11-Beta hydroxysteroid dehydrogenase type 2 in human adult and fetal lung and its regulation by sex steroids.

11-Beta hydroxysteroid dehydrogenase type 2 (HSD2) oxidizes the biologically active glucocorticoid (GC), cortisol, to inactive cortisone. We characterized HSD2 gene expression and activity in human adult and fetal lung tissues and in cultured fetal lung explants, and examined the potential regulation of HSD2 in the fetal lung by sex steroids. Human adult lung, fetal lung, and cultured fetal lung explant tissues contained similar amounts of HSD2 mRNA. However, higher levels of HSD2 protein were detected in human fetal lung tissue than in adult lung, with expression being restricted to a subset of epithelial cells in the fetal lung tissue. Differentiated fetal lung explants maintained in culture expressed higher levels of HSD2 protein and enzymatic activity than undifferentiated fetal lung tissues. Finally, HSD2 protein levels were decreased in male, but not female, fetal lung explants treated with 17-beta estradiol. In contrast, 5-alpha dihydrotestosterone did not significantly affect HSD2 levels. These data indicate that HSD2 protein and activity levels increase in parallel with the differentiation of alveolar type II epithelial cells in vitro, and that HSD2 protein levels are regulated by 17-beta estradiol in male fetal lung tissue.

Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram.

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.

11Beta-hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites.

Glucocorticoid (GC) metabolism by the 11beta-hydroxysteroid dehydrogenase (HSD) system is an important prereceptor regulator of GC action. The HSD enzymes catalyze the interconversion of the endogenous, biologically active GC cortisol and its inactive 11-dehydro metabolite cortisone. The role of the HSD enzymes in the metabolism of synthetic GCs, such as dexamethasone (Dex), is more complex. The human lung is a classic GC-sensitive organ; however, the roles of the HSD enzymes (HSD1 and HSD2) in the human lung are poorly understood. In the present study, we examined the expression of the HSD enzymes in human adult and fetal lung tissues and the human lung epithelial cell line NCI-H441. We observed that human adult and fetal lung tissues, as well as H441 cells, express HSD2 protein and that it is upregulated by Dex (10(-7) M). By contrast, HSD1 protein was undetectable. We also show that the Dex-mediated regulation of surfactant protein A is attenuated by inhibition of HSD2 activity. Furthermore, we demonstrate that unlike the inactive, 11-dehydro metabolite of cortisol (i.e., cortisone), the 11-dehydro metabolite of Dex, 11-dehydro-Dex, competes for binding to the GC receptor (GR) in human lung epithelial cells and retains GR agonist activity. Together, these data suggest that differences exist in the biological activities of the metabolites of cortisol and Dex.

Inflammatory mediators down-regulate 11beta-hydroxysteroid dehydrogenase type 2 in a human lung epithelial cell line BEAS-2B and the rat lung.

In the lung, anti-inflammatory actions of glucocorticoids would be determined by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the microsomal enzyme responsible for the breakdown of bio-active glucocorticoids. However, regulation of 11beta-HSD2 under inflammatory conditions such as acute lung injury is not well understood. In the present study, we examined whether inflammatory substances would influence the activity and mRNA expression of 11beta-HSD2 in the lung. In a human bronchial epithelial cell line BEAS-2B, endotoxin inhibited 11beta-HSD2 enzyme activity in a dose-dependent manner over 48 h with a significant decrease in the mRNA expression. Likewise, tumor necrosis factor (TNF)-alpha inhibited both activity and mRNA expression of 11beta-HSD2. The TNF-alpha-dependent decrease in the enzyme activity was completely blocked by anti-TNF-alpha antibody, while antibody alone showed no significant influence on the enzyme activity. An nitric oxide donor (NO) sodium nitropusside or a cGMP analog 8-br-cGMP caused moderate but significant decreases in both activity and mRNA expression of 11beta-HSD2. Importantly, treatment of rats with endotoxin significantly decreased both activity and mRNA expression of 11beta-HSD2 in the lung tissue. We conclude that lung inflammation reduces local glucocorticoid breakdown and augments glucocorticoid action in the lung by down-regulating 11beta-HSD2 via multiple mechanisms.

Aldosterone enhances 11beta-hydroxysteroid dehydrogenase type 2 expression in colonic epithelial cells in vivo.

OBJECTIVE: [corrected] 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) metabolizes glucocorticoids, thus enabling aldosterone to bind to the mineralocorticoid receptor. However, little is known about the regulatory mechanism of epithelial 11beta-HSD2 expression in the gut. MATERIALS AND METHODS: Sprague-Dawley rats were maintained on a sodium-depleted diet or subjected to continuous aldosterone infusion for 4 weeks. Plasma aldosterone and arginine-vasopressin (AVP) levels were measured by radioimmunoassay. Expression of 11beta-HSD2 in colonic epithelia was evaluated by Northern blotting and immunohistochemistry. T84 and Caco2 cells were stimulated with aldosterone, dexamethasone and AVP alone or in combination, and 11beta-HSD2 mRNA was measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Sodium-depleted and aldosterone-infused rats showed an increase of plasma aldosterone and AVP. Both treatments resulted in induction of 11beta-HSD2 in the colonic epithelia at mRNA and protein levels. Positive immunoreactivity was detected in the cytoplasm of the surface epithelia in control rats. In contrast, epithelial cells in the crypt also showed immunoreactivity for 11beta-HSD2 in the proximal colon of dietary sodium-depleted and aldosterone-infused rats. Induction of 11beta-HSD2 mRNA was observed when T84 cells were stimulated with corticosteroids plus AVP. CONCLUSIONS: Aldosterone has a pivotal role by increasing expression of 11beta-HSD2 in epithelial cells of the colon. AVP may act as a synergistic hormone in aldosterone-mediated 11beta-HSD2 induction.

Aromatase-deficient (ArKO) mice have reduced blood pressure and baroreflex sensitivity.

Aromatase-deficient (ArKO) mice are deficient in estrogens due to deletion of the aromatase gene. We hypothesized that there may be changes in the cardiovascular system of ArKO mice because of evidence linking estrogens with improved cardiovascular outcomes and the induction of the glucocorticoid-metabolizing enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), gene in the kidney, which is important for the regulation of blood pressure (BP). BP and baroreflex sensitivity (BRS) in female conscious ArKO mice were compared with those in age- and weight-matched wild-type (WT) mice. Power spectral analysis was used to determine cardiovascular variability and BRS. Although systolic BP was similar in the two groups, diastolic and mean BPs were lower in the ArKO mice (-6.3 +/- 1.9 and -4.6 +/- 2.1 mm Hg, respectively). Heart rate (HR) was greater in the ArKO mice (+36 +/- 6 beats/min). The mean BP in WT mice was 105 mm Hg, and the HR was 481 beats/min. In the autonomic frequency range, BP variability was 74% greater, and HR variability was only 26% that in WT mice. The BRS of ArKO mice was 46% of the value observed in WT mice. 11betaHSD2 levels were unaltered in ArKO mice, except in the kidney, where they were only 10% of WT levels. Estradiol administration to ArKO mice restored renal 11betaHSD2 to WT levels. The results show that ArKO mice have lower diastolic BP, but increased BP variability, perhaps due to an impaired BRS. Thus, aromatase activity is critical for normal autonomic control of the heart and, hence, for reducing the deleterious effects of high BP variability.

Localization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the male reproductive tract.

The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes, 11beta-HSD1 and 11beta-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11beta-HSD enzymes in the male reproductive tract of the adult rat. 11beta-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11beta-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11beta-HSD1) and at approximately 48 kDa. 11beta-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11beta-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11beta-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11beta-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11beta-HSD1 (i.e. clear cells showing intense 11beta-HSD2 staining but principal cells devoid of signal). 11beta-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11beta-HSD1 and 11beta-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.

Clinical relevance of airway 11beta-hydroxysteroid dehydrogenase type II enzyme in asthma.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) are responsible for the conversion of bioactive glucocorticoids to and from inactive metabolites. 11beta-HSD2 is generally considered a high-affinity inactivator of natural glucocorticoids, although its activity with synthetic compounds in vivo is unknown. Inhaled corticosteroids (ICS) remain the primary antiinflammatory agents for treating asthma, but little is known about their metabolism in the lung. The aims of this study were to determine whether the 11beta-HSD2 enzyme can be localized to human airway tissue and whether differential expression of this enzyme relates to asthma severity and ICS needs. We studied airway biopsy specimens from 22 asthmatic subjects, in two groups: (1) a group not treated with ICS (n = 7); and (2) a group treated with ICS (range: 200 to 1,500 microg/d; n = 15). A control population consisted of nine nonasthmatic subjects. Immunostaining was done with an immunopurified antibody to human 11beta-HSD2. Immunoreactivity was generally localized to the endothelium of vessels in the lamina propria and to airway epithelium both in asthmatic patients and nonasthmatic controls. There was a statistically significant inverse relationship between the ICS dose required for effective treatment and the extent of epithelial 11beta-HSD2 staining (r = -0.44; p = 0.04). This is consistent with 11beta-HSD2 acting as an oxidoreductase that regenerates rather than inactivates ICS. This study suggests that glucocorticoid sensitivity in the lung is not determined by ICS breakdown, but may be related to 11beta-HSD2 sustaining the activation of synthetic glucocorticoids.

11beta-hydroxysteroid dehydrogenase type 2 is expressed in the human kidney glomerulus.

Our previous study demonstrated that the GR is expressed in the human kidney glomerulus. The function of the GR of glomerular cells might be affected by the concentration of intracellular glucocorticoids, which is modulated by 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2). Because the expression of 11betaHSD2 in the glomerular cells remains unclear, we used competitive RT-PCR and immunoblotting to detect the expression of 11betaHSD2 mRNA and protein in isolated human glomeruli, in whole kidney cortex as a positive control, and in a human glomerular visceral epithelial cell line. 11betaHSD2 mRNA was detected in all samples. Specific antihuman 11betaHSD2 antibody recognized a single band at 41 kDa, consistent with the molecular mass of human 11betaHSD2, in the samples of the isolated glomeruli and whole kidney cortex. Furthermore, definite 11betaHSD2 enzymatic activity was also determined with the sample of isolated glomeruli. We also performed immunohistochemistry by light and electron microscopy to determine the cellular and subcellular localization of 11betaHSD2 in the human glomeruli. Immunoreactivity of the enzyme was clearly observed in the glomerular visceral epithelial cells and endothelial cells as well as in the distal convoluted tubules and collecting ducts. The subcellular localization of 11betaHSD2 was shown to be endoplasmic reticulum. These results suggest that 11betaHSD2 might play a crucial role in modulating the intracellular concentration of glucocorticoids in human glomerular cells.