(Received for publication, October 17, 1994)
From the
The rat genome contains four P450c11 genes. One of these (CYP11B1) encodes P450c11, 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.
The synthesis of 11-deoxycorticosterone (DOC) ()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.
Full-length P450c11B3 and P450c11 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.
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, 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.
To assess the validity of
this approach we prepared pure mRNAs for P450c11 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.
Figure 3:
RNase
protection assay of P450c11 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.
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 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.
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
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.
Figure 7:
Dark
field photomicrographs of in situ hybridization of 18 day rat
adrenals. Rat adrenals from day 18 were hybridized with either an 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.
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 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.
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 nor P450c11AS mRNA from male or female rat
adrenals was affected by ACTH treatment.
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 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.
Figure 10:
Autoradiogram of a thin layer
chromatography of 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.
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 or the aldosterone produced by P450c11AS have not be
described in the rat.
While the bovine genome also contains multiple
P450c11 genes(22, 23) , it is not clear if they are
all functional. Bovine P450c11 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.
The
regions of difference and similarity among P450c11, 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.
Our
results demonstrate that P450c11B3 produces more 18-OH
11-deoxycorticosterone (18-OH DOC) than does P450c11. 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.
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 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.
Since we found marked differences in the
regulation of P450c11 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.
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 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17082[GenBank].