©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
P450c11B3 mRNA, Transcribed from a Third P450c11 Gene, Is Expressed in a Tissue-specific, Developmentally, and Hormonally Regulated Fashion in the Rodent Adrenal and Encodes a Protein with Both 11-Hydroxylase and 18-Hydroxylase Activities (*)

(Received for publication, October 17, 1994)

Synthia H. Mellon (§) Susanna R. Bair Helena Monis

From the Department of Obstetrics, Gynecology and Reproductive Sciences and The Metabolic Research Unit, University of California, San Francisco, California 94143-0556

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rat genome contains four P450c11 genes. One of these (CYP11B1) encodes P450c11beta, which is the steroid 11beta-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, 11beta-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 P450c11beta 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 P450c11beta 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 P450c11beta 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 P450c11beta. 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 11beta- and 18-hydroxylation and thus has a spectrum of activities midway between P450c11beta and P450c11AS.


INTRODUCTION

The synthesis of 11-deoxycorticosterone (DOC) (^1)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, P450c11beta and P45011AS(2, 3, 4) . The CYP11B1 gene encoding P450c11beta is regulated by ACTH, is expressed solely in the fasciculata/reticularis, and encodes an 11beta 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 P450c11beta in nucleotide (96% identical) and amino acid (94% identical) sequences. Even in exon 5, where P450c11beta and P450c11AS sequences differ the most (65% identical), P450c11B3 is 97% identical to P450c11beta. P450c11B4 appears to be a pseudogene. Although it is 95% identical to P450c11beta 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 P450c11beta. 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 P450c11beta and P450c11AS.


MATERIALS AND METHODS

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 P450c11beta (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). beta-Actin mRNA protects a 149-base fragment of the 195-base beta-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 P450c11beta cRNA: P450c11beta 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 times 10^6 cpm of P-labeled P450 cRNA probe and with 1 times 10^6 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 P450c11beta-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.

Full-length P450c11B3 and P450c11beta 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(4) precipitation. After 48 h, enzymatic activity was assayed by incubating cells with 40,000 cpm/ml [^14C]11-deoxycorticosterone for 24 h. Medium was collected, extracted with 5 volumes of isooctane, dried under N(2), 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.


RESULTS

Cloning of P450c11B3 cDNA

Two P450c11 genes and their transcripts have been characterized in rodents and human beings: P450c11beta, encoded by the CYP11B1 gene, encodes a steroid 11beta 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 P450c11beta 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.


Figure 1: Sequence of rat adrenal P450c11B3 cDNA. The amino acids encoded by the cDNA are shown above the cDNA sequence.



Detection of Three Forms of P450c11 with a Single Probe

To distinguish P450c11beta, 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 P450c11beta (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 P450c11beta for the probe. The probe contains 219 bases of P450c11beta sequences and 61 bases of vector sequences. Hybridization of this probe to P450c11beta mRNA protects a 219-nt fragment, as expected. Hybridization of this P450c11beta 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 P450c11beta 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 singlestranded 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.


Figure 2: Alignment of P450c11beta, P450c11AS, and P450c11B3 mRNA sequences and the P450c11beta 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 c11beta sequence. All other bases in P450c11AS and P450c11B3 mRNAs are identical to those in P450c11beta. The asterisks under nucleotide 225 and 326 show the P450c11B3:P450c11beta 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:P450c11beta 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 P450c11beta and P450c11B3 by in vitro transcription and assayed these both separately and as a mixture using our P450c11beta 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 P450c11beta 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 P450c11beta-specific fragment is present in adrenal RNA from all three rats, demonstrating that adrenal samples from all three rats contain P450c11beta mRNA. Thus, the time of expression of P450c11B3 mRNA appears to be restricted.


Figure 3: RNase protection assay of P450c11beta and P450c11B3 mRNAs produced by in vitro transcription. P450c11beta and P450c11B3 mRNAs (250 pg) were hybridized overnight, individually or together, with the 280-base P-labeled P450c11beta cRNA probe, digested with RNase A, and separated on 5% acrylamide, 7.5 M urea sequencing gels. P450c11beta mRNA (lane c11beta) 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 c11beta + 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 P450c11beta-specific 219-nt fragment. Adrenal RNA from an 18-day-old rat (1 µg) (lane 18 day) protects the P450c11beta-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 P450c11beta probe.



Expression of P450c11beta and P450c11B3 mRNAs in the Neonatal Adrenal

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 P450c11beta 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 P450c11beta mRNA, barely detectable P450c11AS mRNA, and no detectable P450c11B3 mRNA. By 10 days of age this pattern changes dramatically. P450c11beta 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.


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 times 10^6 cpm of P-labeled P450c11beta 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 P450c11beta probe; lane P2 contains the rat beta-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 times 10^6 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 P450c11beta 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 P450c11beta 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 P450c11beta-specific probes shows that this is the same pattern of expression as that for P450c11beta mRNA (Fig. 7A).


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 P450c11beta 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 P450c11beta and P450c11B3 mRNAs by ACTH

Previous studies from our laboratory demonstrated that P450c11beta and P450scc mRNAs are unresponsive to ACTH treatment of adult rats in vivo(6) . To determine if P450scc and P450c11beta 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. P450c11beta 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 P450c11beta 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.


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 times 10^6 cpm of P-labeled P450c11beta cRNA probe plus 1 times 10^6 cpm of P-labeled rat actin cRNA probe (A) or with 1 times 10^6 cpm of P-labeled P450scc cRNA probe plus 1 times 10^6 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 P450c11beta 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 P450c11beta 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 times 10^6 cpm of P-labeled P450c11beta cRNA probe plus 1 times 10^6 cpm of P-labeled rat beta 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 P450c11beta 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 P450c11beta 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 [^14C]DOC for 24 h, we analyzed the resulting steroidal products by TLC (Fig. 10). Cells transfected with a vector expressing P450c11beta converted DOC primarily to corticosterone, consistent with the predominant 11beta-hydroxylase activity of P450c11beta. 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 P450c11beta. 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 P450c11beta and P450c11AS.


Figure 10: Autoradiogram of a thin layer chromatography of ^14C-labeled steroids extracted from MA-10 cells transfected with eukaryotic expression vectors containing P450c11 cDNAs. MA-10 cells were transfected with either P450c11beta (lane c11beta) or P450c11B3 (lane c11B3) cDNAs and incubated with [^14C]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.




DISCUSSION

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 P450c11beta 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 P450c11beta has both 11beta-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 P450c11beta, 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 P450c11beta (Table 2). These residues may be important for the 18-hydroxylase activity found in P450c11B3 and P450c11AS but not in P450c11beta. In human beings, several mutations in both P450c11beta and in P450c11AS result in dramatic changes in enzymatic activities(25, 26, 27) . A mutation in P450c11beta 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 P450c11beta 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 P450c11beta. 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 P450c11beta 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 P450c11beta 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 P450c11beta 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 P450c11beta 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 P450c11beta. In both P450c11B3 and in the Dahl R rat, these two amino acids have been changed from amino acids normally found in P450c11beta to those found in P450c11AS. Amino acid 381, but not 351, has been suggested to affect the ratio of 18/11-hydroxylase activities of P450c11beta, 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 P450c11beta 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 P450c11beta 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 P450c11beta 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.


FOOTNOTES

*
This work was supported by Grant HD27970 (to S. H. M.) from the National Institutes of Health; Grants 91-115 and 93-222 from the American Heart Association, California Affiliate; Grant 91019540 (to S. H. M.) from the American Heart Association, National Center; and by Core Grant HD11979 to the Reproductive Endocrinology Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17082[GenBank].

§
To whom all correspondence should be addressed: Dept. of OB/GYN and Reproductive Sciences, University of California, San Francisco, CA 94143-0556. Tel.: 415-476-5329; Fax: 415-753-3271.

(^1)
The abbreviations used are: DOC, 11-deoxycorticosterone; nt, nucleotide(s); bp, base pair(s); PCR, polymerase chain reaction.


REFERENCES

  1. Miller, W. L. (1988) Endocr. Rev. 9, 295-318 [Medline] [Order article via Infotrieve]
  2. Lauber, M., and Muller, J. (1989) Arch. Biochem. Biophys. 274, 109-119 [Medline] [Order article via Infotrieve]
  3. Ogishima, T., Mitani, F., and Ishimura, Y. (1989) J. Biol. Chem. 264, 10935-10938 [Abstract/Free Full Text]
  4. Malee, M., and Mellon, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4731-4735 [Abstract]
  5. Ogishima, T., Suzuki, H., Hata, J., Mitani, F., and Ishimura, Y. (1992) Endocrinology 130, 2971-2977 [Abstract]
  6. Sander, M., Ganten, D., and Mellon, S. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 148-152 [Abstract]
  7. Domalik, L. J., Chaplin, D. D., Kirkman, M. S., Wu, R. C., Liu, W., Howard, T. A., Seldin, M. F., and Parker, K. L. (1991) Mol. Endocrinol. 5, 1853-1861 [Abstract]
  8. Curnow, K. M., Tusie-Luna, M. T., Pascoe, L., Natarajan, R., Gu, J. L. Nadler, J. L., and White, P. C. (1991) Mol. Endocrinol. 5, 1513-1522 [Abstract]
  9. Tremblay, A., Parker, K. L., and Lehoux, J. G. (1992) Endocrinology 130, 3152-3158 [Abstract]
  10. Quinn, S. J., and Williams, G. H. (1988) Annu. Rev. Physiol. 50, 409-426 [CrossRef][Medline] [Order article via Infotrieve]
  11. Muller, J., Meuli, C., Schmid, C., and Lauber, M. (1989) J. Steriod Biochem. 34, 271-277
  12. Mukai, K., Imai, M., Shimada, H., and Ishimura, Y. (1993) J. Biol. Chem. 268, 9130-9137 [Abstract/Free Full Text]
  13. Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H., and Okamoto, M. (1989) FEBS Lett. 255, 21-26 [CrossRef][Medline] [Order article via Infotrieve]
  14. Mellon, S. H., Kushner, J. A., and Vaisse, C. (1991) DNA Cell Biol. 10, 339-347 [Medline] [Order article via Infotrieve]
  15. Oonk, R. B., Krasnow, J. S., Beattie, W. G., and Richards, J. S. (1989) J. Biol. Chem. 264, 21934-21942 [Abstract/Free Full Text]
  16. Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy, Z., and Yaffe, D. (1983) Nucleic Acids Res. 11, 1759-1771 [Abstract]
  17. Cicila, G. T., Rapp, J. P., Wang, J.-M., St. Lezin, E., Ng, S. C., and Kurtz, T. W. (1993) Nature Genet. 3, 346-353 [Medline] [Order article via Infotrieve]
  18. Imai, M., Shimada, H., Okada, Y., Matsushima-Hibiya, Y., Ogishima, T., and Ishimura, Y. (1990) FEBS Lett. 263, 299-302 [CrossRef][Medline] [Order article via Infotrieve]
  19. Myers, R. M., Larin, Z., and Maniatis, T. (1985) Science 230, 1242-1246 [Medline] [Order article via Infotrieve]
  20. Townsend, S. F., Dallman, M. F., and Miller, W. L. (1990) J. Biol. Chem. 265, 22117-22122 [Abstract/Free Full Text]
  21. Mornet, E., Dupont, J., Vitek, A., and White, P. C. (1989) J. Biol. Chem. 264, 20961-20967 [Abstract/Free Full Text]
  22. Kirita, S., Hashimoto, T., Kitajima, M., Honda, S., Morohashi, K., and Omura, T. (1990) J. Biochem. ( Tokyo ) 108, 1030-1041 [Abstract]
  23. Morohashi, K., Nonaka, Y., Kirita, S., Hatano, O., Takakusu, A., Okamoto, M., and Omura, T. (1990) J. Biochem. ( Tokyo ) 107, 635-640 [Abstract]
  24. Yanagibashi, K., Haniu, M., Shively, J. E., Shen, W. H., and Hall, P. (1986) J. Biol. Chem. 261, 3556-3562 [Abstract/Free Full Text]
  25. White, P. C., Dupont, J., New, M. I., Leiberman, E., Hochberg, Z., and Rosler, A. (1991) J. Clin. Invest. 87, 1664-1667 [Medline] [Order article via Infotrieve]
  26. Pascoe, L., Curnow, K. M., Slutsker, L., Connell, J. M., Speiser, P. W., New, M. I., and White, P. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8327-8331 [Abstract]
  27. Curnow, K. M., Slutsker, L., Vitek, J., Cole, T., Speiser, P. W., New, M. I., White, P. C., and Pascoe, L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4552-4556 [Abstract]
  28. Komiya, I., Yamada, T., Aizawa, T., Takasu, N., Niwa, A., Maruyama, Y., and Ogawa, A. (1991) Cardiology 78, 99-110 [Medline] [Order article via Infotrieve]
  29. Biglieri, E. G., and Kater, C. E. (1991) Clin. Chem. 37, 1843-1848 [Abstract]
  30. Dahl, L. K., Heine, M., and Tassinari, L. (1962) J. Exp. Med. 115, 1173-1190
  31. Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami, H., Ogihara, T., and Okamoto, M. (1993) J. Biol. Chem. 268, 9117-9121 [Abstract/Free Full Text]
  32. Ozols, J., Heinemann, F. S., and Johnson, E. F. (1981) J. Biol. Chem. 256, 11405-11408 [Abstract/Free Full Text]
  33. Schapiro, S. (1962) Endocrinology 71, 986-988
  34. Walker, C. D., Perrin, M., Vale, W., and Rivier, C. (1986) Endocrinology 118, 1445-1451 [Abstract]
  35. Itoh, S., and Hirota, R. (1976) Folia Endocrinol. Jpn. 52, 1220-1229
  36. Gallant, S., Alfano, J., Charpin, M., and Brownie, A. C. (1992) Endocr. Res. 18, 145-161 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.