Robin E. Smith

Cloning and tissue distribution of the human 1 lβ-hydroxysteroid dehydrogenase type 2 enzyme

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) is thought to protect the non-selective mineralocorticoid receptor from occupation by glucocorticoids, and to modulate access of glucocorticoids to glucocorticoid receptors resulting in protection of the fetus and gonads. A ubiquitous low affinity NADP+ dependent enzyme (11 beta HSD1) and a tissue specific, high affinity NAD+ dependent form (11 beta HSD2) of 11 beta HSD exist. We now report the isolation of a cDNA coding for human 11 beta HSD2. The new isoform is NAD+ dependent, exclusively dehydrogenase in directionality, inhibited by glycyrrhetinic acid and metabolizes the synthetic glucocorticoid dexamethasone; it displays Km values for corticosterone and cortisol of 5.1 nM and 47 nM, respectively. Sequence alignment shows that 11 beta HSD2 shares 35% identity with 17 beta HSD2, but is only 14% identical with 11 beta HSD1. The 11 beta HSD2 gene is highly expressed in kidney, colon, pancreas and placenta and the message is also present in the ovary, prostate and testis. These data suggest that 11 beta HSD2 plays an important role in modulating mineralocorticoid and glucocorticoid receptor occupancy by glucocorticoids.

The type I and type II 11β-hydroxysteroid dehydrogenase enzymes

Abstract Local tissue concentrations of glucocorticoids are modulated by the enzyme 11 β -hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11 β -HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11 β -HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11 β -HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11 β -HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11 β -HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11 β -HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11 β -HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11 β -HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β -sheets, and an α -helix (active site), respectively.

Immunohistochemical and Molecular Characterization of the Rat 11β-Hydroxysteroid Dehydrogenase Type II Enzyme1

Mineralocorticoid action is facilitated by 11β-hydroxysteroid dehydrogenase type II (11βHSD2), which metabolizes glucocorticoids and allows aldosterone to bind to the nonselective mineralocorticoid receptor. We have recently demonstrated the presence of the 11βHSD2 protein in a wide range of human epithelia, suggesting that it is the sole isoform endowing specificity in man. In the present study we have used an immunopurified polyclonal antibody (RAH23) raised against a C-terminal peptide derived from the cloned rat 11βHSD2 protein to perform immunohistochemical and molecular analysis in rat tissues. In frozen sections of rat kidney, strong staining was seen with the RAH23 antibody in the distal tubule; weaker staining was observed in the thick ascending loop of Henle and the medullary and papillary collecting ducts. Punctate cortical staining was observed in the fetus at 20 days gestation and in 8-day-old rats, with a noticeable increase in the staining pattern at 16 days of age. The kidney did not attai...

Rat 11β-hydroxysteroid dehydrogenase type 2 enzyme is expressed at low levels in the placenta and is modulated by adrenal steroids in the kidney

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) protects the non-discriminating mineralocorticoid receptor from occupation by glucocorticoids. In man the enzyme is also highly expressed in the placenta where it is thought to also protect the fetus from the high circulating levels of maternal glucocorticoids. Mutations in the HSD11B2 gene have recently been shown to account for the syndrome of apparent mineralocorticoid excess. In the present study we have used a rat 11 beta HSD2 cDNA to study the distribution and regulation of this enzyme. The rat protein is highly homologous to the mouse, rabbit and human enzymes, except for the carboxy-terminal region which displays extensive divergence between species beyond residue 382. Northern blot analysis of rat total RNA showed that the single copy gene is highly expressed in kidney and adrenal with lower levels in the colon; surprisingly, there was no detectable signal in the placenta. There was also no detectable mRNA in the liver, heart, hippocampus, testis, thymus and pancreas. Nuclease protection analysis revealed the presence of moderate 11 beta HSD2 message levels in the parotid and exceedingly low levels in the placenta. Regulation studies showed that administration of dexamethasone, deoxycorticosterone and 9 alpha-fluorocortisol to adrenalectomized rats for 7 days increased renal enzyme activity 33%-50%, while message levels decreased 35%-70%, suggesting that the increased enzyme activity may represent activation of latent enzyme.

The glucocorticoid receptor is essential for maintaining basal and dexamethasone-induced repression of the murine corticosteroid-binding globulin gene.

We have investigated hepatic expression and glucocorticoid regulation of the corticosteroid-binding globulin (CBG) gene in mice lacking a functional glucocorticoid receptor (GR). GR-/- mice show impaired negative feedback in the hypothalamic-pituitary-adrenal axis, resulting in elevated circulating levels of ACTH and corticosterone. This is seen in the neonatal period and continues into adulthood where ACTH and corticosterone levels are increased up to 4-5 fold. Despite high elevation of corticosterone we find no change in mean arterial blood pressure in GR-/- mice and no change in the renal activity of the glucocorticoid-metabolising enzymes 11beta-hydroxysteroid dehydrogenase type-1 (HSD1) and type-2 (HSD2). We do find markedly increased hepatic expression of CBG with a 50% increase in plasma CBG levels. Increased expression of CBG was detected in adult GR-/- mice and also at birth with a greater than 10-fold increase in CBG hepatic mRNA in day-18.5 embryonic GR-/- mice. Adult GR-/- mice were also resistant to dexamethasone-induced repression of CBG expression in the liver. These results indicate that in mice, GR is essential for maintaining the basal level of CBG gene expression in the liver, and is also required for dexamethasone-induced repression of the CBG gene in the adult.

Characterization of 11β-HSD1B gene expression and enzymatic activity

Abstract Nuclease protection analysis has been used to study the distribution of an mRNA that has been predicted to give rise to a truncated form of the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD1B). The 11β-HSD1B mRNA was found exclusively in the kidney, predominantly localized within the medulla with low amounts of the message in the cortex. Neither the renal papilla nor a range of peripheral and central tissues showed evidence of 11β-HSD1B mRNA. Expression of the whole (11β-HSD1A) and truncated enzymes in COS cells resulted in immunoreactive 34 kDa and 26 kDa proteins respectively, while kidney homogenates showed 34 kDa and 40 kDa species. The 11β-HSD1A enzyme converted corticosterone and cortisol to their respective 11-dehydro metabolites with an absolute dependence on NADP as co-factor, while the 26 kDa 11β-HSD1B protein was unable to metabolize either steroid. These results suggest that transcription of the 11β-HSD1B mRNA is associated with a mechanism down-regulating production of 11β-HSD1 activity.

Sex- and tissue- specific regulation of 11β-hydroxysteroid dehydrogenase mRNA

Abstract In the present study we have investigated the effect of hypophysectomy on 11βHSD1 gene expression in a variety of tissues from male and female rats. Of the tissues analysed significant changes were observed only in the male and female livers, and in the female kidney. Hypophysectomy (hypox) increased hepatic message levels in the female 20-fold, and male levels rose 2-fold. Male renal 11βHSD1 message levels remained constant following hypophysectomy, whereas female renal 11βHSD1 mRNA levels increased approximately 2-fold. In intact animals hepatic 11βHSD1 mRNA levels are 18-fold lower in the female than the male, while renal levels are 1.4-fold higher in the male than the female. Hepatic 11βHSD1 levels in the female rat appear critically responsive to the pattern of growth hormone (GH) administration. Twice daily injections of GH had no significant effect on hepatic message levels in the hypox female rat, whereas constant infusions of GH, via an osmotic mini-pump produced a marked decrease. Renal 11βHSD1 mRNA levels were demonstrated to be under the regulation of estrogen, and 11βHSD1 enzyme activity in both liver and kidney correlated with the observed changes in mRNA levels. 11βHSD1 gene expression is thus regulated in a tissue-specific and sexually dimorphic manner.

The human 11β-hydroxysteroid dehydrogenase type II enzyme: Comparisons with other species and localization to the distal nephron

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

CLONING AND EXPRESSION OF A NOVEL TISSUE SPECIFIC 17BETA -HYDROXYSTEROID DEHYDROGENASE

The 11beta-hydroxysteroid dehydrogenases (11betaHSD) modulate intracellular glucocorticoid levels, with 11betaHSD1 converting cortisone to cortisol mainly in the liver, and 11betaHSD2 performing the reverse reaction in sodium transporting epithelia and placenta. We have attempted to expand the 11betaHSD subfamily by isolating homologous cDNAs. Expressed Sequence Tag databases were screen with segments of the 11betaHSD1 enzyme amino acid sequence and Pan1b identified as a new member of the short chain alcohol dehydrogenase superfamily. Northern blot analysis of total RNA from human tissues showed a single band at 1.9 kb and a tissue specific pattern of expression with high levels in the liver, adrenal carcinoma, lung and small intestine, and much lower levels in the kidney, heart and placenta. Expression studies in a Chinese hamster ovary cell line (CHOP) showed that Pan1b did not metabolize glucocorticoids. However, preliminary studies on a range of substrates revealed that Pan1b acted as a dehydrogenase on 17beta-hydroxysteroids, although further kinetic analysis was confounded by large amounts of endogenous oxidoreductase activity in CHOP cells. These studies suggest the existence of a novel human 17betaHSD enzyme.

Expression of the 11β-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells ☆

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11betaHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11betaHSD2 in 66% of the breast tumor samples. The 11betaHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11betaHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.