ABSTRACT

11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11β-HSD exist. One isoform (11β-HSD₁) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11β-HSD₁ cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 β-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11β-HSD₁ does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11β-HSD isoform. 11β-HSD₁ is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11β-HSD₁ activity in intact mammalian cells, and the possible role of 11β-HSD in regulating glucocorticoid access to GRs, we transfected rat 11β-HSD₁ cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11β-HSD activity or mRNA expression.

Homogenates of COS-7 cells transfected with increasing amounts of 11β-HSD cDNA exhibited a dose-related increase in 11 β-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11β-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11β-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11β-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11β-HSD, but induced MMTV-LTR luciferase activity in the presence of 11β-HSD. These results indicate that rat 11β-HSD₁ can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11β-HSD₁ may regulate ligand access to GRs by reactivating inert glucocorticoids.

Understanding of the principal pathways of steroid hormone biosynthesis was established over two decades ago through advances in steroid radioisotopic and chromatographic techniques. When the enzymes of individual pathways could be examined in more detail, the dissection of the complex pattern of enzyme activities began. At many points, separate pathways employ precisely the same enzyme for equivalent catalytic steps, e.g. for 21-hydroxylase, 11 β-hydroxylase, aromatase and several dehydrogenases (Orth et al. 1992). A further economy was found for 17α-hydroxylase and 17,20-lyase activities, which co-purify with the same P450c17 polypeptide. This enzyme was later cloned and expressed in tissue culture cells, revealing that, contrary to the enzyme in rat, human and cattle, 17α-hydroxylase cannot convert 17α-hydroxyprogesterone to androstenedione (Bradshaw et al. 1987, Fevold et al. 1989). Further complexity emerged with the existence of multiple tissue-specific forms of 5α-reductase (Wilson et al. 1993), and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases, most of which

ABSTRACT

A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). Circulating concentrations of Δ⁵ steroids and Δ⁵ urinary steroid metabolites were elevated and remained elevated after orchidectomy. There was no evidence of salt loss, plasma renin being within normal limits, and no detectable glucocorticoid abnormality. The coding sequences of the genes for 3β-HSD types I and II were amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis (DGGE) and manual and automatic DNA sequencing. A mutation in the gene for 3β-HSD type II was observed at codon 173 (CTA→CGA), leading in the affected patient to a homozygous substitution in which the leucine at residue 173 was altered to an arginine (L173R). The propositus's 2-year-old XX sister was also homozygous for L173R and showed the biochemical characteristics of partial 3β-HSD deficiency without clinical symptoms or signs. The mutation segregated as an autosomal recessive. Three related heterozygous adult females showed evidence of a small over-production of Δ⁵ steroids and steroid metabolites and a variable reduction in ovarian function. Concentrations of Δ⁵ steroids and steroid metabolites in the heterozygous father of the propositus were within the normal range.

These data are discussed in relation to the endocrine causes of pseudohermaphroditism and hirsutism. Evidence for tight linkage between the genes for 3β-HSD types I and II was obtained using a microsatellite polymorphism in the third intron of the gene for 3β-HSD type II and synonymous and non-synonymous mutations and polymorphisms in the gene for 3β-HSD type I. The latter polymorphisms were located 88 bp apart at the 3′ end of the type I coding sequence and could be physically resolved as haplotypes using DGGE. The application of DGGE to the analysis of mutations in members of a multigene family is discussed.

ABSTRACT

The PCR technique and highly degenerate oligonucleotide primers were used to amplify a 282 bp fragment of the horse (Equus caballus) epidermal growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60–70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4·9 kb transcript in horse kidney and endometrial RNA which was of a similar size to the mature EGF transcript found in other mammalian species.

The horse cDNA clone was used in Northern blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term=330–340 days). The level of expression increased from day 33 and showed a further dramatic increase between days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. The horse DNA sequence was used to design sense and antisense oligonucleotide probes (45-mers) for in situ hybridization studies. The antisense probe showed specific hybridization to the glandular, but not lumenal, epithelial cells of the endometrium and there was no signal in fetal membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot analysis. This dramatic increase in EGF expression in the glandular epithelium of the mare's endometrium during pregnancy may provide a mitogenic stimulus to the endometrium and/or trophoblast to facilitate placental differentiation and attachment. Alternatively, the precursor could be involved in the endometrial gland secretory process which is necessary to produce uterine milk for fetal sustenance.

The PCR cloning methods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.