Disorders of steroid 11 beta-hydroxylase isozymes.

The most active corticosteroids are 11 beta-hydroxylated. Humans have two isozymes with 11 beta-hydroxylase activity that are respectively required for cortisol and aldosterone synthesis. CYP11B1 (11 beta-hydroxylase) is expressed at high levels and is regulated by ACTH, whereas CYP11B2 (aldosterone synthase) is normally expressed at low levels and is regulated by angiotensin II. In addition to 11 beta-hydroxylase activity, the latter enzyme has 18-hydroxylase and 18-oxidase activities and thus can synthesize aldosterone from deoxycorticosterone. Insights into the normal functioning of these enzymes are gained from studies of disorders involving them. Mutations in the CYP11B1 gene cause steroid 11 beta-hydroxylase deficiency, a form of congenital adrenal hyperplasia characterized by signs of androgen excess and by hypertension. Mutations in CYP11B2 result in aldosterone synthase (corticosterone methyloxidase) deficiency, an isolated defect in aldosterone biosynthesis that can cause hyponatremia, hyperkalemia, and hypovolemic shock in infancy and failure to thrive in childhood. These are both recessive disorders. Unequal crossing over between the CYP11B genes can generate a duplicated chimeric gene with the transcriptional regulatory region of CYP11B1 but sufficient coding sequences from CYP11B2 so that the encoded enzyme has aldosterone synthase (i.e. 18-oxidase) activity. This results in aldosterone biosynthesis being regulated by ACTH, a condition termed glucocorticoid-suppressible hyperaldosteronism. This form of genetic hypertension is inherited in an autosomal dominant manner.

Glucocorticoid-suppressible hyperaldosteronism and adrenal tumors occurring in a single French pedigree.

Glucocorticoid-suppressible hyperaldosteronism is a dominantly inherited form of hypertension believed to be caused by the presence of a hybrid CYP11B1/CYP11B2 gene which has arisen from an unequal crossing over between the two CYP11B genes in a previous meiosis. We have studied a French pedigree with seven affected individuals in which two affected individuals also have adrenal tumors and two others have micronodular adrenal hyperplasia. One of the adrenal tumors and the surrounding adrenal tissue has been removed, giving a rare opportunity to study the regulation and action of the hybrid gene causing the disease. The hybrid CYP11B gene was demonstrated to be expressed at higher levels than either CYP11B1 or CYP11B2 in the cortex of the adrenal by RT-PCR and Northern blot analysis. In situ hybridization showed that both CYP11B1 and the hybrid gene were expressed in all three zones of the cortex. In cell culture experiments hybrid gene expression was stimulated by ACTH leading to increased production of aldosterone and the hybrid steroids characteristic of glucocorticoid-suppressible hyperaldosteronism. The genetic basis of the adrenal pathologies in this family is not known but may be related to the duplication causing the hyperaldosteronism.

Aldosterone secretion: a molecular perspective.

The major mineralocorticoid hormone aldosterone is secreted from the zona glomerulosa of the adrenal cortex. Aldosterone is synthesized from cholesterol via a series of hydroxylations and oxidations. The enzymes involved in these reactions are mostly members of the cytochrome P450 superfamily. The final steps of this pathway, the conversion of 11-deoxycorticosterone (DOC) to aldosterone, require conversion via the intermediates 18-hydroxy-DOC or corticosterone and 18-hydroxycorticosterone. There are significant differences between species in the number of genes that encode the P450(11beta)-related enzymes (CYP11B) involved in these steps and the zonal distribution of their expression. One enzyme is capable of 11-hydroxylation, 18-hydroxylation, and 18-oxidation of DOC to aldosterone. The genetic basis of four diseases-congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency, glucocorticoid-remediable aldosteronism, aldosterone synthase deficiency type I and type II-is explicable by mutations in these cytochrome P450(11beta)-related genes.

Genetic analysis of the human type-1 angiotensin II receptor.

Angiotensin II is a potent pressor hormone and a primary regulator of aldosterone secretion. It acts through at least two types of receptors termed AT1 and AT2. We analyzed cDNA and genomic clones encoding the human angiotensin II type-1 receptor, AT1. The human AT1 gene was mapped to chromosome 3q by polymerase chain reaction analysis of DNA from a panel of human-hamster somatic cell hybrids. The predicted amino acid sequence is 95% identical to the corresponding rat and bovine receptors and 25% and 22% identical, respectively, to the receptors encoded by the RTA and MAS genes. Characterization of several human cDNA clones demonstrated the existence of two alternate 5'-untranslated regions (UTRs) that contain a common initial sequence but differ by the presence or absence of an insertion of 84 base pairs. In the genomic sequence, the coding sequences are contained in a single exon, with an intron occurring in the 5'-UTR at the position of insertion of the 84-base pair sequence. The exons encoding the alternate 5'-UTRs are located at least 3.8 kilobases away from the exon encoding the protein. Reverse transcription-polymerase chain reaction analysis showed that both forms of 5'-UTR are present in approximately equal abundance in a range of tissues expressing AT1. The reagents developed in this work may be useful in testing the hypothesis that genetic variations in angiotensin II receptor function are associated with a tendency to develop hypertension.

Transcripts originating in intron 1 of the HSD11 (11 beta-hydroxysteroid dehydrogenase) gene encode a truncated polypeptide that is enzymatically inactive.

There is evidence for the presence in the kidney of more than one isoform of 11 beta-hydroxysteroid dehydrogenase (11HSD), an enzyme that interconverts cortisol and cortisone (in rodents, corticosterone and 11-dehydrocorticosterone). A specific isoform might arise from transcripts in the kidney that are known to originate in intron 1; translation from these transcripts is predicted to initiate at the codon that in the full-length rat enzyme encodes Met27. Alignment of the full-length rat and human 11HSD sequences with other members of the short chain dehydrogenase family suggests that initiation of translation at Met27 might yield a functional enzyme, since the amino-termini of most of these enzymes occur at equivalent positions. We confirmed that short transcripts are found in the kidney and are detectable at lower levels in the liver and lung. In vitro transcription and translation of short cDNA demonstrated that the AUG encoding Met27 is indeed a functional initiation codon. However, Chinese hamster ovary cells transfected with short cDNA in the pCMV4 vector expressed apparently low levels of the corresponding truncated polypeptide and had no 11HSD activity. Thus, the functional significance of transcripts originating in intron 1 is unclear.

Alternatively spliced human type 1 angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms.

The peptide hormone angiotensin II (AngII) plays a principal role in regulating blood pressure and fluid homeostasis. Most of its known effects are mediated by a guanine nucleotide-regulatory protein (G protein)-coupled receptor pharmacologically defined as the type-1 AngII receptor or AT1. Characterization of cDNA and genomic clones shows that the human AT1 gene contains five exons and encodes two receptor isoforms as a result of alternative splicing. Exon 5 contains the previously characterized open reading frame for AT1, and exons 1 to 3 are alternatively spliced upstream of it to generate several mRNA species, while transcripts containing exon 4 are of minor abundance. In an in vitro translation system, the presence of exon 1 was found to be extremely inhibitory to translation, probably because it can form a stable secondary structure at the RNA level. The alternatively spliced second exon also had a strong inhibitory effect on translation, presumably because it contains a minicistron commencing with an ATG in an optimal context for translation initiation. Exon 2 was similarly inhibitory to protein production in transfected cells, but exon 1 was found to enhance protein synthesis in this system. Transcripts containing exon 3 and 5, which comprise up to one-third of AT1 mRNAs in all tissues examined, encode a receptor with an amino-terminal extension of 32-35 amino acids. These transcripts were translated into a larger receptor isoform in vitro and produced a functional receptor with normal ligand binding and signaling properties in transfected cells.

Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+ exchangers define a cation binding domain.

cDNA clones encoding a novel putative G protein-coupled receptor have been characterized. The receptor is widely expressed in normal solid tissues. Consisting of 1967 amino acid residues, this receptor is one of the largest known and is therefore referred to as a very large G protein-coupled receptor, or VLGR1. It is most closely related to the secretin family of G protein-coupled receptors based on similarity of the sequences of its transmembrane segments. As demonstrated by cell surface labeling with a biotin derivative, the recombinant protein is expressed on the surface of transfected mammalian cells. Whereas several other recently described receptors in this family also have large extracellular domains, the large extracellular domain of VLGR1 has a unique structure. It has nine imperfectly repeated units that are rich in acidic residues and are spaced at intervals of approximately 120 amino acid residues. These repeats resemble the regulatory domains of Na+/Ca2+ exchangers as well as a component of an extracellular aggregation factor of marine sponges. Bacterial fusion proteins containing two or four repeats specifically bind 45Ca in overlay experiments; binding is competed poorly by Mg2+ but competed well by neomycin, Al3+, and Gd3+. These results define a consensus cation binding motif employed in several widely divergent types of proteins. The ligand for VLGR1, its function, and the signaling pathway(s) it employs remain to be defined.

The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex.

The steroid 11 beta-hydroxylase (P450c11) enzyme is responsible for the conversion of 11-deoxycortisol to cortisol in the zona fasciculata of the adrenal cortex. Animal studies have suggested that this enzyme or a closely related isozyme is also responsible for the successive 11 beta- and 18-hydroxylation and 18-oxidation of deoxycorticosterone required for aldosterone synthesis in the zona glomerulosa. There are two distinct 11 beta-hydroxylase genes in man, CYP11B1 and CYP11B2, which are predicted to encode proteins with 93% amino acid identity. We used a sensitive assay based on the polymerase chain reaction to analyze the expression of the CYP11B1 and B2 genes. Transcripts of CYP11B1 were detected at high levels in surgical specimens of normal adrenals and also in an aldosterone-secreting adrenal tumor. Transcripts of CYP11B2 were found at low levels in normal adrenals, but at a much higher level in the aldosterone-secreting tumor. CYP11B2 mRNA levels were increased in cultured zona glomerulosa cells by physiological levels of angiotensin-II. The entire coding regions of both CYP11B1 and B2 cDNAs were cloned from the tumor mRNA. Expression of these cDNAs in cultured COS-1 cells demonstrated that the CYP11B1 product could only 11 beta-hydroxylate 11-deoxycortisol or deoxycorticosterone, whereas the CYP11B2 product could also 18-hydroxylate cortisol or corticosterone. A small amount of aldosterone was synthesized from deoxycorticosterone only in cells expressing CYP11B2 cDNA. These data demonstrate that the product of CYP11B2 is required for the final steps in the synthesis of aldosterone.

Deletion hybrid genes, due to unequal crossing over between CYP11B1 (11beta-hydroxylase) and CYP11B2(aldosterone synthase) cause steroid 11beta-hydroxylase deficiency and congenital adrenal hyperplasia.

Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11beta-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1-6 of CYP11B2 and exons 7-9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11beta-hydroxylase and aldosterone synthase activities. Apparently the 11beta-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.

Recombinant CYP11B genes encode enzymes that can catalyze conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol.

CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) are 93% identical mitochondrial enzymes that both catalyze 11beta-hydroxylation of steroid hormones. CYP11B2 has the additional 18-hydroxylase and 18-oxidase activities required for conversion of 11-deoxycorticosterone to aldosterone. These two additional C18 conversions can be catalyzed by CYP11B1 if serine-288 and valine-320 are replaced by the corresponding CYP11B2 residues, glycine and alanine. Here we show that such a hybrid enzyme also catalyzes conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol. These latter two steroids are present at elevated levels in individuals with glucocorticoid suppressible hyperaldosteronism (GSH) and some forms of primary aldosteronism. Their production by the recombinant CYP11B enzyme is enhanced by substitution of further amino acids encoded in exons 4, 5, and 6 of CYP11B2. A converted CYP11B1 gene, containing these exons from CYP11B2, would be regulated like CYP11B1, yet encode an enzyme with the activities of CYP11B2, thus causing GSH or essential hypertension. In a sample of 103 low renin hypertensive patients, 218 patients with primary aldosteronism, and 90 normotensive individuals, we found a high level of conversion of CYP11B genes and four cases of GSH caused by unequal crossing over but no gene conversions of the type expected to cause GSH.