeks.
This stress hyporesponsive period, occurs from days 4-20 of life
and is marked by a reduced capacity of the animal to secrete ACTH and
corticosterone in response to stressful stimuli. Females, but not
males, may be responsive to ether stress at day 12 (35) . This
correlates with our finding that rat P450c11B3 is negatively regulated
by ACTH in males but not in females. Consistent with our results,
others have found that testosterone can decrease P450c11 The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U17082[GenBank].
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1643-1649
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, which is the steroid
11-hydroxylase found solely in the adrenal zona
fasciculata/reticularis, and is responsible for the conversion of
11-deoxycorticosterone to corticosterone. A second P450c11 gene (CYP11B2) encodes P450c11AS, which is the aldosterone synthase
found solely in the adrenal zona glomerulosa. P450c11AS has three
activities, 11-hydroxylase, 18-hydroxylase, and 18-oxidase, and is
responsible for the conversion of 11-deoxycorticosterone to
aldosterone. Recently, two more rat P450c11 genes, P450c11B3 and
P450c11B4, were cloned. P450c11B4 appears to be a pseudogene, as two
exons are replaced by unrelated DNA. P450c11B3 closely resembles
P450c11 in mRNA and encoded amino acid sequences, predicting a
protein of 498 amino acids. However, the expression of this mRNA and
protein have not been demonstrated to date. We now demonstrate that
this P450c11B3 mRNA is expressed in the adrenal gland several days
after birth and is not expressed during fetal development or in the
adult rat adrenal. Like P450c11 mRNA, P450c11B3 mRNA is expressed
in the zona fasciculata/reticularis and not in the zona glomerulosa.
However, the regulation of P450c11B3 mRNA expression is different from
that of P450c11 mRNA, in that its abundance is decreased by ACTH
in a sex-dependent fashion. Transfection of eukaryotic cells with a
vector expressing P450c11B3 shows that this form of P450c11 can convert
11-deoxycorticosterone (DOC) to corticosterone and thus has the same
enzymatic activity as P450c11. In addition, P450c11B3 can convert
DOC to 18-OH DOC and corticosterone to 18-OH corticosterone and thus
has 18-hydroxylase activity similar to P450c11AS, but it lacks
detectable 18-oxidase activity. Thus, P450c11B3 catalyzes 11- and
18-hydroxylation and thus has a spectrum of activities midway between
P450c11 and P450c11AS.
)from
cholesterol uses the same adrenal enzymes in both the adrenal
glomerulosa and fasciculata/reticularis(1) . DOC is then
converted to mineralocorticoids in the glomerulosa and to
glucocorticoids in the fasciculata/reticularis by the zone-specific
expression of two P450c11 enzymes, P450c11 and
P45011AS(2, 3, 4) . The CYP11B1 gene
encoding P450c11 is regulated by ACTH, is expressed solely in the
fasciculata/reticularis, and encodes an 11 hydroxylase that
converts DOC to corticosterone or to 18-OH DOC(5, 6) .
The CYP11B2 gene encoding P450c11AS is regulated by sodium and
potassium via the renin-angiotensin system, is expressed solely in the
zona glomerulosa, and encodes aldosterone synthase that converts DOC to
aldosterone(4, 6, 7, 8, 9, 10, 11) .
Two other P450c11 genes, called CYP11B3 and CYP11B4 (which we call P450c11B3 and P450c11B4) were recently cloned from
a rat genomic library(12) . Expression of the CYP11B3 gene could result in the formation of P450c11B3 mRNA encoding a
protein of 498 amino acids. P450c11B3 closely resembles P450c11 in
nucleotide (96% identical) and amino acid (94% identical) sequences.
Even in exon 5, where P450c11 and P450c11AS sequences differ the
most (65% identical), P450c11B3 is 97% identical to P450c11.
P450c11B4 appears to be a pseudogene. Although it is 95% identical to
P450c11 in nucleotide sequence, it has a 598-nt insert between the
end of exon 2 and the 41st nucleotide of exon 4, which bears no
similarity to exons 3 or 4 of the other P450c11 genes, and contains
some sequences identical to intron 2 of P450c11. To date, it has
not been known if P450c11B3 is expressed. We now demonstrate the
zone-specific, developmentally regulated, and hormonally regulated
expression of this mRNA and show that this mRNA encodes an enzyme with
activities intermediate between those of P450c11 and P450c11AS.
Animals
Sprague-Dawley rats were used in all
experiments; the day of birth of the pups is referred to as Day 1. In
experiments in which animals were given ACTH, eight rats (four male,
four female) were injected with 4 units of ACTH (Acthargel,
Rhône-Poulenc-Rhorer Pharmaceuticals,
Collegeville, PA) and eight rats (four male, four female) were injected
with saline (control) and killed 24 h later by cervical transsection
under Metofane (methoxyflurane) (Pitman-Moore, Inc., Washington
Crossing, NJ) anesthesia.Probes
Probes were single-stranded riboprobes
labeled with [P]UTP prepared from specific cDNAs
by transcription of linearized pKS plasmids using T7 RNA polymerase
(Stratagene) as described(4, 6) . A 219-bp fragment of
rat P450c11
(bases 213-432 (13) )(4, 13) , a 241-bp EcoRI/PvuII fragment of rat
P450scc(14, 15) , and a 149-bp EcoRI/Pvu II fragment of rat actin (bases 2066-2216 of rat cytoplasmic
actin cDNA (16) ) (6, 16) were cloned into pKS
(Stratagene). -Actin mRNA protects a 149-base fragment of the
195-base -actin probe. P450scc mRNA protects a 233-base fragment
of the 296-base P450scc probe. Because of the high degree of sequence
identity among the rat P450c11 mRNA species, all three can be detected
by the 280-base P450c11 cRNA: P450c11 mRNA protects a
219-base fragment, P450c11AS mRNA protects a 192-base fragment and
P450c11B3 mRNA protects a 207-base fragment.Analysis of RNA
RNA was isolated from individual
adrenals by guanidinium isothiocyanate homogenization and purification
by CsCl ultracentrifugation. RNA was quantitated by absorbance at 260
nm and was analyzed by RNase protection assays, as
described(4, 6) . In these assays, one microgram of
adrenal RNA was combined with 1 
10
cpm of P-labeled P450 cRNA probe and with 1
10 cpm of P-labeled actin cRNA probe, hybridized
overnight at 42 °C, treated with 20 µg/ml RNase A in buffer
containing 10 mM Tris, pH 7.5, 5 mM EDTA, and 300
mM NaCl, for 30 min. Protected RNA fragments were separated on
5% acrylamide, 7.5 M urea sequencing gels, and gels were
autoradiographed overnight.
In Situ Hybridization
In situ hybridization assays were performed on 10-µm frozen sections
of adrenals from day 18 rats, as described(4, 6) ,
using a P450c11-specific probe(6) , and using the
P450c11B3-specific 19-mer 5` ACTCCAGCGTCATCCGACG 3` (bases
324-342(12) ) as probe (Table 1).
Reverse Transcriptase/PCR Amplification of Adrenal
mRNA
One microgram of adrenal RNA was transcribed into cDNA at
42 °C using reverse transcriptase and a mixture of random hexamers
and poly(dT) primers. Primers used for PCR are shown in Table 1.
The differences among the three different P450c11 cDNA sequences are
included. One-tenth microgram of cDNA was amplified using
P450c11B3-specific primers: 3` primer, 5` AGTGTCCTTTCCACACCT 3` (bases
749-655(12) ); 5` primer, 5` CGTCGGATGACGCTGGAGT 3`
(bases 322-340); amplification conditions were 95 °C for 30
s, 52 °C for 30 s, and 72 °C for 1 min. PCR products were
separated by agarose gel electrophoresis, and the 444-bp fragment was
eluted from the gel and cloned into pKS. Several positive clones were
sequenced by double-stranded DNA dideoxy sequencing.Cloning and Expression of P450c11B3 cDNA
P450c11B3
cDNA was cloned by Reverse Transcriptase/PCR amplification of adrenal
RNA from an 18-day-old rat. The primers used are shown in Table 1. One-tenth microgram of cDNA was amplified for 50 cycles
using the following conditions: 95 °C for 45 s and 72 °C for 90
s. PCR products were digested with HindIII/XhoI,
separated by agarose gel electrophoresis, and the 1497-bp fragment was
cloned into the HindIII/XhoI sites of pKS
(Stratagene). The cDNA was sequenced in its entirety by double-stranded
dideoxy sequencing. cDNAs (17) were cloned into HindIII/XhoI and HindIII/XbaI sites, respectively, of pCM8
(Invitrogen), and were transfected into mouse Leydig MA-10 cells by
CaPO
precipitation. After 48 h, enzymatic activity was
assayed by incubating cells with 40,000 cpm/ml
[
C]11-deoxycorticosterone for 24 h. Medium was
collected, extracted with 5 volumes of isooctane, dried under
N
, and analyzed by TLC, using methylene
chloride:methanol:water (300:20:1, v:v:v) as mobile phase(8) .
Cold steroid standards were used to determine the mobility of steroid
products.
Cloning of P450c11B3 cDNA
Two P450c11 genes and
their transcripts have been characterized in rodents and human beings:
P450c11, encoded by the CYP11B1 gene, encodes a steroid
11 hydroxylase expressed in the adrenal fasciculata/reticularis,
and P450c11AS, encoded by the CYPB2 gene, encodes and an
aldosterone synthase expressed in the adrenal zona
glomerulosa(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
These two P450s appear to account for the enzymatic activities of the
adrenal. However, Mukai et al.(12) recently described
the presence of two additional CYP11 genes in the rat: CYP11B3 and CYP11B4 (which we call P450c11B3 and
P450c11B4), of which only P450c11B3 appears capable of encoding a P450
enzyme. To determine if the P450c11B3 gene is expressed in the rat
adrenal, we designed P450c11B3 sequence-specific oligonucleotides and
used these to amplify cDNA synthesized from adrenal mRNA from
18-day-old rats. These oligonucleotides precisely span the coding
region of the presumed P450c11B3 mRNA, from the ATG translational
initiator to the TAG stop codon (Table 1). This PCR amplification
produced a single product of about 1500 bp (not shown) which was then
cloned into pKS and sequenced. The 1497-bp sequence (Fig. 1) is
identical to the coding region of the gene (12) . It encodes a
498-amino acid protein that bears 94% amino acid identity to
P450c11 and 84% amino acid identity to P450c11AS. Therefore this
protein is a closely related member of this family and is expected to
have a similar tissue distribution of expression and activity.
Detection of Three Forms of P450c11 with a Single
Probe
To distinguish P450c11, P450c11AS, and P450c11B3
mRNAs, we designed an RNase protection probe that could distinguish
these mRNAs in the same sample on a single gel. The nucleotide
sequences of the cloned genes and cDNAs for P450c11 (12, 13) and P450c11AS (12, 18) as
well as the genomic sequence for P450c11B3 (12) are known. We
chose a region corresponding to bases 213-432 (13) of
P450c11 for the probe. The probe contains 219 bases of
P450c11 sequences and 61 bases of vector sequences. Hybridization
of this probe to P450c11 mRNA protects a 219-nt fragment, as
expected. Hybridization of this P450c11 probe to P450c11AS mRNA is
expected to yield a protected fragment of 192 nt due to RNase A
digestion at base 405 (Fig. 2). Hybridization of this probe to
P450c11B3 mRNA is expected to yield a protected fragment of 207 nt due
to RNase A digestion at base 225. Although there are
sequence mismatches between P450c11B3 and the P450c11
probe at
bases 334, 336 and 343 that might appear to result in
smaller fragments, these bases are all Gs in the probe, and RNase A may
not digest single
stranded RNA at purines. RNase A does not
often detect U:G mismatches(19) , and thus only about half of
the hybrids are digested at the U:G mismatch at base 326. When this
mismatch is not digested, the 207-nt fragment results; when it is
digested, two fragments of 101 and 106 nt result. None of these
fragments could result from hybridization to a hypothetical P450c11B4
transcript as P450c11B4 lacks 37 bp at the region of the 5` end of the
probe (nucleotides underlined in Fig. 2), which could
result in a protected fragment of 183 nt.
, P450c11AS, and
P450c11B3 mRNA sequences and the P450c11 cRNA used as probe in the
RNase protection assays. The sequences are from nucleotide 213 to 432,
as numbered by Mukai et al.(12) . Differences in 7
nucleotides in the P450c11B3 sequence and differences in 8 nucleotides
in the P450c11AS sequence are noted below the c11 sequence. All
other bases in P450c11AS and P450c11B3 mRNAs are identical to those in
P450c11. The asterisks under nucleotide 225 and 326 show
the P450c11B3:P450c11 mismatched bases that are cleaved by RNase A
digestion. Digestion at base 225 yields a 207-nt P450c11B3-specific
fragment, and further digestion at base 326 yields two
P450c11B3-specific fragments of 101 and 106 nt. The # symbol under base
405 shows the P450c11AS:P450c11 mismatched base that is cleaved by
RNase A digestion. Digestion at base 405 yields a 192-nt
P450c11AS-specific fragment. The underlined bases(396-432) show the bases missing in the P450c11B4
gene.
and P450c11B3 by in vitro transcription and assayed these both separately and
as a mixture using our P450c11 probe. We also analyzed samples of
RNA from rat adrenals 2 and 18 days old, as well as from adrenals from
adult rats. As shown in Fig. 3, the two forms of P450c11 mRNA
protected fragments of the predicted sizes. Furthermore, when
P450c11 and P450c11B3 mRNAs are present together, the same correct
sizes are seen, indicating that the mRNAs do not interfere with each
other. This figure also shows that the 207, 106, and 101
P450c11B3-specific fragments are only present in adrenal RNA from
18-day-old rats, and not in adrenal RNA from 2-day-old or adult rats,
demonstrating that P450c11B3 mRNA is only present in adrenal RNA from
18-day-old and not from 2-day-old or adult rats. The 219-nt
P450c11-specific fragment is present in adrenal RNA from all three
rats, demonstrating that adrenal samples from all three rats contain
P450c11 mRNA. Thus, the time of expression of P450c11B3 mRNA
appears to be restricted.
and P450c11B3 mRNAs produced by in
vitro transcription. P450c11 and P450c11B3 mRNAs (250 pg)
were hybridized overnight, individually or together, with the 280-base P-labeled P450c11
cRNA probe, digested with RNase A,
and separated on 5% acrylamide, 7.5 M urea sequencing gels.
P450c11 mRNA (lane c11) protects a 219-nt fragment
and P450c11B3 (lane c11B3) protects a 207-nt fragment, as well
as 106- and 101-nt fragments. These patterns do not change when both
mRNAs are hybridized together with the probe (lane c11 + c11B3). Adrenal RNA (1 µg) from a 2-day-old rat (lane 2
day) and from an adult rat (1 µg) (lane adult)
protect the P450c11-specific 219-nt fragment. Adrenal RNA from an
18-day-old rat (1 µg) (lane 18 day) protects the
P450c11-specific 219-nt fragment and protects the
P450c11B3-specfic 207-, 106-, and 101-nt fragments. Markers, M, are P-labeled MspI pBR322 DNA
fragments. The lane tRNA contained 50 µg of tRNA and
P-labeled probe, and the lane Probe RNased contained only probe, treated identically to samples containing
adrenal RNA plus probe. Lane P contains the P450c11
probe.
Expression of P450c11
The regulation of adrenal steroidogenesis in
the fetus is independent of the regulation of adrenal steroidogenesis
in the mother. Previous studies on the ontogeny of P450c11 mRNA
expression in the fetal adrenal demonstrated that P450c11 and P450c11B3 mRNAs in the
Neonatal Adrenal mRNA is
expressed at concentrations comparable to those in the adult adrenal,
whereas P450c11AS mRNA is barely detected in the fetal adrenal.
However, manipulations that regulate expression of these mRNAs in the
mother's adrenal do not regulate the expression of these mRNAs in
the fetal adrenal. To determine the ontogenic pattern of expression and
relative abundance of these mRNAs we analyzed adrenal RNA from 2-, 10-,
12-, and 18-day-old rats (Fig. 4). Adrenals of the 2-day-old
newborn rat contain abundant P450c11 mRNA, barely detectable
P450c11AS mRNA, and no detectable P450c11B3 mRNA. By 10 days of age
this pattern changes dramatically. P450c11 is reduced by about
half, P450c11AS remains low and P450c11B3, which was undetectable at 2
days, now predominates. This pattern persists to 12 and 18 days in both
males and females. Thus, P450c11B3 expression is turned on between 2
and 10 days and becomes the predominant form of P450c11 mRNA in
immature rats of both sexes.
10 cpm of P-labeled P450c11
cRNA probe, hybridized
overnight, digested with RNase A, and separated on 5% acrylamide, 7.5 M urea sequencing gels. Markers (M) are P-labeled MspI pBR322 DNA fragments. The lane
tRNA contained 50 µg of tRNA and
P-labeled probe,
and the lane Probe RNased contained only probe, treated
identically to samples containing adrenal RNA plus probe. Lane P1 contains the P450c11
probe; lane P2 contains the rat
-actin probe.
10 cpm of P-labeled P450scc cRNA probe,
hybridized overnight, digested with RNase A, and separated on 5%
acrylamide, 7.5 M urea sequencing gels. Markers are
P-labeled MspI pBR322 DNA fragments. The lane
tRNA contained 50 µg of tRNA and
P-labeled probe,
and the lane Probe RNased contained only probe, treated
identically to samples containing adrenal RNA plus
probe.
Zone-specific Expression of P450c11B3
To determine
where P450c11B3 mRNA is expressed, we performed in situ hybridization histochemistry on 18-day-old rat adrenals. Although
P450c11B3 mRNA is highly homologous to P450c11 mRNA, we were able
to design oligonucleotide probes specific for P450c11B3 sequences. The
probe used (Table 1) (bases 322-340) differs from the
corresponding sequence of P450c11 at four nucleotides and from the
corresponding region of P450c11AS at another four nucleotides as shown.
Using this P450c11B3-specific probe, we found that P450c11B3 mRNA is
expressed only in the zona fasciculata/reticularis and not in the zona
glomerulosa (Fig. 7B). The corresponding
P450c11-specific probes shows that this is the same pattern of
expression as that for P450c11 mRNA (Fig. 7A).
S-labeled P450c11
cDNA probe (A) or a P-labeled 19-mer P450c11B3-specific oligonucleotide probe (B). Positive signals appear as white grains on a black background. g represents the zona glomerulosa, f/r represents the zona fasciculata/reticularis, and m represents the adrenal medulla. Bars indicate 100
µm.
Regulation of P450c11
Previous studies from our laboratory demonstrated that
P450c11 and P450c11B3 mRNAs by
ACTH and P450scc mRNAs are unresponsive to ACTH treatment of
adult rats in vivo(6) . To determine if P450scc and
P450c11 mRNAs are unresponsive to ACTH in the neonatal adrenal,
and to determine if neonatal P450c11B3 mRNA is regulated by ACTH, we
analyzed adrenal RNA from 12- and 18-day-old rats 24 h after they were
given an injection of long acting ACTH. P450c11 mRNA was
unresponsive to ACTH at either 12 or 18 days, as is seen in the adult
rat adrenal (Fig. 8A), and a longer exposure of the gel
indicates that P450c11 AS mRNA is also unaffected by ACTH (not shown).
As we (6) and others (20) have previously shown in the
adult rat adrenal, P450scc mRNA is also unaffected by ACTH either at
day 12 or day 18 (Fig. 8B). However, unlike P450scc and
the P450c11 and P450c11AS mRNAs, P450c11B3 is regulated by ACTH
treatment in vivo. P450c11B3 mRNA from day 12 or 18 male rat
adrenals is reduced substantially (Fig. 8A) and is
almost undetectable by 24 h after treatment.
10 cpm of P-labeled P450c11
cRNA probe plus 1 10 cpm of P-labeled rat actin cRNA probe (A)
or with 1
10 cpm of P-labeled P450scc
cRNA probe plus 1
10 cpm of P-labeled
rat actin cRNA probe (B), hybridized, digested with RNase A,
and separated on 5% acrylamide, 7.5 M urea gels, as described
in the legend to Fig. 1. In A, P1 contains the
P450c11
probe, and P2 contains the rat actin probe, and in B, P1 contains the P450scc probe, and P2 contains the rat
actin probe. Molecular weight markers are P-labeled MspI pBR322 DNA. The lane t contained 50 µg of
tRNA and
P-labeled probe, treated identically to samples
containing adrenal RNA plus probe.
nor P450c11AS mRNA from male or female rat
adrenals was affected by ACTH treatment.
10 cpm
of P-labeled P450c11
cRNA probe plus 1
10 cpm of P-labeled rat
actin cRNA
probe, hybridized, digested with RNase A, and separated on 5%
acrylamide, 7.5 M urea gels, as described in the legend to Fig. 1. P1 contains the P450c11 probe, and P2 contains the
rat actin probe. Molecular weight markers (lane M) are P-labeled MspI-digested pBR322 DNA. The lane
t contained 50 µg of tRNA and
P-labeled probe,
treated identically to samples containing adrenal RNA plus
probe.
Analysis of P450c11B3 Enzymatic Activity
To
determine the enzymatic activity of the P450c11B3 protein, we cloned
full-length P450c11B3 and P450c11 cDNAs into vectors that will
express the encoded proteins in eukaryotic cells. We transfected these
plasmids into mouse Leydig MA-10 cells, which lack endogenous P450c11
activity, but do contain adrenodoxin and adrenodoxin reductase, two
mitochondrial electron transfer proteins needed for P450c11 activity.
After incubating transfected cells with [C]DOC
for 24 h, we analyzed the resulting steroidal products by TLC (Fig. 10). Cells transfected with a vector expressing
P450c11 converted DOC primarily to corticosterone, consistent with
the predominant 11-hydroxylase activity of P450c11. Cells
transfected with a vector expressing P450c11B3 also convert DOC to
corticosterone; however, these cells also convert DOC to 18-OH DOC,
corticosterone, and 18-OH corticosterone. Thus, P450c11B3, like
P450c11AS, has substantial 18-hydroxylase activity not present in
P450c11. However, cells expressing P450c11B3 did not produce any
aldosterone detectable by the TLC assay (Fig. 10) or by
radioimmunoassay (not shown). Thus, P450c11B3, unlike P450c11AS, has no
18-oxidase activity. Thus, P450c11B3 has a spectrum of activities
intermediate between P450c11 and P450c11AS.
C-labeled steroids extracted from MA-10
cells transfected with eukaryotic expression vectors containing P450c11
cDNAs. MA-10 cells were transfected with either P450c11 (lane
c11) or P450c11B3 (lane c11B3) cDNAs and incubated
with [C]11-deoxycorticosterone. Migration of
authentic steroid standards, DOC, 11-dehydrocorticosterone,
corticosterone, 18-OH DOC, aldosterone, and 18-OH corticosterone, in
parallel lanes, are noted on the side of the TLC. The identities of
some radioactive compounds were not determined and are indicated by
question marks.
or the aldosterone produced by P450c11AS have not be
described in the rat. has both 11-hydroxylase
activity as well as 18-hydroxylase and aldosterone synthase activities,
and thus only one enzyme in the cow may be necessary for the synthesis
of both mineralocorticoids and glucocorticoids(24) . Thus it is
not known if a human or bovine counterpart to P450c11B3 exists., P450c11B3, and
P450c11AS may provide information about the amino acids important for
enzymatic activity. There are 17 amino acids that are identical in
P450c11B3 and P450c11AS but that differ between P450c11B3 and
P450c11 (Table 2). These residues may be important for the
18-hydroxylase activity found in P450c11B3 and P450c11AS but not in
P450c11. In human beings, several mutations in both P450c11
and in P450c11AS result in dramatic changes in enzymatic
activities(25, 26, 27) . A mutation in
P450c11 at amino acid 448 (Arg His), within the heme
binding domain, results in a marked reduction in 11-hydroxylase
activity(25) , whereas mutations in P450c11AS (amino acid 181,
Arg
Trp) abolishes both 18-hydroxylase and 18-oxidase
activities, and very conservative mutation at amino acid 386 (Val
Ala) results in a slight decrease in 18-hydroxylase
activity(26) . The rat P450c11B3 gene has changes in amino
acids 187 and 381, from the amino acids found in P450c11
to the
amino acids found in P450c11AS; these are the two regions that were
found to have profound effects on P450c11AS enzymatic activity in human
beings(24) . These regions of the protein may be important for
the 18-hydroxylase activity of both P450c11B3 and P450c11AS.
. Because
18-OH DOC has mineralocorticoid activity, expression of P450c11B3 could
alter blood pressure in the absence of changes in P450c11AS expression
and consequent aldosterone synthesis. Increases in plasma 18-OH DOC,
without concomitant increases in aldosterone in human beings, have been
associated with a subtype of essential
hypertension(28, 29) . Since a human counterpart for
P450c11B3 has not been identified, it is unclear if increases in plasma
18-OH DOC concentrations are due to expression of P450c11 or
P450c11B3. Thus some cases of human essential hypertension might be due
to the expression of an as yet uncharacterized form of P450c11.
Alternatively, subtle changes in the amino acid sequence of
P450c11 could result in an enzyme with greater 18-hydroxylase
activity. Thus, patients with this subtype of essential hypertension
could have mutations or conversions in their P450c11 gene that
result in P450c11AS amino acid sequences. in the Dahl
R, but not the Dahl S rat, encodes five amino acid
substitutions(17, 31) . Two of these are at amino
acids 351 and 381, the location of two differences in amino acid codons
between P450c11B3 and P450c11. In both P450c11B3 and in the Dahl R
rat, these two amino acids have been changed from amino acids normally
found in P450c11 to those found in P450c11AS. Amino acid 381, but
not 351, has been suggested to affect the ratio of 18/11-hydroxylase
activities of P450c11, i.e. the ratio of 18-OH
DOC/corticosterone (31) . These amino acids are located near
but not in the Ozols' ligand-binding region (32) and
suggest that these amino acid substitutions may result in altered
ligand binding. The expression and regulation of P450c11B3 in the Dahl
rat is unknown, but may play a role in the etiology of hypertension
development in this rat. and P450c11B3 by ACTH, it is interesting to
compare the 5` regulatory regions of both these genes. In 500 bases of
5`-flanking DNA, there are 33 nucleotide differences. Of these
difference, two occur within a putative CRE at -70/-60.
These differences may be responsible for the differential regulation of
these two genes by ACTH. However, in cell transfection experiments
aimed at analyzing promoter elements, others found that 500 bp of
5`-flanking DNA of both the P450c11 and P450c11B3 genes could
confer similar cAMP transcriptional induction (12) . It is thus
unclear how these genes are differentially regulated in vivo,
but suggests that other factors play a role in the regulation of these
genes by ACTH. mRNA,
without affecting P450scc mRNA(36) . The reason for this
apparent sexual dimorphism as well as the role of P450c11B3 in the
development of the adrenal and hypothalamic/pituitary/adrenal axis
remains unknown.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
