Modulation of transalveolar fluid absorption by endogenous aldosterone in adult rats.

We examined whether transalveolar fluid transport is modulated by aldosterone in adult rats. Because colocalization of mineralocorticoid receptors (MR) with 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is important for aldosterone specific action, we first determined the immunohistochemical distribution of MR and 11betaHSD2 in the lung. We found that alveolar epithelial cells express both MR and 11betaHSD2. Reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that rat alveolar type II epithelial cells express both MR and 11betaHSD2. We then measured alveolar fluid clearance in rats treated with chronic low-sodium diet. A low-sodium diet (0.1% NaCl for 12 to 14 days) caused hyperaldosteronism accompanied by hypokalemia, whereas serum corticosterone and adrenaline levels remained normal. We found that hyperaldosteronism was associated with significantly higher alveolar fluid clearance and that this increase was related to the amiloride-sensitive component. In addition, the increase in alveolar fluid clearance was inhibited by spironolactone. Our results show that aldosterone is able to stimulate Na+ channels of alveolar epithelial cells. We conclude that alveolar epithelium is a physiological target tissue for aldosterone and transalveolar fluid absorption could in part be modulated by endogenous aldosterone acting via MR.

Immunohistochemical distribution of 11beta-hydroxysteroid dehydrogenase in human eye.

11beta-hydroxysteroid dehydrogenase (11beta-HSD) regulates local actions of corticosteroids at glucocorticoid and mineralocorticoid receptors. Corticosteroids are thought to play important roles in ocular function. However, mechanisms of intraocular corticosteroid action are still unclear. Therefore, in this study, we examined the immunohistochemical localization of 11beta-HSD type 1 (11beta-HSD1), 11beta-HSD type 2 (11beta-HSD2), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in human ocular tissues from patients (6 months to 78 years of age; n = 10) retrieved from surgical pathology files. Both 11beta-HSD2 and MR immunoreactivity was detected only in non-pigmented epithelium of the ciliary body, but was undetectable in cornea, lens, iris, retina, choroid and sclera, in all the cases examined. GR was detected in all cell types in the human eye. 11beta-HSD1 immunoreactivity was not detected in the human eye in this study. These results suggest that 11beta-HSD2 play an important role in human ocular mineralocorticoid action, such as the production of aqueous humor, in the ciliary body. The widespread expression of GR suggests that glucocorticoids may play an important role in the function and homeostasis of the human eye.

Novel nuclear corticosteroid binding in rat small intestinal epithelia.

When small intestinal epithelial cells are incubated with [(3)H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [(3)H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [(3)H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/microg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11beta-dehydrogenase activity, suggesting that binding may be to an 11beta-hydroxysteroid dehydrogenase (11betaHSD) isoform, although 11betaHSD1 is not present in small intestinal epithelia and 11betaHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [(3)H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11betaHSD2 but is distinguishable from the latter by its distinct intracellular localization.

Expression of 11 beta-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor in primary lung carcinomas.

We examined the immunohistochemical distribution of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) and mineralocorticoid receptor (MR) in 63 primary lung carcinomas. Immunoreactivity of 11 beta HSD2 and MR was detected in 37 cases and in 32 cases of 42 adenocarcinomas, respectively. There was a significant correlation between 11 beta HSD2 and MR immunoreactivity. In three adenosquamous carcinomas, both 11 beta HSD2 and MR were detected only in adenocarcinomatous components. Neither 15 squamous cell carcinomas, 2 small cell carcinomas nor 1 large cell carcinoma expressed 11 beta HSD2 or MR. In papillary and acinar adenocarcinomas, both 11 beta HSD2 and MR immunoreactivity was significantly correlated with the grade of histological differentiation. The patterns of 11 beta HSD2 and MR expression in 10 lymph-node metastases were similar to those determined in the primary lesions. These data suggest that the patterns of 11 beta HSD2 and MR expression may reflect cellular origin and differentiation status of primary lung adenocarcinomas and serve as a new useful marker of differentiation.

11Beta-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human placenta.

In mineralocorticoid target organs, 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) confers specificity on the mineralocorticoid receptor (MR) by converting biologically active glucocorticoids to inactive metabolites. Placental 11beta-HSD2 is also thought to protect the fetus from high levels of circulating maternal glucocorticoid. In this study, we examined the immunoreactivity of 11beta-HSD2 and MR in human placenta from 5 weeks gestation to full term using immunohistochemistry, 11beta-HSD2 messenger RNA (mRNA) expression using Northern blot analysis, and MR mRNA expression using RT-PCR analysis. Marked 11beta-HSD2 immunoreactivity was detected in placental syncytiotrophoblasts at all gestational stages. MR immunoreactivity was moderately detected in syncytiotrophoblasts, some cytotrophoblasts, and interstitial cells of the villous core. Marked mRNA expression of 11beta-HSD2 was detected in placenta by Northern analysis. RT-PCR analysis of MR in placental tissues showed an amplified product consistent in length with the primers selected. These results suggest that placental 11beta-HSD2 is involved in not only regulating the passage of maternal active glucocorticoids into the fetal circulation but also in regulation of maternal-fetal electrolyte and water transport in the placenta, as in other mineralocorticoid target organs.

Mineralocorticoid receptors in the mammalian olfactory mucosa.

Mineralocorticoid hormones regulate secretion and absorption in a wide variety of epithelial tissues, although specific mechanisms in the olfactory mucosa are currently unknown. Utilizing reverse transcription-polymerase chain reaction (RT-PCR) analysis, we have demonstrated the expression of mineralocorticoid (type I) receptor messenger RNA in the rodent olfactory mucosa. Amplification products of predicted size were obtained with nucleotide sequences corresponding to respective mineralocorticoid receptor (MR) kidney transcripts. Immunocytochemistry, using an antibody with known specificity for MRs, was then utilized in order to localize the cellular site(s) of MR protein expression in the olfactory mucosa. The highest levels of MR immunoreactivity were localized to the supranuclear region of sustentacular cells, as well as the acinar cells of the Bowman's glands. The respiratory regions of the nasal cavity were devoid of appreciable MR immunoreactivity. This study demonstrates both MR transcript and protein expression in the olfactory mucosa. We hypothesize that the mineralocorticoid hormones may have a role in modulation of olfactory secretion and/or sensory transduction in the peripheral olfactory system.

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

11 beta-Hydroxysteroid dehydrogenase.

In mammalian tissues, at least two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyze the interconversion of hormonally active C11-hydroxylated corticosteroids (cortisol, corticosterone) and their inactive C11-keto metabolites (cortisone, 11-dehydrocorticosterone). The type 1 and type 2 11 beta-HSD isozymes share only 14% homology and are separate gene products with different physiological roles, regulation, and tissue distribution. 11 beta-HSD2 is a high affinity NAD-dependent dehydrogenase that protects the mineralocorticoid receptor from glucocorticoid excess; mutations in the HSD11B2 gene explain an inherited form of hypertension, the syndrome of apparent mineralocorticoid excess in which cortisol acts as a potent mineralocorticoid. By contrast, 11 beta-HSD1 acts predominantly as a reductase in vivo, facilitating glucocorticoid hormone action in key target tissues such as liver and adipose tissue. Over the 10 years, 11 beta-HSD has progressed from an enzyme merely involved in the peripheral metabolism of cortisol to a crucial pre-receptor signaling pathway in the analysis of corticosteroid hormone action. This review details the enzymology, molecular biology, distribution, regulation, and function of the 11 beta-HSD isozymes and highlights the clinical consequences of altered enzyme expression.

11Beta-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor in human fetal development.

11Beta-Hydroxysteroid dehydrogenase type II (11betaHSD2) confers specificity on the mineralocorticoid receptor (MR) by converting biologically active glucocorticoids to inactive 11-keto metabolites. The biological significance of 11betaHSD2 activity during fetal development is currently being explored, but the temporal and spatial distributions of the enzyme and receptor have not been examined. We therefore examined their distributions during various stages of human fetal development using immunohistochemistry. Both 11betaHSD2 and MR immunoreactivity were detected in the distal convoluted and collecting tubules of the kidney from early in gestation. Fetal skin, intermediate layer of the epidermis, peridermal cells, and hair follicles were positive for both 11betaHSD2 and MR. Weak 11betaHSD2 and MR immunoreactivity was detected in the superficial ciliated epithelium of the esophagus, the deep layer of gastric epithelial cells, and the superficial epithelium of the small intestine. Columnar epithelium in the terminal bronchiolar budding component of fetal lung and tracheal and bronchial ciliated epithelium were also positive for MR and 11betaHSD2 from early gestation. Colonic epithelium and pancreatic exocrine duct cells, which demonstrated marked immunoreactivity of both MR and 11betaHSD2 in the adult, did not express MR and 11betaHSD2 until very late in gestation. These results imply that mineralocorticoid action in the upper fetal gastrointestinal tract, kidney, skin, and lung is facilitated by 11betaHSD2 and is involved in water and electrolyte transport between fetus and amniotic fluid as well as fetal urine production.

Type II 11beta-hydroxysteroid dehydrogenase expression in human colonic epithelial cells of inflammatory bowel disease.

Type IIbeta-hydroxysteroid dehydrogenase endows specificity on the mineralocorticoid receptor by metabolizing cortisol and regulates sodium absorption in renal and colonic epithelium. Altered expression of this enzyme may be associated with impaired sodium absorption often seen in colonic mucosa of inflammatory bowel disease. The aim of this study was to investigate possible abnormality of 11beta-hydroxysteroid dehydrogenase protein and mRNA expression in inflammatory bowel disease. In Crohn's disease, the colonic epithelium showed comparable levels of immunoreactivity and mRNA expression to those of control, except for the decreased immunoreactivity in severe inflamed lesions with deep ulcer. In contrary, a lack or decrease of immunoreactivity was relevant in ulcerative colitis regardless of the histological degree of inflammation. The mRNA expression was also significantly decreased in ulcerative colitis. This study demonstrates that abnormality of epithelial cells in ulcerative colitis includes the enzyme that regulates water and sodium absorption, which are physiologically essential.

Structural analysis and evaluation of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) gene in human essential hypertension.

AIM: Mutations of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) gene cause the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive form of hypertension. We therefore investigated the question of whether variants of the 11beta-HSD2 gene can contribute to genetic susceptibility to essential hypertension. SUBJECTS AND METHODS: We performed a linkage study in 162 French hypertensive sibships using the affected sib-pair method on 347 sibling pairs and a polymorphic microsatellite marker that we identified in a 30 kb cosmid clone containing the 11beta-HSD2 gene. The coding sequence, introns 2-4 and 350 bp of the 5'-flanking region of the 11beta-HSD2 gene were screened for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism, and a single polymorphism, Glu178/Glu (G534A), was identified in exon 3, which did not change the encoded amino acid sequence. A case-control study was conducted on 370 hypertensive subjects with a positive family history of hypertension and 783 French subjects with hypertension with or without a family history of hypertension, compared with 313 normotensive control subjects, all of whom were analyzed for the newly identified bi-allelic polymorphism. RESULTS: Statistical analyses using the affected sib-pair method did not show significant linkage between the 11beta-HSD2 microsatellite marker and hypertension. Furthermore, no positive association with hypertension was found with the Glu178/Glu (G534A) polymorphism. CONCLUSION: Our data do not suggest that variants of the 11beta-HSD2 gene contribute substantially to essential hypertension in Caucasians.

11 beta-hydroxysteroid dehydrogenase type II in human colon: a new marker of fetal development and differentiation in neoplasms.

The colon plays an important role in water and electrolyte homeostasis and is a major target tissue for aldosterone. 11 beta-Hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) confers specificity to the non-selective mineralocorticoid receptor by inactivating glucocorticoids, thus allowing binding by mineralocorticoids. Using immunohisto/cytochemistry and Northern blot analysis, we examined 11 beta-HSD2 expression in human fetal colon (23 cases ranging in age from 15 to 40 weeks gestation), neonatal colon (2 cases, 6 and 12 months old), normal adult colon (15), adenoma (35), adenocarcinoma (34) and the human colonic epithelial cell line T84, in the presence or absence of sodium butyrate, to study the correlation between enzyme expression and cellular or neoplastic differentiation. In fetal colon, weak 11 beta-HSD2 immunoreactivity was detectable in superficial epithelium from 25 weeks gestation, but normal adult levels were apparent only after 40 weeks gestation, suggesting that fully developed aldosterone induced electrolyte transport can occur only at late gestational stages. In adult normal colon, superficial absorptive cells at the top of crypts were strongly positive for 11 beta-HSD2, whereas immunoreactivity was weak in adenomas and carcinomas, and the pattern of localization varied among patients. Northern blot analysis performed on 4 cases of adenocarcinoma also demonstrated lower levels of mRNA than autologous non-neoplastic colonic mucosa. In carcinomas, 11 beta-HSD2 immunoreactivity was more frequently detected in differentiated structures, such as those forming glandular or tubular structures. Studies with T84 cells showed that expression of 11 beta-HSD2 was markedly enhanced with the addition of sodium butyrate, a well known inducer of cell differentiation in colonic epithelia. These results suggest that the expression of 11 beta-HSD2 is associated with differentiation or maturation in human colonic epithelia and that the enzyme may serve as a useful marker in development and disease.

11Beta-hydroxysteroid dehydrogenase type 2 in human lung: possible regulator of mineralocorticoid action.

11Beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) catalyzes the conversion of cortisol to biologically inactive cortisone and is thought to confer specificity on mineralocorticoid receptors (MR). Cortisol is a prerequisite for surfactant synthesis and fetal lung maturation. Recently, expression of 11betaHSD2 was demonstrated in human fetal lung, but its localization and possible biological roles remain unknown. Therefore, in this study, we examined immunohistochemical localization of 11betaHSD2, MR, and glucocorticoid receptor (GR) in nonpathological human lungs from fetus to adult (8 weeks gestation to 55 yr of age; n = 40) retrieved from pathology files. Both 11betaHSD2 and MR immunoreactivities were detected in airway epithelia, from bronchiole to trachea and in fetal and neonatal ciliated collecting duct cells of tracheal and bronchial glands, but were undetectable in alveoli. On the other hand, GR was detected in all cell types. These results indicate that 11betaHSD2 colocalizes with MR in human airway epithelia and suggest that 11betaHSD2 play an important role in pulmonary mineralocorticoid activity such as sodium and fluid transport.

Serines at the active site of 11 beta-hydroxysteroid dehydrogenase type I determine the rate of catalysis.

Short chain alcohol dehydrogenases have an invariant YXXXK motif at the active site. Database analysis of 116 superfamily members showed that 92% also contain a Serine or Threonine residue at the Y + 1 or Y + 3 positions, a pattern we previously described as the ST rule. In the present study we have mutated Serines in the active site, YSASK, motif of 11 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studies were facilitated by the generation of a new specific polyclonal antibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34 kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active site Serines resulted in modest increases of Km values for corticosterone from 325 nM for 11 beta HSD1 to 512 nM-588 nM for the S1 (YAASK), S2 (YSAGK) and S3 (YAAGK) mutants in homogenates of transfected CHOP cells. However, far greater effects were observed on the first order rate constants with mutants displaying 10%, 1% and 1% of the wild type activity, respectively. When the oxidoreductase reaction was studied in whole cells mutagenesis again had a minimal effect on the Km value but dramatically lowered first order rate constants to 34%, 5% and 6%, respectively, of the wild type. These data show that Serines at the active site of 11 beta HSD1 play an important role in determining the rate of catalysis. Coexpression of wild type and mutant enzymes did not lower wild type activity suggesting that the active site of the multimeric enzyme is not a composite of active site Serines from different subunits.

A genetic defect resulting in mild low-renin hypertension.

Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (HSD11B2) encoding the 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD2) cause AME. Typical patients with AME have defective 11beta-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-3H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the HSD11B2 gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately elevated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0-6% in typical patients with AME whereas the normal conversion is 90-95%. Molecular analysis of the HSD11B2 gene of this patient showed a homozygous C-->T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation. In vitro expression studies showed an increase in the Km (300 nM) over normal (54 nM). Because approximately 40% of patients with essential hypertension demonstrate low renin, we suggest that such patients should undergo genetic analysis of the HSD11B2 gene.

Steroid specificity of the putative DHB receptor: evidence that the receptor is not 11betaHSD.

Recently, we identified a novel putative nuclear receptor in colonic crypt cells distinct from both mineralocorticoid receptor and glucocorticoid receptor, with high affinity for 11-dehydrocorticosterone (11-DHB) (33). In the present study, competitive nuclear binding assays demonstrated that this site has a unique steroid binding specificity that distinguishes it from other steroid receptors. Western blot analysis showed the presence of 11beta-hydroxysteroid dehydrogenase-2 (11betaHSD2) but not 11betaHSD1 in colonic crypt cells and showed that 11betaHSD2 was present in the nuclear pellet. Differences in steroid specificity between the putative DHB receptor and inhibition of 11betaHSD activity indicate that binding is not to the enzyme. Furthermore, modified Chinese hamster ovary cells transfected with the 11betaHSD2 gene express nuclear 11betaHSD2 but not a nuclear DHB binding site. In conclusion, these data support the existence of a novel nuclear DHB receptor in rat colon that is distinct from the classic steroid receptors and from both 11betaHSD1 and 11betaHSD2.

Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.

Examination of genotype and phenotype relationships in 14 patients with apparent mineralocorticoid excess.

Apparent mineralocorticoid excess (AME) is a genetic disorder causing pre- and postnatal growth failure, juvenile hypertension, hypokalemic metabolic alkalosis, and hyporeninemic hypoaldosteronism due to a deficiency of 11 beta-hydroxysteroid dehydrogenase type 2 enzyme activity (11 beta HSD2). The 11 beta HSD2 enzyme is responsible for the conversion of cortisol to the inactive metabolite cortisone and therefore protects the mineralocorticoid receptors from cortisol intoxication. Several homozygous mutations are associated with this potentially fatal disease. We have examined the phenotype, biochemical features, and genotype of 14 patients with AME. All of the patients had characteristic signs of a severe 11 beta HSD2 defect. Birth weights were significantly lower than those of their unaffected sibs. The patients were short, underweight, and hypertensive for age. Variable damage of one or more organs (kidneys, retina, heart, and central nervous system) was found in all of the patients except one. The follow-up studies of end-organ damage after 2-13 yr of treatment in six patients demonstrated significant improvement in all patients. The urinary metabolites of cortisol demonstrated an abnormal ratio with predominance of cortisol metabolites, i.e. tetrahydrocortisol plus 5 alpha-tetrahydrocortisol/tetrahydrocortisone was 6.7-33, whereas the normal ratio is 1.0. Infusion of [11-3H]cortisol resulted in little release of tritiated water, indicating the failure of the conversion of cortisol to cortisone. Thirteen mutations in the HSD11B2 gene have been previously published, and we report three new genetic mutations in two patients, one of whom was previously unreported. All of the patients had homozygous defects except one, who was a compound heterozygote. Our first case had one of the most severe mutations, resulting in the truncation of the enzyme 11 beta HSD2, and died at the age of 16 yr while receiving treatment. Three patients with identical homozygous mutations from different families had varying degrees of severity of clinical and biochemical features. Due to the small number of patients with identical mutations, it is difficult to correlate genotype with phenotype. In some cases, early and vigilant treatment of AME patients may prevent or improve the morbidity and mortality of end-organ damage such as renal or cardiovascular damage and retinopathy. The outcome of treatment in more patients may establish the efficacy of treatment.

Immunolocalization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in rat uterus: variation across the estrous cycle and regulation by estrogen and progesterone.

Glucocorticoid hormone action in several target tissues is dependent not only on the expression of the glucocorticoid receptor, but also on that of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes, 11betaHSD-1 and -2. In the uterus, glucocorticoids can exert inhibitory effects on a range of important functions, particularly in relation to the effects of estrogen. Therefore, the present study examined immunolocalization of the two 11betaHSD enzymes in the rat uterus at each stage of the estrous cycle and after ovariectomy with or without estrogen/progesterone replacement. In cycling rats 11betaHSD-1 was localized to luminal and glandular epithelial cells and to eosinophils in both the endometrial stroma and myometrium. In contrast, 11betaHSD-2 immunostaining was localized to endometrial stromal cells and myometrial cells, with no staining evident in epithelial cells or eosinophils. Immunostaining for both enzymes was cycle dependent, being maximal at proestrus and minimal at diestrus. Western blot analysis of whole uterus at proestrus showed the presence of 34- and 40-kDa immunoreactive species for 11betaHSD-1 and -2, respectively. These immunoreactive signals were almost abolished by ovariectomy, but this effect was reversed for both enzymes by estrogen replacement with or without progesterone. These effects of ovariectomy and steroid replacement were confirmed by immunocytochemical analysis, with the exception that progesterone appeared to enhance the stimulatory effects of estrogen on 11betaHSD-2 specifically within the endometrial stroma. In conclusion, these results establish the presence of both 11betaHSD-1 and -2 in the nonpregnant rat uterus and show distinct distributions for the two enzymes and cyclic variation related to positive regulation by ovarian steroids. The physiological implications of these patterns of 11betaHSD expression will ultimately depend on the reaction direction for each enzyme, but 11betaHSD-2 is likely to limit disruptive effects of glucocorticoids on the endometrial stroma, and 11betaHSD-1 may then serve to selectively reactivate glucocorticoids in epithelial cells.

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.