Expression and Regulation of 11- ß Hydroxysteroid Dehydrogenase Type 2 Enzyme Activity in the Glucocorticoid-Sensitive CEM-C7 Human Leukemic Cell Line.

Glucocorticoids are commonly used in the first-line treatment of hematological malignancies, such as acute lymphoblastic leukemia, due to the ability of these steroids to activate pro-apoptotic pathways in human lymphocytes. The goal of the current study was to examine the gene expression and enzyme activity of the microsomal enzyme, 11- ß hydroxysteroid dehydrogenase type 2 (HSD11B2, HSD2), which is responsible for the oxidation of bioactive glucocorticoids to their inert metabolites. Using the glucocorticoid-sensitive human leukemic cell line, CEM-C7, we were able to detect the expression of HSD2 at the level of mRNA (via RT-PCR), protein (via immunohistochemistry and immunoblotting), and enzyme activity (via conversion of tritiated cortisol to cortisone). Furthermore, we observed that HSD2 enzyme activity is down regulated in CEM-C7 cells that were pretreated with the synthetic glucocorticoid, dexamethasone (100 nM, <15 hours), and that this down regulation of enzyme activity is blocked by the administration of the glucocorticoid receptor antagonist, RU-486. Taken collectively, these data raise the possibility that the effectiveness of glucocorticoids in the treatment of human leukemias may be influenced by: (1) the ability of these neoplastic cells to metabolize glucocorticoids via HSD2 and (2) the ability of these steroids to regulate the expression of this key enzyme.

Placental HSD2 Expression and Activity Is Unaffected by Maternal Protein Consumption or Gender in C57BL/6 Mice.

The placenta acts as a physiological barrier, preventing the transfer of maternal glucocorticoids to the developing fetus. This is accomplished via the oxidation, and subsequent inactivation, of endogenous glucocorticoids by the 11- ß hydroxysteroid dehydrogenase type 2 enzyme (HSD2). Maternal protein restriction during pregnancy has been shown to result in a decrease in placental HSD2 expression and fetal glucocorticoid overexposure, especially late in gestation, resulting in low birth weight and "fetal programming" of the offspring. This dietary intervention impairs fetal growth and cardiovascular function in adult C57BL/6 offspring, but the impact on placental HSD2 has not been defined. The goal of the current study was to examine the effects of a maternal low-protein diet (18% versus 9% protein) on placental HSD2 gene expression and enzyme activity in mice during late gestation. In contrast to previous studies in rats, a maternal low-protein diet did not affect HSD2 protein or enzyme activity levels in the placentas of C57BL/6 mice and this was irrespective of the gender of the offspring. These data suggest that the effects of maternal protein restriction on adult phenotypes in C57BL/6 mice depend upon a mechanism that may be independent of placental HSD2 or possibly occurs earlier in gestation.

Relaxation breathing improves human glycemic response.

OBJECTIVES: This study evaluated a simple relaxation breathing exercise for acute improvement of postprandial glycemic and insulinemic status. DESIGN: Healthy human subjects were randomized to control breathing (CB; n=13) or a relaxation breathing exercise (RB; n=13) that was repeated every 10 minutes for the 30 minutes before and 90 minutes after consuming a glucose challenge (oral glucose tolerance test; OGTT; 75 g/240 mL). Blood samples were collected before, and 30, 60, and 90 minutes post OGTT for glucose and insulin analysis. RESULTS: Blood glucose at 0 minutes (pre-OGTT), and 30, 60, and 90 minutes post-OGTT with continued RB was 93.7±1.9, 136.5±8.1, 165.7±8.1, and 130.2±6.9 mg/dL, and 97.1±2.4, 173.1±8.4, 158.7±11.1, and 137.1±10.1 with CB, respectively. RB blood glucose was significantly lower at 30 minutes than CB. Glucose area under the curve (AUC) for CB and RB were not significantly different. Plasma insulin for both CB and RB was significantly increased relative to baseline at 30, 60, and 90 minutes. Insulin values for RB tended to be higher than CB at 30 and 60 minutes, although the difference was not statistically significant. Insulin AUC for CB and RB was not significantly different. CONCLUSIONS: Relaxation breathing acutely improves the glycemic response of healthy subjects, and breathing pattern could be important for interpretation of glycemic index measurements.

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.

Glucocorticoid metabolism in the human fetal lung: implications for lung development and the pulmonary surfactant system.

It has been nearly 35 years since Liggins and Howie first reported the benefits of antenatal glucocorticoid (GC) treatment to promote the maturation of the human fetal lung, and nearly that long since Pasqualini and colleagues demonstrated that the human fetal lung actively metabolizes GCs. Since that time, our understanding of the effects of GCs on fetal lung maturation and pulmonary surfactant production has increased dramatically. Similarly, characterization of the enzymes involved in GC metabolism has greatly expanded our understanding of GC signaling in target tissues. In man, the biologically active GC (cortisol) and the biologically inactive GC (cortisone) are interconverted by the tissue-specific expression of the type 1 and type 2 11beta-hydroxysteroid dehydrogenase enzymes (HSD1 and HSD2). Much of the research on GC metabolism in peripheral target tissues has focused on the role of HSD1 in amplifying the effects of GCs in liver and adipose tissue or on the role of HSD2 in blocking the effects of GCs in the kidney and placenta. In contrast, the role of GC metabolism in modulating the effects of GCs on fetal lung maturation and the pulmonary surfactant system in humans is less understood. The goal of this review article is to present a brief overview of the role of GCs in human fetal lung maturation and pulmonary surfactant production, and to familiarize the reader with the biochemistry of the metabolism of natural and synthetic GCs by the HSD enzymes. In addition, we will review data concerning the expression and activity of the HSD enzymes in the human fetal lung and contrast this to what is known about the HSD enzymes in the fetal rodent lung. Although rodents, rabbits, sheep, and several primates have been invaluable model systems for the study of fetal lung development, we have chosen to largely focus this review on human lung, since there are significant differences in GC metabolism between humans and other species.

Zygotic control of maternal cyclin A1 translation and mRNA stability.

Cyclin mRNAs are unstable in the adult cell cycle yet are stable during the first 12 cell divisions in Xenopus laevis. We recently reported that cyclin A1 and B2 maternal mRNAs are deadenylated upon completion of the 12th division (Audic et al. [2001] Mol. Cell Biol. 21:1662-1671). Deadenylation is mediated by the 3' untranslated region (UTR) of the mRNA and precedes the terminal disappearance of the cyclin proteins, with both processes requiring zygotic transcription. The purpose of the current study was (1) to ask whether deadenylation leads to translational repression and/or destabilization of endogenous cyclin A1 and B2 mRNAs, and (2) to further characterize the regulatory sequences required. We show that zygote-driven deadenylation leads to translational repression and mRNA destabilization. A 99-nucleotide region of the 3'UTR of the cyclin A1 mRNA mediates both deadenylation and destabilization. Surprisingly, two AU-rich consensus elements within this region are dispensable for this activity. These results suggest that zygote-dependent deadenylation, translational repression, and mRNA destabilization by means of novel 3'UTR elements contribute to the disappearance of maternal cyclins. They also suggest that translational control of cyclins may play a role in the transition to the adult cell cycle. These data concur with previous studies in Drosophila showing that zygote-mediated degradation of maternal cdc25 mRNA may be a general mechanism whereby transition to the adult cell cycle proceeds.