11beta-hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex

Although oxidation of cortisol or corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11beta-HSD type 2 (11beta-HSD2) gene, and found a marked 11beta-HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11-dehydrocorticosterone (DH-B), the inactive forms to which the former glucocorticoids are converted by 11beta-HSD. Addition of the 11beta-HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH-B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH-B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration-dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11beta-hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11beta-HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra-adrenal concentration of non-11beta-hydroxylated steroid hormones.     

Development of 11 beta-hydroxysteroid dehydrogenase expression in the rat cochlea

Spontaneous coronary dissection is rare and the diagnosis is usually post-mortem. Less than 60 cases have been diagnosed at coronary angiography. The authors report, to the best of their knowledge, the first case of multiple spontaneous coronary artery dissections in a type IV Ehlers-Danlos syndrome in a young woman admitted to hospital for acute myocardial infarction. She had a previous history of regressive complete tetraplegia due to dissection of the basilar artery and episodes of dizziness related to a dissecting aneurysm of the left vertebral artery. The diagnosis of type IV Ehlers-Danlos syndrome was established after skin biopsy had shown typical histological changes. The patient died several months later after an acute abdominal syndrome probably related to dissection of the aorta. An autopsy was refused by her family. The authors believe this to be the first case of spontaneous coronary dissection related to a type IV Ehlers-Danlos syndrome.     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.     

Functional activity of 3beta-hydroxysteroid dehydrogenase/isomerase

3Beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding the human wild-type I (placental) enzyme and the human type I mutant- Y253F. The wild-type and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells. Ultraviolet (UV) spectral analyses showed that the wild-type enzyme induced changes in the UV spectrum of the competitive isomerase inhibitor, 19-nortestosterone, and the Y253F mutant did not. The wild-type isomerase required activation by coenzyme to produce the spectral shift. Activation of isomerase by NADH produced a greater change in the 19-nortestosterone spectrum than activation by NAD+. These observations provide direct evidence that Tyr253 functions as the general acid (proton donor) in the isomerase reaction mechanism. Furthermore, the coenzyme-activation profiles support our proposed two-step enzyme mechanism in which NADH produced by the 3beta-HSD activity induces the enzyme to assume the isomerase conformation.     

IGFBP-2 expression in a human cell line is associated with increased IGFBP-3 proteolysis, decreased IGFBP-1 expression and increased tumorigenicity

Insulin-like growth factors (IGF-I and -II) play an active role in cell proliferation. In biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP-2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP-1 and -3 and small amounts of IGFBP-4, but no IGFBP-2, was stably transfected with an expression vector comprising IGFBP-2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP-2-expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP-2 expression in these tumours, were a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis, both of which increase the bioavailability of the IGF-II produced by the cells. The increased IGFBP-3 proteolysis most probably resulted from amplified expression of tissue-type plasminogen activator (t-PA) and depression of its inhibitor (PAI-I) observed in IGFBP-2-expressing xenografts. Our findings suggest that IGFBP-2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF-II bioavailability.     

Development of decompensated dilated cardiomyopathy is associated with decreased gene expression and activity of the milrinone-sensitive cAMP phosphodiesterase PDE3A

BACKGROUND: Phosphodiesterase III (PDE3) inhibitors are inotropic agents used to treat congestive heart failure (CHF) and are less effective in patients with severe CHF. Little is known about relative changes in PDE3 activity or gene expression during the evolution of cardiomyopathy. METHODS AND RESULTS: In the present study, we evaluated temporal changes in PDE3A gene expression before and after pacing-induced CHF in nine mongrel dogs. Three weeks of left ventricular (LV) pacing produced LV end-diastolic pressures of 15+/-1.7 mm Hg, whereas overt CHF at 4 to 5 weeks was associated with LV end-diastolic pressures of 24+/-1.7 mm Hg; prepacing values were 6.6+/-0.6 mm Hg. Total RNA isolated from LV tissues was analyzed on Northern blots; 10 unpaced normal hearts served as tissue controls. Signals for PDE3A mRNAs (7, 8, and 10 kb) or PDE4D (7.6 kb) were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal 18S RNA. Before the onset of CHF, PDE3A/GAPDH ratios were not different between the control and 3-week paced groups. In contrast, all PDE3A/GAPDH ratios were selectively reduced by 52%, and PDE3A/18S was reduced by 70% (P<.05) in CHF; PDE4D/GAPDH (or 18S) was unchanged. LV tissues from four control and four CHF dogs were also processed to isolate cytosolic and microsomal membrane protein for cAMP PDE3 activity assays. CHF was associated with a significant 54% reduction (P<.05) in microsomal but not cytosolic PDE3 activity. CONCLUSIONS: Selective downregulation of PDE3A may account in part for the ineffectiveness of milrinone in the treatment of severe CHF.     

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 cDNA's. 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.     

Renal 11 beta-hydroxysteroid dehydrogenase activity is enhanced by ramipril and captopril

Changes of renal 11 beta-hydroxysteroid dehydrogenase activity may contribute to variations of sodium excretion by modulating inactivation of cortisol or corticosterone and thus their access to mineralocorticoid receptors. Angiotensin-converting enzyme inhibitors enhance sodium excretion but by mechanisms still incompletely understood. To test the hypothesis that the angiotensin-converting enzyme inhibitors ramipril and captopril act in part by enhancing renal 11 beta-hydroxysteroid dehydrogenase activity, the effects of these agents in slices of rat renal outer medulla were examined. Conversion of 3H-corticosterone to 3H-11-dehydrocorticosterone was 58% greater in tissue from fasted rats than from fed rats (mean +/- SE 2467 +/- 146 vs. 1584 +/- 102 pmol/mg protein.h, P < 0.01). Incubation of tissue from fed rats with physiological concentrations of ramiprilat, the active form of ramipril, enhanced activity (1497 +/- 76) to fasted levels (2323 +/- 120, P < 0.02). Captopril had a similar in vitro effect (1557 +/- 92 to 2109 +/- 116, P < 0.01). Ramipril given in vivo to fed rats also increased activity to fasted levels (1716 +/- 101 to 2737 +/- 396, P < 0.05). Angiotensin II incubated with renal tissue from fasted rats suppressed activity to fed levels, but this effect was prevented by the presence of ramiprilat. Both ramipril and captopril enhance renal 11 beta-hydroxysteroid dehydrogenase activity, and this effect is only partly explained by limitation of endogenous angiotensin II production.     

Ultrastructural demonstration of 3 beta-hydroxysteroid dehydrogenase activity using potassium ferricyanide

Sections of Rat and Amphibian adrenocortical tissue fixed in a mixture of 1% formaldehyde and 0.25% glutaraldehyde, are incubated in a medium containing namely a 3 beta-hydroxysteroid (substrate), NAD, potassium ferricyanide (hydrogen acceptor) and copper sulfate. A hyaloplasmic copper ferrocyanide precipitate is observed in the immediate vicinity of the smooth endoplasmic reticulum membranes, or in close contact with them. This reaction, which does not occur in media lacking the substrate or containing cyanoketone, is a result of 3 beta-hydroxysteroid dehydrogenase activity. Problems concerning the precise localization of this enzyme are discussed.     

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

During early neonatal life, important changes occur in the gut. The intestine is challenged by both milk and a microbial flora. Later on, at weaning, the diet of mice changes from milk to pelleted food leading to changes in microbial contents. This period seems essential for a complete development of the mucosal immune system. We investigated the development of both intraepithelial (IEL) and lamina propria lymphocytes (LPL), from day 5, and every 5 days, up to day 30 after birth. IEL and LPL were isolated from the small intestine and the phenotype was assessed by FACS analyses, using antibodies for detection of T-cell markers CD3, TCR alpha beta, TCR gamma delta, CD4, CD8 alpha, CD8 beta, CD5, CD18, CD54, and CD49d. Our data show a clear increase in the number of LPL just before weaning, while the number of IEL increased after day 15. A more mature pattern of membrane antigen expression of both IEL and LPL was observed at weaning. The adhesion molecules CD18, CD54, and CD49d, essential for cellular communication of lymphocytes, showed an expression peak at weaning. In conclusion, the mouse mucosal immune system develops during the first 3 weeks of neonatal life leading to the formation of a more mature immune system at weaning.     

Activation of the K-ras oncogene in colorectal neoplasms is associated with decreased apoptosis

BACKGROUND: Recent in vitro data indicate that the oncogenic effects of activated ras genes may be mediated, at least in part, through inhibition of apoptotic cell death. To examine this proposition in vivo, the relationship between mutations of the K-ras gene and the frequency of apoptosis was studied in a series of 69 sporadic colorectal neoplasms (11 adenomas and 58 carcinomas). METHODS: Mutations in codon 12 of K-ras were determined by a single tube, enriched polymerase chain reaction. Apoptotic cells in tumor sections were identified by in situ end-labeling of fragmented DNA, whereas levels of bcl-2 and p53 proteins were determined by immunohistochemistry. RESULTS: Tumors with mutant K-ras had a significantly lower apoptotic index than those with the wild-type allele (P < 0.05). They were also more likely to exhibit positive bcl-2 staining (P < 0.05). Adenomas showed significantly greater bcl-2 positivity than carcinomas (89% and 51%, respectively; P < 0.05). The frequency of apoptosis in these tumors was not related to either bcl-2 positivity or p53 status. CONCLUSIONS: These findings suggest that activation of K-ras in colorectal carcinoma may inhibit apoptosis and thus favor tumor progression. Alternatively, this association may reflect an accumulation of K-ras mutations in cells in which normal apoptotic pathways have been impaired.     

Ultracytochemical demonstration and probable localization of 3beta-hydroxysteroid dehydrogenase activity with a ferricyanide technique

In order to localize 3beta-hydroxysteriod dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3beta-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium. A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. the addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.     

Dihydropyrimidine dehydrogenase but not thymidylate synthase expression is associated with resistance to 5-fluorouracil in colorectal cancer

BACKGROUND/AIMS: In planning adjuvant treatment of colorectal cancer, it is of critical importance to optimize the treatment by identifying subsets of patients that will respond or not to chemotherapy. Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are key enzymes involved in the biochemical functions of the antimetabolite 5-fluorouracil (5-FU). In searching for the factors determining the 5-FU sensitivity of colorectal cancer, TS and DPD were analyzed in relation to the inhibitory effect of 5-FU on cell proliferation in a series of human colorectal cancer cell lines. METHODOLOGY: TS and DPD protein expressions were quantified in 5 human colorectal cancer cell lines, using TS binding assay and Western blotting, respectively. Cellular growth inhibition was assessed by MTT assay after 48 hours of continuous exposure to 5-FU or cisplatin (CDDP). RESULTS: TS protein expression was detected in all but one of the cell lines studied and varied within a 17-fold range, while DPD protein expression was detectable in only one cell line (CaR1). CaR1, which expressed the highest level of DPD and no detectable TS, showed remarkable resistance to 5-FU. The other colorectal cancer cell lines with undetectable DPD expression were sensitive to 5-FU. There was no correlation between TS expression and 5-FU sensitivity. All of the cell lines studied showed similar sensitivity to CDDP. CONCLUSIONS: These data suggest that DPD, but not TS, expression predicts 5-FU sensitivity in colorectal cancer cell lines.     

N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn394-Leu395-Ser396) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.     

Cloning of zebrafish cDNA for 3beta-hydroxysteroid dehydrogenase and P450scc

P450scc and 3beta-HSD cDNA were isolated from a zebrafish lambda gt10 cDNA library using trout SCC and 3beta-HSD cDNA as the probes. The zebrafish SCC cDNA encodes a protein of 509 amino acids, which shares a 78% similarities with the trout SCC and 58% with the human SCC. As for 3beta-HSD, two forms of cDNA were isolated, termed HSD 5 and HSD 17, which may have resulted from alternative splicing. HSD 5 and HSD 17 encode proteins of 374 and 341 amino acids respectively. Both share 77% amino acid similarities with trout 3beta-HSD and 53% similarities with the mouse 3beta-HSD. Zebrafish has been increasingly used as a genetic model system to study organ development and to investigate human diseases. The cloning and the characterization of zebrafish P450scc and 3beta-HSD should facilitate study of steroidogenesis and human disease associated with steroid imbalance.     

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.