Expression Of 11β-hydroxysteroid Dehydrogenase Type Research Articles

Peripheral mechanisms contributing to the glucocorticoid hypersensitivity in proopiomelanocortin null mice treated with corticosterone.

Proopiomelanocortin (POMC) deficiency causes severe obesity through hyperphagia of hypothalamic origin. However, low glucocorticoid levels caused by adrenal insufficiency mitigate against insulin resistance, hyperphagia and fat accretion in Pomc−/− mice. Upon exogenous glucocorticoid replacement, corticosterone-supplemented (CORT) Pomc−/− mice show exaggerated responses, including excessive fat accumulation, hyperleptinaemia and insulin resistance. To investigate the peripheral mechanisms underlying this glucocorticoid hypersensitivity, we examined the expression levels of key determinants and targets of glucocorticoid action in adipose tissue and liver. Despite lower basal expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which generates active glucocorticoids within cells, CORT-mediated induction of 11β-HSD1 mRNA levels was more pronounced in adipose tissues of Pomc−/− mice. Similarly, CORT treatment increased lipoprotein lipase mRNA levels in all fat depots in Pomc−/− mice, consistent with exaggerated fat accumulation. Glucocorticoid receptor (GR) mRNA levels were selectively elevated in liver and retroperitoneal fat of Pomc−/− mice but were corrected by CORT in the latter depot. In liver, CORT increased phosphoenolpyruvate carboxykinase mRNA levels specifically in Pomc−/− mice, consistent with their insulin-resistant phenotype. Furthermore, CORT induced hypertension in Pomc−/− mice, independently of adipose or liver renin–angiotensin system activation. These data suggest that CORT-inducible 11β-HSD1 expression in fat contributes to the adverse cardiometabolic effects of CORT in POMC deficiency, whereas higher GR levels may be more important in liver.

Depot-specific messenger RNA expression of 11β-hydroxysteroid dehydrogenase type 1 and leptin in adipose tissue of children and adults

To compare expression of messenger RNA (mRNA) coding for the cortisol regenerating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), and the adipocytokines leptin and resistin in paired biopsies of subcutaneous adipose tissue (SC) and omental adipose tissue (OM) from children. Paired biopsies (SC and OM) were obtained from 54 children (age 0.17-16 years, body mass index (BMI) 12.5-28.3 kg/m(2), BMI standard deviation score (SDS) -2.5-4.5) and 16 adults (age 27-79 years, BMI 19-46 kg/m(2)) undergoing open abdominal surgery. mRNA levels of 11beta-HSD1, leptin and resistin were measured using quantitative real-time polymerase chain reaction (PCR). 11beta-HSD1 mRNA level was higher in OM than in SC (P<0.05), whereas leptin mRNA was higher in SC than in OM (P<0.001). There was no difference in the resistin mRNA level between SC and OM. These results were consistent in children and adults. In children, 11beta-HSD1 mRNA in SC was positively associated with BMI SDS (P<0.05), whereas in OM it was positively associated with age (P<0.05). The association between 11beta-HSD1 expression and age remained significant after adjustment for BMI SDS and gender. Leptin mRNA was positively associated with BMI SDS (SC: P<0.001, OM: P<0.001) but not with age in children. In multiple regression analyses, including anthropometric variables and age, BMI SDS was independently associated with mRNA levels of 11beta-HSD1 (P<0.05) and leptin (P<0.001) in SC. When normal weight and overweight children were analyzed separately, 11beta-HSD1 mRNA levels were positively associated with leptin in OM in the overweight group (P<0.05). There are depot-specific differences in mRNA levels of 11beta-HSD1 and leptin in children and adults. The positive association of 11beta-HSD1 mRNA in OM with age may reflect a causal role in visceral fat accumulation during growth. Increasing 11beta-HSD1 and leptin mRNA in SC with increasing BMI SDS could suggest that the risk of metabolic consequences of obesity may be established early in life.

Effect of voluntary exercise on peripheral tissue glucocorticoid receptor content and the expression and activity of 11β-HSD1 in the Syrian hamster

Recent findings indicate that elevated levels of glucocorticoids (GC), governed by the expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and GC receptors (GR), in visceral adipose tissue and skeletal muscle lead to increased insulin resistance and the metabolic syndrome. Paradoxically, evidence indicates that aerobic exercise attenuates the development of the metabolic syndrome even though it stimulates acute increases in circulating GC levels. To investigate the hypothesis that training alters peripheral GC action to maintain insulin sensitivity, young male hamsters were randomly divided into sedentary (S) and trained (T) groups (n = 8 in each). The T group had 24-h access to running wheels over 4 wk of study. In muscle, T hamsters had lower 11beta-HSD1 protein expression (19.2 +/- 1.40 vs. 22.2 +/- 0.96 optical density, P < 0.05), similar 11beta-HSD1 enzyme activity (0.9 +/- 0.27% vs. 1.1 +/- 0.26), and lower GR protein expression (9.7 +/- 1.86 vs. 15.1 +/- 1.78 optical density, P < 0.01) than S hamsters. In liver, 11beta-HSD1 protein expression tended to be lower in T compared with S (19.2 +/- 0.56 vs. 21.4 +/- 1.05, P = 0.07), whereas both enzyme activity and GR protein expression were similar. In contrast, visceral adipose tissue contained approximately 2.7-fold higher 11beta-HSD1 enzyme activity in T compared with S (12.9 +/- 3.3 vs. 4.8 +/- 1.5% conversion, P < 0.05) but was considerably smaller in mass (0.24 +/- 0.02 vs. 0.71 +/- 0.06 g). Thus the intracellular adaptation of GC regulators to exercise is tissue specific, resulting in decreases in GC action in skeletal muscle and increases in GC action in visceral fat. These adaptations may have important implications in explaining the protective effects of aerobic exercise on insulin resistance and other symptoms of the metabolic syndrome.

Expression of 11β-hydroxysteroid dehydrogenase type 1 permits regulation of glucocorticoid bioavailability by human dendritic cells

AbstractGlucocorticoids (GCs) exert powerful anti-inflammatory effects that may relate in part to their ability to restrict the differentiation and function of dendritic cells (DCs). Although these inhibitory effects are dependent upon GCs binding to nuclear glucocorticoid receptors (GRs), fine-tuning of GR signaling is achieved by prereceptor interconversion of cortisol that binds GRs with high affinity and cortisone that does not. We show for the first time that human monocyte-derived DCs are able to generate cortisol as a consequence of up-regulated expression of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Immature DCs demonstrate selective enhancement of 11β-HSD1 reductase activity, leading to increased conversion of inactive cortisone to active cortisol. Enhancement of GC bioavailability is maintained or increased upon terminal differentiation induced by signals associated with innate immune activation. In marked contrast, maturation induced by CD40 ligation leads to a sharp reduction in cortisol generation by DCs. The differentiation of DCs from monocyte precursors is inhibited at physiologic concentrations of inactive cortisone, an effect that requires activity of the 11β-HSD1 enzyme. In conclusion, prereceptor regulation of endogenous GCs appears to be an important determinant of DC function and represents a potential target for therapeutic manipulation.

The Expression of 11β-Hydroxysteroid Dehydrogenase Type I by Lymphocytes Provides a Novel Means for Intracrine Regulation of Glucocorticoid Activities

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes control the interconversion of active glucocorticoids (GCS) and their inactive 11-keto metabolites, a process commonly referred to as the cortisone/cortisol shuttle. Although the prereceptor metabolism of GCS by 11beta-HSD is well documented in a variety of cells and tissues, it has not yet been carefully investigated in the major cell types of the immune system. In this study, we demonstrate that 11beta-HSD1 transcripts, protein, and enzyme activities are actively expressed in murine CD4(+), CD8(+), and B220(+) lymphocytes, as well as CD11c(+) dendritic cells. Only reductase activity was observed in living cells, evidenced by the restricted conversion of cortisone to cortisol. Activation of CD4(+) T cells increased their 11beta-HSD1 activity, as did their polarization into Th1 or Th2 cells. CD4(+) T cells isolated from aged donors (>16 mo) had increased 11beta-HSD1 protein and an elevated capacity to convert cortisone to cortisol. The GCS generated in murine CD4(+) T cells from their inactive 11-keto metabolites could activate the GCS receptor, demonstrated by an up-regulation of IL-7Ralpha and GCS-induced leucine zipper gene expression. The presence of a functional 11beta-HSD1 provides lymphocytes with a novel intracrine regulatory mechanism that could influence such processes as lymphocyte development, effector function, and susceptibility to apoptosis. Thus, the presence of 11beta-HSD1 provides an additional means to facilitate GCS influences over lymphocyte activities, uncoupled from the plasma concentration of GCS.

Induction of 11β-hydroxysteroid dehydrogenase type 2 and hyperaldosteronism are essential for enhanced sodium absorption after total colectomy in rats

Background Patients who undergo total colectomy with ileopouch anal reconstruction often have persistent diarrhea and frequent bowel movements. Analysis of the intestinal adaptation after total colectomy may lead to developing novel therapies for postoperative diarrhea. Methods Sprague-Dawley rats underwent total colectomy with ileoanal reconstruction and were sacrificed 4 and 8 weeks later. Mucosal response to aldosterone was evaluated with the use of ileal mucosa in an Ussing chamber by measuring short circuit current after in vitro stimulation with aldosterone. We investigated the expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD 2) in intestinal epithelial cells. To examine the role of hyperaldosteronism, we also evaluated rats treated with a sodium-deficient diet or subcutaneous aldosterone infusion. Results Aldosterone levels increased 80-fold after total colectomy. A comparable amount of aldosterone dramatically increased aldosterone-mediated, amiloride-sensitive short circuit current in the mucosa from colectomized rats, but not in control rats. We measured an increase in 11β-HSD 2 messenger RNA and protein in the distal ileum from colectomized rats. Circulating aldosterone appears to be essential for these functional and molecular changes because similar results were obtained by using the mucosa from both dietary sodium–depleted and aldosterone-infused rats. Conclusions Induction of 11β-HSD 2 is essential for enhanced mineralocorticoid action in the remnant ileum after total colectomy in rats.

Glucocorticoids Stimulate the Expression of 11β-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Placental Trophoblast Cells

Proper glucocorticoid exposure in utero is vital for normal fetal organ growth and maturation. The placental enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), plays a pivotal role in controlling fetal exposure to high levels of maternal glucocorticoid by converting cortisol into its inactive metabolite, cortisone. The present study was designed to determine whether glucocorticoids auto-regulate 11beta-HSD2 in the human placenta using cultured trophoblast cells as a model system. Trophoblasts were isolated from uncomplicated term placentas and treated with glucocorticoids. The synthetic glucocorticoid dexamethasone increased 11beta-HSD2 activity in a time- and concentration-dependent manner; this effect was accompanied by a corresponding increase in 11beta-HSD2 mRNA. Furthermore, the glucocorticoid receptor antagonist, RU-486, abrogated the dexamethasone-induced increase in 11beta-HSD2 activity, suggesting that the effect of dexamethasone is mediated through the glucocorticoid receptor. Results from transient transfection and mRNA decay experiments indicate that the glucocorticoid-induced increase in 11beta-HSD2 expression is mediated at both the transcriptional and posttranscriptional levels. In conclusion, the present study demonstrates that in cultured human trophoblasts, 11beta-HSD2 is subject to auto-regulation by glucocorticoids. If this occurs in the human placenta in vivo, the glucocorticoid-induced up-regulation of placental 11beta-HSD2 would represent an important safeguard mechanism by which the fetus may be protected from detrimental exposure to elevated levels of maternal glucocorticoids.

Differential expression of 11β-hydroxysteroid dehydrogenase types 1 and 2 mRNA and glucocorticoid receptor protein during mouse embryonic development

Accumulating evidence suggests that the actions of glucocorticoids in target tissues are critically determined by the expression of not only the glucocorticoid receptor (GR) but also the glucocorticoid-metabolizing enzymes, known as 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1 and 11β-HSD2). To gain insight into the role of glucocorticoids in fetal development, the expression patterns of the two distinct 11β-HSD isozymes and GR were studied in the mouse embryo from embryonic day 12.5 (E12.5, term = E19) to postnatal day 0.5 (P0.5) by in situ hybridization and immunohistochemistry, respectively. 11β-HSD1 mRNA was detected in the heart as early as E12.5 and maintained thereafter. In the lung and liver, 11β-HSD1 mRNA was first detected between E14.5 and E16.5, increased to high levels towards term and maintained after birth. Relatively low levels of 11β-HSD1 mRNA were also detected in the kidney, adrenal glands and gastrointestinal tract at E18.5. However, the mRNA for 11β-HSD1 was undetectable in all other embryonic tissues including the brain. In contrast, kidney was the only organ that expressed appreciable levels of 11β-HSD2 mRNA during embryonic life. The level of 11β-HSD2 mRNA in the kidney increased dramatically in the newborn, which coincided with expression of 11β-HSD2 mRNA in the whisker follicle, tooth and salivary gland. Distinct from the profiles of 11β-HSD1 and 11β-HSD2 mRNA, GR protein was detectable in all tissues at all ages studied except for the thymus, salivary gland, and bone. Taken together, the present study demonstrates that tissue- and developmentally-stage specific expression of 11β-HSD1 and 11β-HSD2 as well as GR occurs in the developing mouse embryo, thus highlighting the importance of these two enzymes and GR in regulating glucocorticoid-mediated maturational events in specific tissues during murine embryonic development.

Expression of 11β-hydroxysteroid dehydrogenase types 1 and 2 in colorectal cancer

Glucocorticoid hormones have been reported to operate as regulators of cell proliferation and differentiation and to inhibit growth of several colon tumors and adenocarcinoma cell lines. The glucocorticoid action is regulated, in part, at the pre-receptor level through the expression of isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD1, 11βHSD2) which are responsible for the interconversion of hormonally active cortisol to cortisone. Since both of these isoforms are expressed in the mammalian colon, we examined whether 11βHSD1 and 11βHSD2 are expressed in human colorectal cancer and whether their expression differs between neoplastic and autologous non-neoplastic tissue. We provide evidence that both isoforms of 11βHSD are expressed in the colon adenocarcinoma, but their expression is not identical in neoplastic and non-neoplastic tissue. There is a significant decrease of 11βHSD2 mRNA abundance and enzyme activity in neoplastic tissue. In contrast, 11βHSD1 activity and mRNA abundance are increased in some but not all tumor samples. The results demonstrate that (1) neoplastic transformation is associated with decreasing steady-state levels of 11βHSD2 mRNA and enzyme activity and in some cases also with increasing expression of 11βHSD1, and (2) colorectal tumor cells have a decreased capability of autocrine inactivation of glucocorticoids.

Transgenic expression of 11beta-hydroxysteroid dehydrogenase type 2 in osteoblasts reveals an anabolic role for endogenous glucocorticoids in bone.

Glucocorticoid excess leads to bone loss, primarily by decreasing bone formation. However, a variety of in vitro models show that glucocorticoids can promote osteogenesis. To elucidate the role of endogenous glucocorticoids in bone metabolism, we developed transgenic (TG) mice in which a 2.3-kb Col1a1 promoter fragment drives 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) expression in mature osteoblasts. 11beta-HSD2 should metabolically inactivate endogenous glucocorticoids in the targeted cells, thereby reducing glucocorticoid signaling. The inhibitory effect of 300 nm hydrocortisone on percent collagen synthesis was blunted in TG calvariae, demonstrating that the transgene was active. Collagen synthesis rates were lower in TG calvarial organ cultures compared with wild-type. Trabecular bone parameters measured by microcomputed tomography were reduced in L3 vertebrae, but not femurs, of 7- and 24-wk-old TG females. These changes were also not seen in males. In addition, histomorphometry showed that osteoid surface was increased in TG female vertebrae, suggesting that mineralization may be impaired. Our data demonstrate that endogenous glucocorticoid signaling is required for normal vertebral trabecular bone volume and architecture in female mice.

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11 beta-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11 beta-HSD2 activity (71.9+/-22.3 pmol/h/mg protein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

Comparative Enzymology of 11β-Hydroxysteroid Dehydrogenase Type 1 from Glucocorticoid Resistant (Guinea Pig) Versus Sensitive (Human) Species

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.

Spatial and temporal patterns of expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 messenger RNA and glucocorticoid receptor protein in the murine placenta and uterus during late pregnancy.

To gain insight into the role of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes and actions of glucocorticoids in the murine placenta and uterus, the expression pattern of the mRNA for 11beta-HSD1 and 11beta-HSD2 and the glucocorticoid receptor (GR) protein were determined from Embryonic Day 12.5 (E12.5, term = E19) to E18.5 by in situ hybridization and immunohistochemistry, respectively. Consistent with its putative role in regulating the transplacental passage of maternal glucocorticoid to the fetus, 11beta-HSD2 mRNA was highly expressed in the labyrinthine zone (the major site of maternal/fetal exchange) at E12.5, and its level decreased dramatically at E16.5, when it became barely detectable. Remarkably, the silencing of 11beta-HSD2 gene expression coincided with the onset of 11beta-HSD1 gene expression in the labyrinth at E16.5 when moderate levels of 11beta-HSD1 mRNA were detected and maintained to E18.5. By contrast, neither 11beta-HSD1 mRNA nor 11beta-HSD2 mRNA were detected in any cell types within the basal zone from E12.5 to E18.5. Moreover, the expression of 11beta-HSD1 and 11beta-HSD2 in the decidua exhibited a high degree of cell specificity in that the mRNA for both 11beta-HSD1 and 11beta-HSD2 was detected in the decidua-stroma but not in the compact decidua. A distinct pattern was also observed within the endometrium where the mRNA for 11beta-HSD1 was expressed in the epithelium, whereas that for 11beta-HSD2 was confined strictly to the stroma. By comparison, the expression of GR in the placenta and uterus was ubiquitous and unremarkable throughout late pregnancy. In conclusion, the present study demonstrates for the first time remarkable spatial and temporal patterns of expression of 11beta-HSD1 and 11beta-HSD2 and GR in the murine placenta and uterus and highlights the intricate control of not only transplacental passage of maternal glucocorticoid to the fetus but also local glucocorticoid action during late pregnancy.

The expression of 11 beta-hydroxysteroid dehydrogenase type 2 is induced during trophoblast differentiation: effects of hypoxia.

The intracellular enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) catalyzes the unidirectional conversion of bioactive glucocorticoids to their inert metabolites. In the human placenta, 11 beta-HSD2 is highly expressed in syncytiotrophoblasts, although cytotrophoblasts also express this enzyme at lower levels. Given that cytotrophoblasts will differentiate into syncytiotrophoblasts in vivo and in vitro, the present study was designed to examine the hypothesis that the expression of 11 beta-HSD2 is induced during in vitro trophoblast differentiation. When Percoll-purified human cytotrophoblast cells were cultured under standard (20% oxygen) conditions, they aggregated and fused to form syncytiotrophoblasts. Within the first 24 h during differentiation, levels of 11 beta-HSD2 protein and activity were increased by 2- to 3-fold, but they did not increase further thereafter. However, when the cells were exposed to hypoxic (1% oxygen) conditions, both the induction of 11 beta-HSD2 and trophoblast differentiation were prevented. Taken together, these results demonstrate for the first time that the expression of 11 beta-HSD2 is induced early during trophoblast differentiation, and hypoxia prevents this induction, indicating that placental 11 beta-HSD2 expression is subjected to regulation by the local oxygen environment. If placental villi respond to hypoxia in a similar fashion in vivo, the present findings would suggest that hypoxia might be a factor contributing to the previously reported decreases in placental 11 beta-HSD2 in pregnancies complicated by intrauterine growth restriction and preeclampsia.

Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines.

Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.

Decreased gene expression of 11beta-hydroxysteroid dehydrogenase type 2 and 15-hydroxyprostaglandin dehydrogenase in human placenta of patients with preeclampsia.

Cortisol reduces the activity of the PG-inactivating enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH) in human placental cells. The objective was to investigate a possible relation between 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), converting cortisol to cortisone, and PGDH gene expression in the placenta of patients with preeclampsia. In placental tissue taken from 20 healthy women with normal pregnancy, 20 premature babies born after labor before term, and 18 neonates after preeclamptic pregnancy, 11beta-HSD2 and PGDH messenger RNA (mRNA) expression was determined using quantitative TaqMan real-time PCR and quantitative competitive PCR. When comparing matched pairs, there were 3-fold lower 11beta-HSD2/glyceraldehyde-3-phosphate dehydrogenase (11beta-HSD2/GAPDH) mRNA levels in placentas of patients with preeclampsia than in controls [0.18 +/- 0.04 relative units (RU) and 0.61 +/- 0.10 RU, P = 0.0003]. We also found a 2-fold reduction in placental PGDH/GAPDH mRNA concentrations (0.28 +/- 0.15 RU and 0.50 +/- 0.18 RU, P = 0.0003). PGDH and 11beta-HSD2 mRNA levels correlated significantly (r = 0.66, P < 0.0001). In term placenta, 11beta-HSD2/GAPDH, but not PGDH, showed a significant correlation to birth weight (r = 0.43, P = 0.01) and to placental weight (r = 0.47, P = 0.01). Results could be confirmed by competitive PCR. We conclude that, in preeclampsia, 11beta-HSD2 mRNA expression is reduced, leading to the known decrease of 11beta-HSD2 activity. By means of an autocrine or paracrine mechanism, the diminished conversion of placental cortisol may lead to reduced PGDH mRNA expression as found in the present study.

Impact of Restriction of Placental and Fetal Growth on Expression of 11β-Hydroxysteroid Dehydrogenase Type 1 and Type 2 Messenger Ribonucleic Acid in the Liver, Kidney, and Adrenal of the Sheep Fetus1

We have investigated the effects of fetal growth restriction, induced by restriction of placental growth and function (PR), on 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1) and 11betaHSD-2 messenger RNA (mRNA) expression in fetal tissues in the sheep, using Northern blot analysis. Fetal liver, kidney, and adrenals were collected from normally grown fetuses at 90 days (n = 6), 125 days (n = 6), and 141-145 days (n = 7) and from PR fetuses at 141-145 days (n = 6). Expression of 11betaHSD-1 mRNA in the fetal liver increased significantly between 125 days (7.4+/-0.8) and 141-145 days gestation (27+/-5.3). There was also an approximately 2-fold increase in the ratio of 11betaHSD-1 mRNA/18S rRNA expression in the PR group (53.8+/-7.9) compared with that in control animals at 141-145 days gestation. There was a significant decrease in 11betaHSD-2 mRNA in fetal adrenals between 125 days (41.6+/-2.4) and 141-145 days (26.7+/-1.1) gestation, but there was no effect of PR on the expression of adrenal 11betaHSD-2 mRNA. 11betaHSD-2 mRNA expression in the fetal kidney increased between 90 days (16.8+/-1.7) and 141-145 days gestation (31.7+/-4.3), but there was no effect of PR on the levels of 11betaHSD-2 mRNA in the fetal kidney. In summary, 11betaHSD-2 mRNA is differentially regulated in the fetal adrenal and kidney in the sheep fetus during late gestation. There is also a specific increase in the expression of 11betaHSD-1 mRNA in the liver of growth-restricted fetuses in late gestation. This suggests that there is increased hepatic exposure to cortisol in the growth-restricted fetus, which may be important in the reprogramming of hepatic physiology that occurs after growth restriction in utero.

Expression of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) in the developing human adrenal gland and human adrenal cortical carcinoma and adenoma

The aim of this study was to investigate the ontogeny of localization of 11βHSD-2 protein in the human adrenal gland. In addition, we have investigated the effects of abnormal adrenal function on 11βHSD-2 by determining the pattern of localization of 11βHSD-2 protein, and the amount and level of expression of 11βHSD-2 mRNA and protein in human adrenal cortical carcinoma and adenoma. In the human foetal adrenal gland 11βHSD-2 immunoreactivity (11βHSD-2-ir) was detected in the foetal zone, whereas in normal adult adrenal glands 11βHSD-2-ir was not detected by immunocytochemistry. In adrenal cortical carcinoma and adenoma, 11βHSD-2-ir was detectable in specific regions, which have been identified as steroid synthesizing cells using 3βHSD-ir as a marker. In adrenal cortical carcinoma and adenoma, 11βHSD-2 mRNA and 11βHSD-2 protein were detected by nuclease protection analysis and by western blot analysis, respectively. In summary, 11βHSD-2-ir was detected in the foetal zone of the mid-gestation human foetal adrenal, whereas, 11βHSD-2-ir was not detectable in the postnatal or normal adult adrenal gland. 11βHSD-2 protein and mRNA was induced in adult human adrenal cortical carcinoma and adenoma. The induction of expression of 11βHSD-2 in the adrenal cortex suggests a possible role in regulating abnormal adrenal steroidogenic function in these patients.

Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.

The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (R(T) approximately 4700 ohm x cm2) and potential difference (P(D) approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 microA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10(-10) to 10(-6) M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5x10(-7) M)-induced Isc (fourfold) was associated with a three- to fivefold increase in alpha-ENaC mRNA (but not in those for beta- or gamma-ENaC) and three- to 10-fold increases in alpha-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Expression of 11β-hydroxysteroid dehydrogenase type 2 in an ACTH-producing small cell lung cancer

Non-pituitary tumors that produce adrenocorticotropic hormone (ACTH) exhibit resistance to the normal feedback effects of glucocorticoids on proopiomelanocortin (POMC) gene expression. This glucocorticoid resistance is typically complete, although some tumors show only relative glucocorticoid resistance in the clinical setting. The molecular mechanisms responsible for these clinical pathophysiologic observations are unknown, but might include glucocorticoid receptor defects or aberrant expression of enzymes or transporters that exclude glucocorticoids from access to their intracellular receptors. We examined whether ACTH-producing non-pituitary tumor cells might express 11β-hydroxysteroid dehydrogenase (11β-HSD), the principal ‘gatekeeper’ enzyme known to metabolize glucocorticoids. 11β-HSD mRNA and enzyme activity were assessed in DMS-79 cells, a line derived from an ACTH-producing small cell lung cancer. RT-PCR studies showed expression of mRNA encoding 11β-HSD2 but not 11β-HSD1 in DMS-79 cells. Control human fibroblasts expressed predominantly 11β-HSD1 but also had detectable 11β-HSD2 mRNA, while HepG2 hepatoma cells also expressed only 11β-HSD2 mRNA. Whole cell assays in DMS-79 cells revealed 11β-HSD activity with a K m for cortisol of 26.1 ± 9.0 nM and V max of 57.0 ± 5.9 pmol/h/mg protein. HepG2 cells expressed a similar high affinity enzyme activity, while control fibroblasts expressed 11β-HSD activity with a K m for cortisol of 652 nM. Conversion of cortisol to cortisone in DMS-79 cells was inhibited to 7% of baseline by addition of 10 μM glycyrrhetinic acid. Dexamethasone (20 nM) was converted to a single product in DMS-79 cells at a rate of 17.2 pmol/h/mg protein; this activity was also inhibited by glycyrrhetinic acid. We conclude that DMS-79 cells express 11β-HSD2. While DMS-79 cells harbor additional defects in glucocorticoid signaling, these data suggest that expression of 11β-HSD2 might contribute to the development of the glucocorticoid-resistant phenotype of some ACTH-producing tumors.