Regulation of sodium-potassium adenosine triphosphate subunit gene expression by corticosteroids and 11 beta-hydroxysteroid dehydrogenase activity.

The induction of Na,K-ATPase plays a vital role in mediating epithelial sodium transport. Although its activity is regulated by corticosteroids, it is uncertain whether this is predominantly by mineralo- or glucocorticoid mechanisms. 11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the interconversion of active corticosterone (B) to inactive 11-dehydrocorticosterone and protects the nonselective mineralocorticoid receptor (MR) from glucocorticoid excess. We have studied the regulation of the alpha 1- and beta 1-subunits of Na,K-ATPase by mineralo- and glucocorticoids in vitro and in vivo, and how this is modulated by 11 beta HSD activity. Cultured rat kidney epithelial cells (NRK 52-E) expressed 11 beta HSD activity, which was inhibited by the licorice derivative glycyrrhetinic acid (GE). Dexamethasone, aldosterone, and high concentrations of B (1-10 microM) increased Na,K-ATPase alpha 1 and beta 1 messenger RNA (mRNA) levels, an effect that was inhibited by coincubation with the MR antagonist RU 26752, but not by the glucocorticoid receptor antagonist RU 38486. GE, which itself reduced Na,K-ATPase alpha 1/beta 1 mRNA levels, potentiated the action of B, so that low concentrations of B (10 nM) increased Na,K-ATPase alpha 1/beta 1 mRNA levels. In contrast, in vivo, RU 26752 and RU 38486 given ip for 4 days (n = 6/group) reduced renal Na,K-ATPase alpha 1 and beta 1 levels. Glycyrrhizic acid also inhibited both renal 11 beta HSD mRNA and activity and levels of Na,K-ATPase alpha 1/beta 1 mRNA. In vivo renal Na,K-ATPase subunit mRNA levels are regulated by both mineralo- and glucocorticoid mechanisms. In vitro, however, although NRK 52-E cells expressed the glucocorticoid receptor, corticosteroid regulation of Na,K-ATPase, even by dexamethasone, occurred exclusively via the MR, suggesting that accessory transcription factors required for glucocorticoid hormone action are absent in this cell line. Finally, although the licorice derivatives GE and glycyrrhizic acid reduced Na,K-ATPase alpha 1/beta 1 mRNA levels, they also potentiated the stimulatory effect of B by inhibiting its metabolism via 11 beta HSD, establishing 11 beta HSD as an important prereceptor modulator of mineralocorticoid hormone action.

Epithelial cell localization of type 2 11 beta-hydroxysteroid dehydrogenase in rat and human colon.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD), by catalyzing the interconversion of active corticosterone (B) to inactive 11-dehydrocorticosterone (A) in the rat and cortisol (F) to cortisone in man, maintains normal in vivo specificity of the mineralocorticoid receptor (MR) in both kidney and distal colon. Two isoforms of 11 beta HSD have been reported: the cloned type I, NADP(H)-dependent 11 beta-dehydrogenase/oxo-reductase, and a high affinity NAD+-dependent 11 beta-dehydrogenase (type 2 isoform). Previous studies indicate that the MR in the distal colon is localized to ion-transporting surface epithelial cells and non-epithelial neuroendocrine cells within the lamina propria. We have now analyzed the expression and activity of 11 beta HSD in specific cells isolated from both rat and human colonic mucosa by a chemical shear and microdissection method. Both isoforms of 11 beta HSD were detected in rat and human colonic mucosa. Type 2 11 beta HSD activity, with an apparent Km (mean +/- SE) of 56.3 +/- 2.2 nM for B in the rat and 35.3 +/- 1.2 nM for F in man, was exclusively localized to surface and crypt epithelial cells. In contrast, the type I isoform in the rat, with an apparent Km of 0.95 +/- 0.14 microM for B, was localized exclusively to specific nonepithelial cells in the lamina propria. Human colon type I 11 beta HSD, however, which has an apparent Km for F of 0.51 +/- 0.04 microM, was present in both the lamina propria and the surface epithelium. Northern blot analysis of rat colonic RNA using a 32P-labeled complementary DNA probe for rat type I 11 beta HSD confirmed the presence of type I 11 beta HSD messenger RNA in intact distal colon mucosa, but failed to detect 11 beta HSD messenger RNA in surface epithelial cells. In conclusion, abundant levels of a high affinity NAD(+)-dependent type 2 11 beta HSD isoform are expressed in both rat and human colon. Colonic type 2 11 beta HSD is kinetically distinct from the low affinity NADP-dependent type I isoform, behaves predominantly as a dehydrogenase, is localized exclusively to the ion-transporting epithelia, and is likely to be the product of a second 11 beta HSD gene. Furthermore, the spatially distinct patterns of expression of these isoforms suggest that in vivo there are two physiologically distinct populations of MR in the colon: the aldosterone selective MR in the epithelium and the nonselective MR in the nonepithelial cells within the lamina propria.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Immunohistochemical localization of type 1 11beta-hydroxysteroid dehydrogenase in human tissues.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11betaHSD) catalyze the interconversion of hormonally active cortisol to inactive cortisone. Activity and messenger ribonucleic acid studies indicate that type 1 11betaHSD (11betaHSD1) is expressed in glucocorticoid target tissues such as liver, gonad, and cerebellum, where it regulates the exposure of cortisol to glucocorticoid receptors. To further understand the role of 11betaHSD1 in human tissues, we have studied the localization of this isozyme using an antibody raised in sheep against amino acids 19-33 of human 11betaHSD1. Western blot analyses indicated that the immunopurified antibody recognized a band of approximately 34 kDa in human liver and decidua. Immunoperoxidase studies on liver, adrenal, ovary, decidua, and adipose tissue indicated positive cytoplasmic staining for 11betaHSD1. 11BetaHSD1 immunoreactivity was observed more intensely around the hepatic central vein, with no staining around the portal vein, hepatic artery, or bile ducts. No staining for 11betaHSD1 was observed in the adrenal medulla, but 11betaHSD1-immunoreactive protein was observed in all three zones of the adrenal cortex, with the most intense staining in the zona reticularis > zona glomerulosa > zona fasciculata. In the human ovary, immunoreactivity was observed in the developing oocyte and the luteinized granulosa cells of the corpus luteum. No staining was observed in granulosa cells, thecal cells, or ovarian stroma, which contrasted with the marked expression of 11betaHSD2 in the granulosa cell layer. Sections of human decidua showed high expression of 11betaHSD1 in decidual cells. In omental adipose tissue, 11betaHSD1 immunoreactivity was observed in both stromal and adipocyte cells. Immunohistochemical localization of 11betaHSD1 in human liver, adrenal, ovary, decidua, and adipose tissue using this novel antiserum provides us with a tool to investigate the role of this isozyme in modulating glucocorticoid hormone action within these tissues.

New insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3betaHSD) deficiency is a form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene and causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. To identify the molecular lesion(s) in the HSD3B2 gene in the 11 patients from the seven new families suffering from classical 3betaHSD deficiency, the complete nucleotide sequence of the whole coding region and exon-intron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics laboratory in France, whereas the two other families were investigated by Peter's group in Germany. Functional characterization studies were performed by Simard's group in Canada. Following transient expression in 293 cells of each of the mutant recombinant proteins generated by site-directed mutagenesis, the effect of the 25 mutations on enzyme activity was assessed by incubating intact cells in culture with 10 nM [14C]-DHEA as substrate. The stability of the mutant proteins has been investigated using a combination of Northern and Western blot analyses, as well as an in vitro transcription/translation assay using rabbit reticulocyte lysates. The present report describes the identification of 8 mutations, in seven new families with individuals suffering from classical 3betaHSD deficiency, thus increasing the number of known HSD3B2 mutations involved in this autosomal recessive disorder to 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 missense mutations). In addition to the mutations reported here in these new families, we have also investigated for the first time the functional significance of previously reported missense mutations and or sequence variants namely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized. Furthermore, their effects have been compared with those of the 10 previously reported mutant enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3betaHSD type 2 isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form of CAH due to classical 3betaHSD deficiency. Whereas the nonsalt-losing form also results from missense mutation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme activity, thus leaving sufficient enzymatic activity to prevent salt wasting. The functional data described in the present study concerning the sequence variants A167V, S213G, K216E and L236S, which were detected with premature pubarche or hyperandrogenic adolescent girls suspected to be affected from nonclassical 3betaHSD deficiency, coupled with the previous studies reporting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in such patients strongly support the conclusion that this disorder does not result from a mutant 3betaHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3betaHSD deficiency involving protein instability and further illustrates the complexity of the genotype-phenotype relationships of this disease, in addition to providing further valuable information concerning the structure-function relationships of the 3betaHSD superfamily.

Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 activity and mRNA in human choriocarcinoma cells.

The type 2 isoform of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2), which catalyzes the conversion of cortisol to hormonally inactive cortisone in man, is principally expressed in the placenta and mineralocorticoid target tissues, kidney and colon. To date, few studies have addressed the regulation of this novel 11 beta-HSD2 isoform. We have characterized the nature and regulation of the 11 beta-HSD activity expressed in a human cytotrophoblastic cell line, the JEG-3 choriocarcinoma cell. The 11 beta-HSD activity in JEG-3 cell homogenates required NAD+ as cofactor with NADP+ ineffective and demonstrated a high affinity for cortisol (apparent Km 31 nM). Incubation of JEG-3 cells with forskolin and dibutyryl cyclic AMP increased 11 beta-HSD2 activity several-fold in a time-dependent manner, while treatment with phorbol ester had little, if any, effect on 11 beta-HSD2 activity. Northern blot analysis of RNA isolated from JEG-3 cells after these treatments demonstrated a marked increase in a 1.9 kb 11 beta-HSD2 mRNA species in cells treated with forskolin for 24 h. We conclude that 11 beta-HSD2 is regulated by activation of the protein kinase A pathway, but not the protein kinase C pathway in human choriocarcinoma cells, and that this regulation occurs at a pretranslational level. JEG-3 cells provide an excellent model for further studies on the regulation of 11 beta-HSD2 gene expression in human trophoblast tissue.

Regulation of 11 beta-hydroxysteroid dehydrogenase type 1 in primary cultures of rat and human hepatocytes.

Two isozymes of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) are responsible for the interconversion of the active glucocorticoid, cortisol in man, (corticosterone in the rodent), to the inactive 11-keto metabolite, cortisone (11-dehydrocorticosterone). We have examined the regulation of type 1 11 beta-HSD (11 beta-HSD1) using primary cultures of rat and human hepatocytes, both of which express only 11 beta-HSD1. Only 11 oxo-reductase activity could be demonstrated in cultured hepatocytes (apparent Km for cortisone 382 +/- 43 nM in human hepatocytes, apparent Km for 11-dehydrocorticosterone 14.6 +/- 1.5 microM in rat hepatocytes). There exists a marked discrepancy between 11 beta-HSD oxo-reductase activity and 11 beta-HSD1 mRNA levels in cultured human hepatocytes and human liver. Thus oxo-reductase specific activity is much higher in the cultured hepatocytes (7.2 +/- 0.01 nmoles cortisol/mg/h vs 0.89 +/- 0.06 for whole liver homogenates) whilst the converse is true for steady state 11 beta-HSD1 mRNA levels (0.78 +/- 0.02 vs 1.94 +/- 0.07 in whole liver, 11 beta-HSD1/18S expressed as arbitrary units). Carbenoxolone has a significant inhibitory effect on 11 oxo-reductase activity in both rat and human hepatocytes. However, there is clear species-specific regulation of 11 oxo-reductase activity by thyroid hormone (tri-iodothyronine (T3)), which increases 11 oxo-reductase activity in rat hepatocytes but has no effect on activity in human hepatocytes, and progesterone which inhibits activity in human hepatocytes, but has no effect on activity in rat hepatocytes. Neither T3 nor progesterone altered 11 beta-HSD1 mRNA levels. A series of growth factors (hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor beta 1) were without effect on 11 oxo-reductase activity in cultured rat hepatocytes. In contrast to homogenates of human liver, cultured hepatocytes express only 11 beta-HSD oxo-reductase activity. This is inhibited by carbenoxolone and shows species-specific regulation by T3 and progesterone. Growth factors do not appear to regulate activity or expression of 11 beta-HSD1. The discrepant enzyme activity data and 11 beta-HSD1 mRNA expression in hepatocytes and whole liver could reflect unstable 11 beta-HSD1 oxo-reductase activity or, alternatively, an additional 11 beta-HSD oxo-reductase isoform in cultured hepatocytes.

Ontogeny and sexual dimorphic expression of mouse type 2 11beta-hydroxysteroid dehydrogenase.

11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the interconversion of cortisol to hormonally inactive cortisone (corticosterone (B) to 11-dehydrocorticosterone (A) in rodents), and as such is established as a pre-receptor signalling pathway for corticosteroid hormone action. To further evaluate the role of this enzyme in adult and fetal life we have characterized two isoforms of 11beta-HSD in mouse tissues. Mouse 'liver' or type 1 11beta-HSD is a bi-directional dehydrogenase/oxo-reductase (K(m) for B 1.9 microM, K(m) for A 0.73 microM). Oxo-reductase activity utilized only NADPH as a co-factor, whilst dehydrogenase activity increased with both NAD or NADP. Mouse 'kidney' or 11beta-HS3D2 activity was NAD-dependent with a K(m) for B of 0.11 microM. Dexamethasone was not a substrate. Using an in-house mouse 11beta-HSD2 cDNA and NAD-dependent activity studies, 11 beta-HSD2 was expressed in epithelial cells of colon, renal collecting ducts, ovary, and adrenal, but was absent in liver, spleen, testis and heart. With the exception of gonadal tissues, activity and mRNA levels were consistently higher in adult male versus female tissues. In fetal kidney and colon there was absent/low levels of 11beta-HSD2 expression from fetal day 15 to term (day 19/20). Placental 11beta-HSD2 mRNA and activity were highest on fetal day 13/14 and fell progressively to undetectable levels by term. Two isoforms of 11beta-HSD are present in mouse tissues in accordance with other mammalian species. The sexual-dimorphic expression 11 beta-HSD2 in kidney and colon may reflect male-female differences in sodium homeostasis, and the absent expression of 11 beta-HSD2 in late gestation may facilitate glucocorticoid-dependent maturation of mouse fetal tissues.

Detection of human 11 beta-hydroxysteroid dehydrogenase isoforms using reverse-transcriptase-polymerase chain reaction and localization of the type 2 isoform to renal collecting ducts.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), responsible for the interconversion of hormonally active cortisol to inactive cortisone, dictates specificity for the mineralocorticoid receptor (MR) in the distal nephron and colon. Two isoforms of human 11 beta-HSD have been cloned, an NADP(H)-dependent (type 1) dehydrogenase/oxo-reductase enzyme, and a high-affinity NAD-dependent (type 2) unidirectional dehydrogenase. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of RNA extracted from human adult tissues, type 1 11 beta-HSD mRNA was found in decidua, placenta, liver, lung, spleen, kidney medulla, cerebellum and pituitary, but was absent in kidney cortex, sigmoid and rectal colon, salivary gland and thyroid. In contrast, type 2 11 beta-HSD mRNA was found only in placenta and in the classical mineralocorticoid target tissues, kidney cortex, kidney medulla, sigmoid and rectal colon, salivary gland, and colonic epithelial cell lines (AAC1 and RGC28). In situ hybridization studies of renal cortex, cortico-medullary junction and medulla using a 35S-labeled antisense cRNA probe for type 2 human 11 beta-HSD, revealed specific localization of type 2 11 beta-HSD mRNA expression exclusively to renal cortical and medullary collecting ducts. Type 1 and type 2 isoforms of human 11 beta-HSD are expressed in a distinct tissue-specific fashion, in keeping with the proposed differences in their physiological roles. Type 2 11 beta-HSD is found predominantly in mineralocorticoid target tissues where it serves to protect the MR in an autocrine fashion.

Regulation of 11beta-hydroxysteroid dehydrogenase type 2 by diuretics and the renin-angiotensin-aldosterone axis.

In the kidney and colon 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) inactivates cortisol to cortisone, thereby protecting the non-selective mineralocorticoid receptor from cortisol. Deficiency of 11beta-HSD2 results in cortisol-mediated sodium retention and hypertension, suggesting that the physiological regulation of 11beta-HSD2 in mineralocorticoid target tissues may be important in modulating sodium homoeostasis and blood pressure control. Using the human epithelial colon cell line SW-620, reverse transcriptase-polymerase chain reaction and enzyme kinetic analysis indicated expression of only 11beta-HSD2 (Km for cortisol 66 nmol/l). Bradykinin (10(-8) to 10(-12) mol/l), frusemide (10(-4) to 10(-9) mol/l), benzamiloride hydrochloride (10(-5) to 10(-10) mol/l) and atrial natriuretic peptide (10(-6) to 10(-10) mol/l) had no effect on 11beta-HSD2 expression. Using a range of concentrations of angiotensin II (2x10(-8) to 2x10(-5) mol/l) a significant reduction in activity was seen but only at supra-physiological concentrations, [e.g. 2x10(-6) mol/l at 4 h pretreatment: 36.7+/-2.0 pmol cortisone. h-1.mg-1 (mean+/-S.E.M.) compared with 45.1+/-1.7 pmol.h-1.mg-1 in control; P<0.05]. The angiotensin-converting enzyme inhibitors captopril, enalapril, lisinopril, perindopril, quinapril and trandolapril at 10(-7) mol/l, but not fosinopril, significantly increased 11beta-HSD2 activity after pretreatment for 16 or 24 h (P<0.05-P<0.01 compared with control). No effects were seen at 4 h pretreatment. Hydrochlorothiazide (10(-7) mol/l) significantly decreased 11beta-HSD2 activity (P<0.05 compared with control) at 4 h pretreatment. Commonly used diuretics, atrial natriuretic peptide and physiological concentrations of angiotensin II and bradykinin do not alter 11beta-HSD2 activity. In contrast, a series of angiotensin-converting enzyme inhibitors significantly increase 11beta-HSD2 activity in vitro. This may explain how intrarenal infusions of angiotensin-converting enzyme inhibitors increase renal sodium excretion independent of circulating concentrations of angiotensin II. The interaction between angiotensin-converting enzyme inhibitors and 11beta-HSD2 may be an additional mechanism by which the former can lower blood pressure.

A new insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) deficiency is a rare form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene, causing varying degrees of salt-loss in both sexes and incomplete masculinization of the external genitalia in genetic males. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing and 23 missense mutations) have been identified in the HSD3B2 gene. Results from functional charaterization studies of the mutant proteins agrees with the prediction that no functional type II 3beta-HSD isoenzyme is expressed in the adrenals and gonads of the patients with the severe salt-losing form, whereas the nonsalt-losing form causes an incomplete loss in enzymatic activity, thereby leaving sufficient enzymatic activity to prevent salt loss. Recent studies have highlighted the fact that various mutations appear to have a drastic effect upon the stability of the protein, therefore providing molecular evidence of a new mechanism involved in classical 3beta-HSD deficiency. Finally, the functional characterization of the missense mutations known to be involved in this autosomal recessive disorder provides valuable information concerning the structure-function relationships of the 3beta-HSD enzyme superfamily.

The synthesis of novel matrix metalloproteinase inhibitors employing the Ireland-Claisen rearrangement.

Matrix metalloproteinase inhibitors of general formula (1) were synthesised by a route involving an Ireland-Claisen rearrangement which enables systematic modification of the substituent alpha to the hydroxamic acid. An analogue (12c) possessing an alpha-cyclopentyl group is a potent broad spectrum inhibitor that displays high and sustained blood levels following oral dosing in both the rat and marmoset ex-vivo bioassays. This compound and analogues are also potent inhibitors of TNF alpha release.

The synthesis and biological evaluation of non-peptidic matrix metalloproteinase inhibitors.

Novel sulfonamide matrix metalloproteinase inhibitors of general formula (9) were synthesised by a route involving a stereoselective conjugate addition reaction. Enzyme selectivity was found to be dependant on the nature of the sulfonamide substituents. Compounds (9f, 9q) are potent selective collagenase inhibitors with good oral bioavailability.