Published ahead of print on February 5, 2003, doi:10.1164/rccm.200210-1139OC
© 2003 American Thoracic Society Dexamethasone Upregulates 11ß-Hydroxysteroid Dehydrogenase Type 2 in BEAS-2B CellsDepartment of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan; Laboratory of Molecular Hypertension, Baker Medical Research Institute, Melbourne, Australia; and Departments of Pathology and of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai, Japan Correspondence and requests for reprints should be addressed to Dr. Satoshi Suzuki, M.D., Department of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Japan 980-8575. E-mail: satoshisuzuki{at}idac.tohoku.ac.jp
The actions of natural and synthetic glucocorticoids are in part determined by 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). We examined whether carbenoxolone, a potent inhibitor of 11ß-HSD, would potentiate the inhibitory action of dexamethasone on interleukin-8 release from BEAS-2B cells, and whether prolonged treatment with dexamethasone at therapeutic doses would upregulate 11ß-HSD2 in the cells. We found that carbenoxolone increased the potency of dexamethasone almost 10-fold. Reverse transcription-polymerase chain reaction and Western blot revealed that BEAS-2B cells expressed 11ß-HSD2, but not 11ß-HSD1. An enzyme activity assay of the cell homogenate demonstrated only NAD+-dependent dehydrogenase activity. The Km value for cortisol in intact BEAS-2B cells was estimated to be 42 nM. When the cells were incubated with dexamethasone for up to 72 hours at increasing concentrations (10-9 to 10-5 M), there were considerable increases in mRNA and protein levels of 11ß-HSD2. Prolonged treatment with dexamethasone also increased the enzyme activity of 11ß-HSD in the cells in a dose- and time-dependent manner, with complete inhibition by RU38486. These results suggest that bronchial epithelial cells possess an autoregulatory system for glucocorticoids in the control of their own bioactive levels by inducing the expression of 11ß-HSD2, and that 11ß-HSD2 in the bronchial epithelium may play a role in the local regulation of inhaled glucocorticoid actions.
Key Words: bronchial epithelial cells • glucocorticoid • interleukin-8 • 11ß-hydroxysteroid dehydrogenase type 2 Glucocorticoids, through interaction with glucocorticoid receptors (GR), exert a diverse array of powerful biological effects such as modulation of cellular metabolism, cellular proliferation and differentiation, water and electrolyte homeostasis, and antiinflammatory actions on target tissues. In the lung, bronchial epithelium is an active target site for glucocorticoids (1), and these potent steroids are, at present, recognized as the most effective antiinflammatory therapeutic agents for the treatment of bronchial inflammatory conditions such as asthma (2). Although bronchial epithelial cells come in direct contact with inhaled glucocorticoids, their biological actions would be determined in part by 11ß-hydroxysteroid dehydrogenase (11ß-HSD), the microsomal enzyme responsible for the interconversion of bioactive glucocorticoids and their receptor-inactive 11-oxo metabolites. To date, two distinct isoforms of 11ß-HSD (11ß-HSD1 and -2) have been cloned and characterized in humans (3, 4). The type 1 isoform (11ß-HSD1) requires NADP+ as a cofactor and, although enzymatic reactions performed in vitro under optimal conditions have demonstrated both dehydrogenase and reductase activities (5), 11ß-HSD1 functions predominantly to convert inactive 11-oxo metabolites to their active forms in vivo (6). In contrast, the type 2 isoform (11ß-HSD2) requires NAD+ as a cofactor and possesses only dehydrogenase activity, thereby inactivating endogenous glucocorticoids (7), although this enzyme may also act as oxidoreductase on some metabolites of synthetic and fluorinated glucocorticoids (8–10). The enzyme activity of 11ß-HSD has been demonstrated in human lung tissues (11) and cell lines (12, 13). Our previous immunohistochemical studies showed that the human bronchial epithelium expresses not only GR and mineralocorticoid receptor (MR), but also 11ß-HSD2 (14). Coexistence of MR with 11ß-HSD2 suggests that 11ß-HSD2 in these cells may be functioning partly to protect MR from higher concentrations of glucocorticoids reaching the airway epithelium (15). In addition, it has been proposed that steroid metabolism in the airway epithelium may regulate glucocorticoid antiinflammatory actions (16). Feinstein and coworkers reported that glycyrrhetinic acid, a potent 11ß-HSD inhibitor, potentiated the inhibitory effects of cortisol on the release of granulocyte macrophage colony-stimulating factor from primary cultured human bronchial epithelial cells after interleukin (IL)-1ß challenge (17). In other cell types, such as rat pituitary GH3 cells, it has also been shown that licorice derivatives potentiated the action of rat glucocorticoid corticosterone on the expression of the glucocorticoid target gene prolactin more than did either agent alone (18). Although these observations seem to indicate that 11ß-HSD2 modulates local actions of natural glucocorticoids, its effect on synthetic glucocorticoids has yet to be defined. Moreover, it is interesting to examine whether prolonged treatment with synthetic glucocorticoids would modulate 11ß-HSD2 expression in the human bronchial epithelial cell. We therefore examined in this study whether carbenoxolone, another potent inhibitor of 11ß-HSD, would potentiate the inhibitory action of a synthetic glucocorticoid dexamethasone on IL-8 release from BEAS-2B cells, an established cell line derived from normal human bronchial epithelial cells (19). We then examined whether prolonged treatment with dexamethasone at therapeutic doses would upregulate 11ß-HSD2 in the cells.
Cell Culture and Preparation of Human Tissues BEAS-2B cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F12 medium (GIBCO-BRL, Rockville, MD) containing 10% fetal bovine serum (GIBCO-BRL) on plastic plates at 37°C in a humidified 95% air–5% CO2 incubator. All cells were grown to 80–90% confluency. The cells in 24-well plastic plates were preincubated with dexamethasone (Sigma, St. Louis, MO) at increasing concentrations (10-9 to 10-5 M) in the presence or absence of 10-6 M carbenoxolone (Sigma) in serum-free medium for 24 hours. The cells were then exposed to tumor necrosis factor (TNF)-  (10 ng/ml; Boehringer Mannheim, Mannheim, Germany) for 10 hours. IL-8 concentration in the medium was determined by enzyme-linked immunosorbent assay (ELISA) (Endogen, Woburn, MA). To examine whether prolonged treatment with dexamethasone would upregulate 11ß-HSD2 in BEAS-2B cells, the cells in six-well plastic plates were exposed to dexamethasone (Sigma) in serum-free medium for up to 72 hours at increasing concentrations (10-9 to 10-5 M). In addition, the cells were also incubated in the presence of RU38486 (10-6 M) (Roche, Paris, France), a potent and specific GR inhibitor. Nonpathological adult human lung, kidney, and liver tissues were obtained from autopsies at Tohoku University Hospital (Sendai, Japan) with approval of the ethics committee of Tohoku University School of Medicine.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Western Blot Cell and tissue sample homogenates (100-µg protein samples) were separated in a polyacrylamide gel containing 10% sodium dodecyl sulfate, electrophoretically transferred to a nitrocellulose membrane, and then reacted with either HUH13 or HUH23 antibody. HUH13 identifies human 11ß-HSD1 as a 34-kD single band in liver tissue homogenates. HUH23 has been shown to identify human 11ß-HSD2 as a 41-kD single band in kidney and placenta homogenates (24). The blotted membrane was visualized on X-ray film, using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). Additional details are provided in the online supplement.
Enzyme Activity Assay
Statistical Analysis
Dexamethasone inhibited IL-8 release from TNF- -stimulated BEAS-2B cells in a dose-dependent manner, and half-inhibition of the maximum effect (IC50) was estimated to be about 10-7 M (Figure 1) . When the cells were preincubated with carbenoxolone (10-6 M), the inhibitory effect of dexamethasone on IL-8 release appeared to be potentiated almost 10-fold (IC50 = 10-8 M), whereas maximum inhibition by dexamethasone was not influenced (Figure 1). Carbenoxolone at 10-6 M did not change cell number in culture.
RT-PCR revealed that BEAS-2B cells and lung tissue expressed, in addition to GR and MR, the mRNA for 11ß-HSD2, but not for 11ß-HSD1 (Figure 2)
. Quantitative RT-PCR showed that dexamethasone treatment for 48 hours increased 11ß-HSD2 mRNA expression in BEAS-2B cells in a dose-dependent manner (Figure 3)
.
Western blot demonstrated 11ß-HSD2 at 41 kD in both lung tissue and BEAS-2B cells, although the expression level in BEAS-2B cells was lower than that in lung tissue (Figure 4) . When the blotted membrane was reacted with HUH13 antibody, 11ß-HSD1 was detected at 34 kD in liver, but was absent in lung tissue or BEAS-2B cells (Figure 4). Treatment with dexamethasone increased 11ß-HSD2 protein expression in BEAS-2B cells in a dose- and time-dependent manner (Figure 5) .
Homogenates of BEAS-2B cells and adult lung tissue displayed only NAD+-dependent dehydrogenase activity, with no significant increase in steroid conversion in the presence of NADP+ cofactor (Figure 6) . The Km value for cortisol in intact BEAS-2B cells was estimated to be 42 nM. Dexamethasone treatment increased the enzyme activity of 11ß-HSD in intact BEAS-2B cells almost twofold over control levels at a dose starting from 10-8 M dexamethasone at 48 hours (Figure 7A) . An increase in enzyme activity was observed after only 12 hours of incubation with 10-6 M dexamethasone (Figure 7B). RU38486 was able to completely block the increased steroid conversion induced by dexamethasone (10-6 M) and cortisol (10-6 M), whereas RU38486 alone did not influence the enzyme activity of 11ß-HSD (Figure 8) .
It has been suggested that actions of natural glucocorticoids in human bronchial epithelium would be in part regulated by 11ß-HSD (16, 17). We found in this study that pretreatment with carbenoxolone potentiated the inhibitory effect of dexamethasone on IL-8 release from TNF- -stimulated BEAS-2B cells almost 10-fold. Carbenoxolone has been used at similar concentrations to inhibit 11ß-HSD activity in rat Leydig cells (26), human placental trophoblast cells (27), and human lung tissues, in which the IC50 for carbenoxolone was estimated to be 1.5 x 10-7 M (15). In BEAS-2B cells, the IC50 for carbenoxolone was estimated to be about 10-7 M and no detectable steroid conversion was observed in the presence of 10-6 M carbenoxolone (data not shown). Although we did not directly measure conversion of dexamethasone in BEAS-2B cells, our results indicate that bronchial epithelial 11ß-HSD is capable of inactivating synthetic glucocorticoids and modulating their antiinflammatory actions.
We then decided to characterize 11ß-HSD in BEAS-2B cells. Although previous studies reported that human lung tissue and bronchial epithelial cells in primary culture displayed enzyme activity of 11ß-HSD (16, 17), they did not measure 11ß-HSD1 and -2 separately. Our RT-PCR revealed, in addition to GR The exclusive 11ß-HSD2 expression was also examined by measuring steroid conversion in the presence of cofactors. Using cortisol as a physiological substrate, we found that cell and lung tissue preparations displayed only NAD+-dependent dehydrogenase activity, suggesting that cortisol is metabolized predominantly via 11ß-HSD2, not 11ß-HSD1. In addition, the Michaelis–Menten plot demonstrated that the enzyme activity of 11ß-HSD in intact BEAS-2B cells showed a high affinity for cortisol (Km = 42 nM). The apparent Km of human 11ß-HSD2 for cortisol has been reported to be about 50 nM, whereas that of 11ß-HSD1 is in the micromolar range (28). Because 11ß-HSD2 is expressed predominantly in the ciliated epithelia of bronchus and bronchiole, but not alveolar epithelial cells, in adult human lung (14), it was expected that BEAS-2B cells, derived from normal human bronchial cells (18), would possess higher expression of 11ß-HSD2 protein than peripheral lung tissue samples. However, the results of Western blots and enzyme activity assays in the present study demonstrated the low expression of 11ß-HSD2 in BEAS-2B cells relative to lung tissue. It has been reported that human bronchial epithelial cells in primary culture display more significant steroid conversion than do BEAS-2B cells (17). Although it is clear that only 11ß-HSD2 is responsible for glucocorticoid breakdown in BEAS-2B cells, this cell line may not provide the best model for the investigation of steroid metabolism in the bronchial epithelium in vivo. We next examined whether prolonged treatment with dexamethasone at the therapeutic doses would upregulate 11ß-HSD2. It has been reported in other cell types, such as Ishikawa cells, a human endometrial adenocarcinoma cell line, that expression of 11ß-HSD2 is upregulated by dexamethasone as well as cortisol (29). Quantitative RT-PCR showed that treatment with dexamethasone for 48 hours increased 11ß-HSD2 mRNA levels in BEAS-2B cells in a dose-dependent manner. Western blot also showed a significant dose- and time-dependent increase in the protein expression of 11ß-HSD2 in dexamethasone-treated BEAS-2B cells. In addition, we observed an approximately twofold increase in enzyme activity of 11ß-HSD in BEAS-2B cells following 48 hours of treatment with increasing doses of dexamethasone. A similar dose- and time-dependent increase in enzyme activity of 11ß-HSD was shown with cortisol treatment (data not shown). Dexamethasone or cortisol in our study appeared to be functioning via GR, because the potent and specific GR inhibitor RU38486 completely inhibited the action of glucocorticoids on the enzyme activity of 11ß-HSD in BEAS-2B cells. Human GR has been shown to be expressed and functional in BEAS-2B cells (30). These results indicate that the stimulatory effects of glucocorticoids were mediated, at least in part, at the gene transcriptional level. We conclude therefore that human bronchial epithelial cells possess a putative autoregulatory system for not only natural, but also synthetic, glucocorticoids in the control of their own bioactive levels by inducing the expression of 11ß-HSD2.
Because of the importance of synthetic glucocorticoids in the suppression of airway inflammation and the potential role of the bronchial epithelium in asthma, and numerous other pathological conditions, the local metabolism of glucocorticoids in the bronchial epithelium may be a significant clinical issue. Inhaled glucocorticoids are effective antiinflammatory agents in the treatment of asthma; however, a small but significant population of patients with asthma requires longer term treatment with higher doses of glucocorticoids (31). It is also unknown whether glucocorticoid-dependent asthma might develop into glucocorticoid-resistant asthma, both being a part of the same disease spectrum with glucocorticoid-resistant asthma simply being a more severe form of the two. Better understanding of the mechanisms leading to prolonged glucocorticoid-dependent and glucocorticoid-resistant conditions is a topical and important issue in establishing new therapeutic approaches, because high-dose glucocorticoid therapy in patients with asthma may be accompanied by serious side effects (32, 33). Studies on the GR in patients with asthma suggest that the increased expression of GRß may explain the resistance to inhaled glucocorticoids (34, 35). However, it has also been reported that persistent airway inflammation in patients with severe asthma is not associated with low GR The expression of MR mRNA in BEAS-2B cells is also an important finding in this study. To date, the current theory states that 11ß-HSD2 confers specificity of MR to aldosterone by inactivating glucocorticoids and thereby regulating transepithelial fluid transport in aldosterone target tissues (40). We have reported that MR and 11ß-HSD2 coexist in both fetal and adult normal human bronchial epithelia (14), and also in their neoplastic forms (41). Coexpression of MR and 11ß-HSD2 in human bronchial epithelial cells, including BEAS-2B cells, may therefore imply that aldosterone has an important role in fluid balance across the airway epithelium. It has been shown that Na+ transport in canine tracheal epithelial cell monolayers is both acutely and chronically regulated by aldosterone (42). The role that MR plays in controlling fluid transport in the lung is most apparent in mice lacking functional MR; these mice are noted to have a marked decrease in Na+ channel function (43). 11ß-HSD2 in the human bronchial epithelium, as in the kidney, may possibly be functioning partly to protect MR from higher concentrations of circulating glucocorticoids reaching the airway epithelium. In summary, our results obtained from BEAS-2B cells provide new evidence that 11ß-HSD2 in the bronchial epithelium may be, at least in part, responsible for the regulation of bioactive glucocorticoids by interacting with the local milieu of this active glucocorticoid target site in the lung. Analyses of 11ß-HSD2 under lung inflammatory conditions will be important to clarify whether the pharmacological manipulation of 11ß-HSD2 would serve as a new therapeutic alternative to amplify glucocorticoid antiinflammatory action in vivo.
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form October 7, 2002; accepted in final form February 2, 2003
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p < 0.05 versus the values without RU38486.
