11beta-hydroxysteroid dehydrogenase 1 and 2 expression in colon from patients with ulcerative colitis

BACKGROUND AND AIM: 11beta-hydroxysteroid dehydrogenase (11betaHSD) is an enzyme responsible for the interconversion of active 11beta-hydroxysteroids (cortisol) into biologically inactive 11-oxosteroids (cortisone). The isoform 11betaHSD1 operates predominantly as a reductase converting cortisone to cortisol, whereas 11betaHSD2 catalyzes oxidation of cortisol to cortisone. This mechanism of peripheral metabolism of glucocorticoids has been suggested to be involved in increasing the availability of anti- inflammatory glucocorticoids as a response to inflammatory stimuli. The aim of this study therefore was to investigate the impact of inflammatory bowel disease on the expression of colonic 11betaHSD1 and 11betaHSD2. METHODS: Quantitative real-time RT-PCR was used to assess messenger RNA for 11betaHSD1 and 11betaHSD2 in bioptic samples taken from patients with ulcerative colitis and in healthy controls, and in colon of rats with colitis induced by dextran sulfate sodium (DSS). Rat colonic fragments were used for assessment of local metabolism of glucocorticoids. RESULTS: In both human and rat specimens colitis up-regulated the expression of colonic 11betaHSD1 mRNA and down-regulated 11betaHSD2 mRNA. A similar pattern was observed at the level of local metabolism of corticosterone. Oxidation of corticosterone to 11-dehydrocorticosterone was decreased and reduction of 11-dehydrocorticosterone to corticosterone was increased in colonic tissue of rats with DSS-colitis. CONCLUSIONS: Colonic inflammation induces local glucocorticoid activation via 11betaHSD1 and impairs glucocorticoid inactivation via 11betaHSD2. The observed changes indicate a role for local metabolism of glucocorticoids in the control of colonic inflammation.     

Colitis up-regulates local glucocorticoid activation and down-regulates inactivation in colonic tissue

BACKGROUND: Pro-inflammatory processes are counteracted by anti-inflammatory factors such as glucocorticoids. The response of target cells to glucocorticoids depends on several factors including prereceptor modulation of glucocorticoid signals via local glucocorticoid metabolism. This is determined by two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD); 11betaHSD1 operates in vivo as a reductase converting inactive 11-oxo glucocorticoids to active glucocorticoids cortisol or corticosterone, whereas 11betaHSD2 catalyses oxidation of active glucocorticoids to their inactive 11-oxo derivatives. The aim of this study was to investigate the changes in local metabolism of glucocorticoids and in the expression of 11betaHSD1 and 11betaHSD2 mRNA during colonic inflammation. METHODS: Acute colitis was induced by intracolonic administration of 2,4,6-trinitrobenzenesulphonic acid (TNBS) or by drinking a dextran sodium sulphate (DSS) solution. Metabolism of glucocorticoids was measured in tissue fragments in vitro and 11betaHSD1 and 11betaHSD2 mRNA abundance was quantified using real-time RT-PCR one week after administration of TNBS and 10 days after drinking the DSS solution. RESULTS: In both models of inflammatory bowel disease we observed down-regulation of corticosterone oxidation to 11-dehydrocorticosterone by 64% (TNBS) and 53% (DSS) and reciprocal stimulation of reduction of 11-dehydrocorticosterone to corticosterone by 83% and 54%, respectively. A similar pattern was observed at the level of mRNA; 11betaHSD1 mRNA was significantly higher (TNBS: increase by 660%; DSS: increase by 760%) and 11betaHSD2 mRNA lower (TNBS: decrease by 85%; DSS: decrease by 60%) during inflammation. CONCLUSIONS: Colitis induces local glucocorticoid activation from 11-oxo steroids and decreases glucocorticoid inactivation; i.e. inflammation increases local tissue ratio of active and inactive glucocorticoids. The results indicate that the changes in local metabolism of glucocorticoids could contribute to the control of an overshoot of inflammation processes in the colon.     

Stimulation of testosterone production in rat Leydig cells by aldosterone is mineralocorticoid receptor mediated

The testis is known to be a site of corticosterone action, and testosterone production in Leydig cells is directly inhibited by glucocorticoids. Glucocorticoids bind to both glucocorticoid receptors (GRs) and to mineralocorticoid receptors (MRs). In Leydig cells, selective mineralocorticoid binding could result from oxidative inactivation of glucocorticoid by type 1 and/or 2 11beta-hydroxysteroid dehydrogenase (11betaHSD), as both isoforms are expressed. However, it remains unclear whether Leydig cells express MRs and respond directly to mineralocorticoid action. Therefore, the aims of the present study were to ascertain: (1) whether MR mRNA, protein and receptor binding are present in Leydig cells; and (2) if the mineralocorticoid modulates testosterone production. The mRNA encoding MR, as well as protein, and binding activity were each observed in adult rat Leydig cells. MR-ligand binding specificity within isolated Leydig cells was evaluated further by measuring displacement of MR binding to aldosterone by corticosterone in the presence and absence of carbenoxolone, an inhibitor of 11betaHSD1 and 2 that decreases conversion to biologically inert 11-dehydrocorticosterone. Carbenoxolone inhibited 11betaHSD oxidative activity, and reduced corticosterone-binding by 50%. Mineralocorticoid effects on steroidogenesis were assessed in the presence of aldosterone (0.01-10 nM) with or without the MR antagonist, RU28318. Aldosterone induced dose-dependent increases in both basal and luteinizing hormone-stimulated testosterone production. RU28318 eliminated the increase, indicating that these effects of aldosterone were mediated by the MR. The effects of aldosterone and luteinizing hormone (0.1 ng/ml) on testosterone production were synergistic, suggesting that the two hormones increased steroidogenesis through separate pathways. We conclude that Leydig cells express MRs and that testosterone production is subject to regulation by aldosterone.     

Light and electron microscopy localization of the 11beta-hydroxysteroid dehydrogenase type I enzyme in the rat

The 11beta-hydroxysteroid dehydrogenase type I enzyme (11betaHSD1) converts cortisone to cortisol in humans, and 11-dehydrocorticosterone to corticosterone in rodents. In the present study we used a new immunopurified polyclonal antibody, RAH113, to localize 11betaHSD1 at the light and electron microscopy levels in a wide range of rat tissues. 11betaHSD1 staining in the liver was of highest intensity around the central vein and decreased radially. In the lung, 11betaHSD1 was found at highest levels in the interstitial fibroblast, with levels in the type II pneumocyte an order of magnitude lower. RAH113 stained proximal tubules of the renal cortex and interstitial cells of the medulla and papilla. Adrenal 11betaHSD1 was confined to the glomerulosa and medulla, whereas the glucocorticoid-inactivating hydroxysteroid dehydrogenase isoform 11betaHSD2 was present in fascilulata/reticularis. 11betaHSD1 was found in parietal cells of the fundic region of the stomach, but not in the antrum. In the heart, 11betaHSD1 was detected in cells resembling interstitial fibroblasts of the endocardium and in the adventitial fibroblasts of blood vessels. Western blot analysis confirmed the presence of an antigen of the correct size (34 kDa) and intensity consistent with levels of enzyme activity previously reported in these tissues. Brain and testis also displayed the 34-kDa protein, confirming the expression of authentic 11betaHSD1 in these tissues. Electron microscopy of lung and kidney interstitial cells showed that 11betaHSD1 was localized both to the endoplasmic reticulum and the nuclear membrane. These results show that 11betaHSD1 is present in discrete cell populations where it may facilitate intracrine and paracrine glucocorticoid action in addition to its classical role of maintaining circulating glucocorticoids via activity in the liver.     

Tissue-specific distribution of the NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) converts the active glucocorticoid corticosterone to inactive 11-dehydrocorticosterone in rat (or cortisol to cortisone in man), thereby protecting renal mineralocorticoid receptors from corticosterone or cortisol and allowing preferential access for aldosterone. Recent work suggests that a nicotinamide adenine dinucleotide (NAD+)-dependent 11 beta-OHSD isoform is expressed in distal renal tubule, in contrast with the hepatic isoform which is NAD(+)-phosphate (NADP+)-dependent. To establish the distribution of the NAD(+)-dependent isoform we measured in vitro conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in homogenized rat tissues in the presence of NADP+ or NAD+. In most tissues (liver, testis, hippocampus, heart, aorta, mesenteric artery) NADP+ increased activity and NAD+ was without effect. However, in whole renal cortex, colon, placenta, and lung both NADP+ and NAD+ increased activity. No difference in cofactor utilization was demonstrated between proximal and distal renal tubules following density gradient separation. This distribution of NAD(+)-dependent activity corresponds with: (i) the distribution of multiple mRNA and/or protein species of 11 beta-OHSD; (ii) the distribution of aldosterone-specific mineralocorticoid receptors; and (iii) the equilibrium between active and inactive glucocorticoids in each tissue. We suggest that the tissue-specific expression of isoforms of 11 beta-OHSD with different kinetic properties confers on them diverse roles in modulating corticosteroid receptor activation.     

Regulation of 11beta-hydroxysteroid dehydrogenase isoforms and glucocorticoid receptor gene expression in the rat uterus

Glucocorticoids are known to have diverse effects on the uterus, generally believed to be mediated by the glucocorticoid receptor (GR). To date, two isoforms of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been identified, namely 11betaHSD1 and 11betaHSD2, which interconvert active and inactive glucocorticoids and regulate local levels of hormones available to the GR in target tissues. The aim of the present study was to examine the uterine expression of 11betaHSD and GR mRNA. The interplay of these parameters is probably an important factor in determining actions of glucocorticoids on the uterus. Using Northern analysis we investigated the uterine expression of 11betaHSD1, 11betaHSD2 and GR mRNA in relation to serum levels of sex steroid hormones and uterine progesterone receptor mRNA expression in an animal model. Immature female rats were treated with 10 IU pregnant mare serum gonadotrophin (PMSG) followed by 10 IU human chorionic gonadotrophin (hCG) 48 h afterwards, and then killed at 0, 3, 6, 9, 12 and 24 h and 5 days after the hCG injection. Expression of both 11betaHSD1 and 11betaHSD2 mRNA in total uterine RNA was found to be up-regulated by more than 50% at 48 h after PMSG injection when oestradiol levels were also high. Following hCG treatment the expression of 11betaHSD1 and 11betaHSD2 further increased to reach maximal levels at 24 and 12 h respectively. GR mRNA expression was down-regulated by more than 50% by PMSG but gradually recovered after hCG injection. The results show that mRNA expression of 11betaHSD1, 11betaHSD2 and GR in the uterus is developmentally regulated, suggesting that these key determinants of glucocorticoid action may play an important role in uterine function.     

11beta-hydroxysteroid dehydrogenase and corticosteroid action in lyon hypertensive rats

Adrenocorticosteroid activity in Lyon hypertensive (LH) and low blood pressure (LL) rat strains differ in several respects. Abnormal activity of 11beta-hydroxysteroid dehydrogenase enzymes (11beta-HSD1 and 11beta-HSD2), which interconvert corticosterone and inactive 11-dehydrocorticosterone, might contribute to the LH phenotype by regulating corticosteroid hormone access to receptors. 11beta-HSD2 (expressed in kidney but not liver) prevents endogenous glucocorticoids from binding to mineralocorticoid receptors. 11beta-HSD1 (expressed in liver and kidney) favors active glucocorticoid formation from 11-dehydrocorticosterone. 11beta-HSD properties in LH and LL have been compared by several approaches: (1) 11betaHSD activities have been measured in vitro as corticosterone dehydrogenation and in vivo as interconversion of injected cortisol and cortisone; (2) the effects of cortisol and cortisone on urine electrolytes and volume have been measured; and (3) 11beta-HSD mRNA expression has been measured by in situ hybridization. 11beta-HSD2 enzyme activities in LH and LL rats were similar and urinary cortisone:cortisol ratios were not different after cortisol injection. Cortisol caused a natriuresis and kaliuresis in both strains, with a slightly reduced response in LH rats. Renal 11beta-HSD2 mRNA expression was slightly lower in LH rats. 11beta-HSD1 was less active in LH than LL rats: enzyme activities were lower in tissue extracts; urinary cortisone:cortisol was lower in LL rats after cortisone injections; cortisone increased urine volume in LL but not LH rats; and mRNA levels tended to be lower in LH tissues. We conclude that 11beta-HSD1 is impaired in LH rats. The LH phenotype of heavier adrenals, raised corticosterone, and reduced thymus weight is similar to that described for 11beta-HSD1 knockout mice.     

Obese Zucker rats have reduced mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 1 expression in hippocampus-implications for dysregulation of the hypothalamic-pituitary-adrenal axis in obesity

Obese Zucker rats have elevated basal corticosterone levels and an increased stress response suggestive of an increased activity of the hypothalamic-pituitary-adrenal (HPA) axis. We hypothesized that altered central expression of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), and/or 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) contribute to these changes. In brains from young adult male rats, in situ hybridization and Western blotting showed that obese rats had normal hippocampal GR mRNA and protein levels. In contrast, in obese rats, 11betaHSD1 mRNA levels were reduced in a subpopulation of hippocampal cells in the main neuronal layers (by 37-47%, P < 0.05), whereas 11betaHSD1 levels in sparse high-expressing cells did not differ. MR mRNA was decreased in all regions of the hippocampus (by 37-49%, P < 0.05 for CA1-2 and P < 0.01 for dentate gyrus) and in frontal cortex (by 16%, P < 0.05) in obese rats. In whole hippocampal homogenates, however, neither the protein concentration of MR by Western blot nor activity of 11betaHSD1 was measurably different between the phenotypes. To test the functional importance of lower central MR expression, groups of lean and obese rats were given spironolactone before restraint stress. In vehicle-treated animals, obese rats had higher plasma corticosterone levels than lean rats after stress (by ANOVA, P < 0.05). Spironolactone markedly increased the corticosterone response in both groups, but the incremental rise was smaller in the obese rats, so that spironolactone abolished the differences between groups. We conclude that lower levels of MR, but not GR, contribute to the increased HPA activity in the obese Zucker rats and that this seems more influential during stress than in the basal state. This may be exacerbated by impaired local regeneration of corticosterone by 11betaHSD1. These abnormalities could contribute to the subtle changes in the HPA axis in rodent and human obesity.     

11Beta-hydroxysteroid dehydrogenases in human endometrium

Key reproductive events, such as menstruation and implantation, are considered to be inflammatory processes and glucocorticoids act as anti-inflammatory agents. The balance of expression of types 1 and 2 11beta-hydroxysteroid dehydrogenases (11betaHSD) controls the availability of cortisol to bind to the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Expression profiles of glucocorticoid-metabolising enzymes and their cognate receptors have been characterized in the reproductive tract. We propose that factors that peripherally promote glucocorticoid action are part of an anti-inflammatory response to tissue remodelling in human endometrium. Protein and mRNA expression in endometrium were investigated using immunohistochemistry and quantitative real-time PCR. There was up-regulated expression of 11betaHSD-1 at menstruation and in first trimester decidua. 11BetaHSD-2 and GR were expressed across the cycle. The MR expression pattern across the cycle and in decidua implies progesterone may also play a regulatory role. The precise roles and interactions of these proteins require further investigation.     

Pituitary expression of type I and type II glucocorticoid receptors during chicken embryonic development and their involvement in growth hormone cell differentiation

Glucocorticoids can induce somatotroph differentiation in vitro and in vivo during chick embryonic and rat fetal development. In the present study, we identified the nuclear receptors involved in somatotroph differentiation and examined their ontogeny and cellular distribution during pituitary development in the chicken embryo. Several steroids were tested for their ability to induce GH cell differentiation. Only glucocorticoids and aldosterone were effective at low nanomolar concentrations, suggesting involvement of both type I (mineralocorticoid) and type II (glucocorticoid) receptors (MR and GR, respectively). ZK98299 and spironolactone (GR and MR antagonists, respectively) when used alone were unable to block corticosterone or aldosterone (2 nm)-induced somatotroph differentiation. However, ZK98299 and spironolactone in combination abolished corticosterone or aldosterone (2 nm)-induced somatotroph differentiation. When used separately, both antagonists attenuated induction of GH mRNA by corticosterone. Spironolactone alone blocked somatotroph differentiation induced by 0.2 nm corticosterone or aldosterone, indicating that corticosteroids at subnanomolar concentrations act only through the MR. GR protein was detected in pituitary extracts as early as embryonic d 8, whereas MR protein was readily detectable only around d 12. GR were expressed in greater than 95% of all pituitary cells, whereas MR were expressed in about 40% of all pituitary cells. Dual-label immunofluorescence revealed that the majority of somatotrophs on d 12 expressed MR. Given the high affinity of corticosteroids for MR and that corticosteroid concentrations during embryonic development are in the subnanomolar range, expression of MR may constitute a significant developmental event during somatotroph differentiation.     

11 beta-hydroxysteroid dehydrogenase in vascular smooth muscle and heart: implications for cardiovascular responses to glucocorticoids

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) converts the active glucocorticoid corticosterone to inactive 11-dehydrocorticosterone in the rat (or cortisol to cortisone in man), thereby protecting renal mineralocorticoid receptors from corticosterone or cortisol and allowing preferential access for aldosterone. We have previously demonstrated that cortisol-induced cutaneous vasoconstriction in man is potentiated by the 11 beta-OHSD inhibitor glycyrrhetinic acid, suggesting that 11 beta-OHSD may protect vascular corticosteroid receptors. In this study we report quantitation of 11 beta-OHSD bioactivity in homogenates of rat aorta, mesenteric artery, caudal artery, and heart, expressed as the percent in vitro conversion of 3H-corticosterone to 3H-11-dehydrocorticosterone. Nicotinamide adenine dinucleotide phosphate (NADP+)-dependent 11 beta-OHSD activity was found in all of these tissues and was significantly higher in resistance vessels than aorta (P less than 0.05) [without NADP+: caudal artery (4.2 +/- 0.2%) greater than mesenteric artery (2.5 +/- 0.7%) = heart (1.67 +/- 0.2%) greater than aorta (0.79 +/- 0.2%); with 200 microM NADP+: caudal artery (43.9 +/- 2.1%) greater than heart (20.6 +/- 1.0%) = mesenteric artery (17.7 +/- 3.1%) = aorta (11.4 +/- 0.4%); heart greater than aorta]. All of these were lower than renal cortex (29.4 +/- 1.8% without NADP+; 82.4 +/- 0.4% with NADP+; P less than 0.001). 3H-11-dehydrocorticosterone was the major metabolite of 3H-corticosterone (greater than 97% of 3H-corticosterone metabolized). Reduction of 3H-11-dehydrocorticosterone to 3H-corticosterone was not detected in these experiments. We also report localization of 11 beta-OHSD-like immunoreactivity by immunohistochemistry using antisera raised against rat liver 11 beta-OHSD, and of 11 beta-OHSD messenger RNA expression by in situ hybridization using complementary RNA probes transcribed from complementary DNA encoding rat liver 11 beta-OHSD. We found 11 beta-OHSD immunoreactivity and messenger RNA expression in vascular and cardiac smooth muscle cytoplasm but not in endothelium. Thus, 11 beta-OHSD is appropriately sited to modulate access of corticosterone to vascular receptors and could influence vascular resistance, cardiac output and thereby blood pressure.     

Expression of glucocorticoid receptor, mineralocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase 1 and 2 in the fetal and postnatal ovine hippocampus: ontogeny and effects of prenatal glucocorticoid exposure

To determine the expression of glucocorticoid metabolizing and action genes in the hippocampus of fetal, neonatal, and adult sheep. Pregnant ewes (or their fetuses) received intramuscular injections of saline or betamethasone (BETA, 0-5 mg/kg) at 104, 111, 118, and/or 125 days of gestation (dG). Hippocampal tissue was collected prior to (75, 84, and 101 dG), during (109 and 116 dG), or after (121, 132, and 146 dG; 6 and 12 postnatal weeks; 3.5 years of age) saline or BETA injections. Hippocampal glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11beta-hydroxysteroid dehydrogenase (11betaHSD)1 and 11betaHSD2 mRNA levels were determined using qRT-PCR. Control animals late in gestation demonstrated a decrease in mRNA encoding GR and 11betaHSD1, whereas 11betaHSD2 was undetectable, consistent with a damping of the negative feedback influence of circulating or locally produced cortisol on the hypothalamic-pituitary-adrenal (HPA) axis. BETA-administration had transient effects on fetal GR and MR, and early in postnatal life (12 weeks of age) 11betaHSD1 mRNA was increased. Hippocampal MR mRNA was elevated in adult offspring exposed to either one or four doses of maternal BETA (P<0.001). Four courses of maternal BETA increased 11betaHSD2 (P<0.05) but not 11betaHSD1 mRNA levels. Late in gestation a reduction in hippocampal GR and 11betaHSD1 mRNA suggests lessening of glucocorticoid negative feedback, facilitating increased preterm HPA activity and parturition. Adult offspring of BETA-treated mothers demonstrated increased MR and 11betaHSD2 mRNA, therefore it appears that exposure of fetus to high levels of synthetic glucocorticoids may have long-lasting effects on the hippocampal expression of HPA-related genes into adulthood.     

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.     

Corticosterone oxidative neutralization by 11-beta hydroxysteroid dehydrogenases in kidney and colon of the domestic fowl

In mammalian organs involved in sodium reabsorption, the 11-beta hydroxysteroid dehydrogenases (11betaHSDs) oxidize glucocorticoids (GC) from their 11-alcohol form to their 11-keto state and therefore prevent their binding to mineralocorticoid (MC) receptors (MR) and the development of a MC excess syndrome. In birds the information about 11betaHSDs and GC metabolism in such organs is scarce. Herein, we report the expression and enzymatic activity of 11betaHSDs in the kidney and colon of chickens. Both organs express 11betaHSD2-like mRNA. With NAD(+), microsomes from both tissues oxidized corticosterone (CS) into 11-dehydrocorticosterone (DHC) with K(m) of 200 and 20nM and V(max) of 13 and 2pmol/mg protein/min in the kidney and colon, respectively. Thiram, a specific 11betaHSD2 inhibitor, suppressed this oxidation in kidney. The expression and action of the putative 11betaHSD3 were also tested. The chicken colon, and to a greater extent the kidney, expressed 11betaHSD3-like mRNA. Microsomal fractions from both tissues oxidized CS into DHC in the presence of NADP(+) with K(m) of 150 and 4nM and V(max) of 5 and 0.3pmol/mg protein/min for the kidney and the colon, respectively. This oxidation was not affected when NADP(+) conversion into NAD(+) was inhibited by excess pyrophosphate or a phosphatase inhibitor cocktail. In microsomes of chicken's duodenum, where 11betaHSD1-like mRNA expression is high, NADP(+)-dependent oxidation of CS into DHC has a low-affinity K(m) of 1130nM. This study documented the expression and activity of two enzymes that convert CS into DHC, one is 11betaHSD2-like and the other is similar to the putative mammalian 11betaHSD3.     

Induction of cardiac fibrosis by aldosterone

An intracardiac aldosterone system which responds to short- and long-term physiological stimuli has been described. This cardiac generated aldosterone has possibly autocrine or paracrine actions. Normal cardiac tissue contains mineralocorticoid receptors (MR) and cardiac high affinity MR are localized in cardiac myocytes and endothelial cells. Data concerning the presence of MR in cardiac fibroblasts are, however, controversial. MR are not specific for aldosterone but they also bind glucocorticoids. Cardiac fibroblasts however contain the enzyme 11beta-hydroxy-steroid dehydrogenase II which converts these glucocorticoids to inactive metabolites. Discordant findings on the in vitro effect of aldosterone on the collagen synthesis in cardiac fibroblasts are reported and can at least partly attributed to the presence of various fibroblasts phenotypes. During chronic aldosterone infusion in uninephrectomized rats on a high-salt diet, a marked accumulation of interstitial and to a lesser extent perivascular collagen occurs in the heart in both ventricles. This cardiac fibrosis in this aldosteronism model is prevented by spironolactone. This effect of aldosterone is crucially dependent on the salt status of the rat. Indeed, rats on a restricted salt intake infused with aldosterone had no cardiac fibrosis above control levels. During the continuous infusion of aldosterone in the rat the appearance of fibrosis was delayed and starts 4 weeks after the beginning of the infusion which argues against a direct effect of aldosterone. The mechanism of aldosterone-salt induced cardiac fibrosis possibly involves angiotensin II acting through upregulated AT1 receptors and the cardiac AT1 receptor is the target for aldosterone. An accumulation of collagen in the heart has also been found in patients with adrenal adenomas and during chronic activation of the renin-angiotensin-aldosterone system such as in surgically induced unilateral renal ischemia, unilateral renal artery banding or renovascular hypertension. Spironolactone prevents aortic collagen accumulation in spontaneously hypertensive rats. In patients with stable chronic heart failure spironolactone treatment in addition to diuretics and angiotensin-converting enzyme (ACE) inhibition reduced circulating levels of procollagen type III N-terminal aminopeptide. Also, in the Randomized Aldactone Evaluation Study spironolactone coadministered with conventional therapy of ACE inhibitors, loop diuretics and digitalis in patients with symptomatic heart failure defined as NYHA classes III-IV reduces total mortality by 30%.     

11beta-hydroxysteroid dehydrogenase type 2 in mouse aorta: localization and influence on response to glucocorticoids

Both isozymes of 11beta-hydroxysteroid dehydrogenase, which interconvert active and inactive glucocorticoids, are expressed in the mouse aortic wall. Mice deficient in 11HSD type 2 (which converts active corticosterone into inert 11-dehydrocorticosterone) have hypertension and impaired endothelial nitric oxide activity. It has been suggested that 11HSD2 influences vascular function directly by limiting glucocorticoid-mediated inhibition of endothelium-derived nitric oxide. This study sought to determine (1) the cellular distribution of the 11HSD isozymes within the mouse aortic wall and (2) the influence of 11HSD2 on direct glucocorticoid-mediated changes in aortic function. Mouse aortas were separated into their component layers and RNA extracted for RT-PCR. Both types of corticosteroid (mineralocorticoid and glucocorticoid) receptors and both 11HSD isozymes were expressed in the aortic wall. 11HSD1 expression colocalized with alpha-smooth muscle actin (a marker for smooth muscle cells), whereas 11HSD2 colocalized with TIE-2 (a marker for endothelial cells). Functional relaxation responses of mouse aortic rings were unaltered after exposure to glucocorticoids for 24 hours. In the presence of l-arginine, glucocorticoids produced an endothelium-independent reduction of contraction; similar results were obtained with aortas from mice with genetic inactivation of 11HSD2. Incubation in medium containing l-arginine reversed the endothelial cell dysfunction associated with 11HSD2 inactivation. Thus, 11HSD2 is appropriately sited to modulate endothelial cell function, but endothelial dysfunction in 11HSD2 knockout mice cannot be explained simply by increased access of corticosterone to endothelial cell corticosteroid receptors. Therefore, additional mechanisms, possibly involving indirect effects of enhanced corticosterone action in the kidney and the resultant hypertension, must be involved.     

The maternal diet during pregnancy programs altered expression of the glucocorticoid receptor and type 2 11beta-hydroxysteroid dehydrogenase: potential molecular mechanisms underlying the programming of hypertension in utero.

Potential mechanisms underlying prenatal programming of hypertension in adult life were investigated using a rat model in which maternal protein intake was restricted to 9% vs. 18% casein (control) during pregnancy. Maternal low protein (MLP) offspring exhibit glucocorticoid-dependent raised systolic blood pressure throughout life (20-30 mm Hg above the control). To determine the molecular mechanisms underlying the role of alterations in glucocorticoid hormone action in the prenatal programming of hypertension in MLP offspring, tissues were analyzed for expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11betaHSD1, 11betaHSD2, and corticosteroid-responsive Na/K-adenosine triphosphatase alpha1 and beta1. GR protein (95 kDa) and messenger RNA (mRNA) expression in kidney, liver, lung, and brain was more than 2-fold greater in MLP vs. control offspring during fetal and neonatal life and was more than 3-fold higher during subsequent juvenile and adult life (P < 0.01). This was associated with increased levels of Na/K-adenosine triphosphatase alpha1- and beta1-subunit mRNA expression. Levels of MR gene expression remained unchanged. Exposure to the MLP diet also resulted in markedly reduced levels of 11betaHSD2 expression in the MLP placenta on days 14 and 20 of gestation (P < 0.001), underpinning similar effects on 11betaHSD2 enzyme activity that we reported previously. Levels were also markedly reduced in the kidney and adrenal of MLP offspring during fetal and postnatal life (P < 0.001). This programmed decline in 11betaHSD2 probably contributes to marked increases in glucocorticoid hormone action in these tissues and potentiates both GR- and MR-mediated induction of raised blood pressure. In contrast, levels of 11betaHSD1 mRNA expression in offspring central and peripheral tissues remained unchanged. In conclusion, we have demonstrated that mild protein restriction during pregnancy programs tissue-specific increases in glucocorticoid hormone action that are mediated by persistently elevated expression of GR and decreased expression of 11betaHSD2 during adult life. As glucocorticoids are potent regulators not only of fetal growth but also of blood pressure, our data suggest important potential molecular mechanisms contributing to the prenatal programming of hypertension by maternal undernutrition in the rat.     

Early inflammatory responses in experimental cardiac hypertrophy and fibrosis: effects of 11 beta-hydroxysteroid dehydrogenase inactivation

In epithelial tissues such as kidney, mineralocorticoid receptors (MR) are protected against glucocorticoid occupancy by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) type 2. If the enzyme is congenitally inactive, or blocked by carbenoxolone, physiologic glucocorticoids act as MR agonists in such tissues. In most nonepithelial tissues, including cardiomyocytes, 11 beta HSD2 is expressed at minimal levels; in these tissues physiologic glucocorticoids act as MR antagonists, with the basis for this tissue selectivity currently unknown. Vascular smooth muscle cells (VSMC) express MR and 11 beta HSD1/2, with 11 beta HSD1 reported to show uncharacteristic oxidase activity, so that VSMC thus constitute a potential physiologic aldosterone target tissue. Because mineralocorticoid/salt administration triggers marked inflammatory responses in coronary vasculature, we reasoned that VSMC (like epithelial) MR may be activated by glucocorticoids if the protective enzyme is blocked. We thus gave uninephrectomized rats 0.9% NaCl solution to drink, and deoxycorticosterone (DOC, as a single 20 mg sc dose) or carbenoxolone (CBX, 2.5 mg/d in the drinking solution). Both DOC and CBX increased systolic blood pressure, heart, and kidney weight, and expression of cyclooxygenase 2, ED-1-positive macrophages, and osteopontin, with effects of both DOC and CBX blocked by the selective MR antagonist eplerenone. These findings suggest that local glucocorticoid excess, reflecting lower VSMC 11 beta HSD1/2 activity may mimic systemic mineralocorticoid excess, and play a direct etiologic role in coronary vascular inflammatory responses under circumstances of a high salt intake.     

Intravascular glucocorticoid metabolism during inflammation and injury in mice

11beta-hydroxysteroid dehydrogenases (11betaHSDs) catalyze interconversion of 11-hydroxy-glucocorticoids with inactive 11-keto metabolites. In blood vessel walls, loss of 11betaHSD1 is thought to reduce local glucocorticoid concentrations, reducing the progression of atheroma and enhancing angiogenesis. Conversely, on the basis that 11betaHSD1 is up-regulated approximately 5-fold by inflammatory cytokines in cultured human vascular smooth muscle cells, it has been proposed that increased 11betaHSD1 during vascular inflammation provides negative feedback suppression of inflammation. We aimed to determine whether inflammation and injury selectively up-regulate 11betaHSD1 reductase activity in vitro and in vivo in intact vascular tissue in mice. In isolated mouse aortae and femoral arteries, reductase activity (converting 11-dehydrocorticosterone to corticosterone) was approximately 10-fold higher than dehydrogenase activity and was entirely accounted for by 11betaHSD1 because it was abolished in vessels from 11betaHSD1(-/-) mice. Although 11betaHSD1 activity was up-regulated by proinflammatory cytokines in cultured murine aortic smooth muscle cells, no such effect was evident in intact aortic rings in vitro. Moreover, after systemic inflammation induced by ip lipopolysaccharide injection, there was only a modest (18%) increase in 11beta-reductase activity in the aorta and no increase in the perfused hindlimb. Furthermore, in femoral arteries in which neointimal proliferation was induced by intraluminal injury, there was no change in basal 11betaHSD1 activity or the sensitivity of 11betaHSD1 to cytokine up-regulation. We conclude that increased generation of glucocorticoids by 11betaHSD1 in the murine vessel wall is unlikely to contribute to feedback regulation of inflammation.