Potentiation of Activity in Hypoxia through Induction of the Glucocorticoid

This information is current as Martin O. Leonard, Catherine Godson, Hugh R. Brady and of September 30, 2021. Cormac T. Taylor J Immunol 2005; 174:2250-2257; ; doi: 10.4049/jimmunol.174.4.2250 http://www.jimmunol.org/content/174/4/2250 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Potentiation of Glucocorticoid Activity in Hypoxia through Induction of the Glucocorticoid Receptor1

Martin O. Leonard,2 Catherine Godson, Hugh R. Brady, and Cormac T. Taylor

Tissue hypoxia is intimately associated with chronic inflammatory disease and may signal to the resolution of inflammatory processes. Glucocorticoid signaling through the (GR) represents a clinically important endogenous anti-inflammatory pathway. Microarray analysis reveals that the GR is transcriptionally up-regulated by hypoxia in human renal proximal tubular epithelial cells. Hypoxic up-regulation of the GR was confirmed at the level of promoter activity, mRNA, and protein expression. Furthermore, functional potentiation of glucocorticoid activity in hypoxia was observed as an enhancement of -induced glucocorticoid response element promoter activity and enhanced dexamethasone-mediated inhibition of IL-1␤-stimulated IL-8 expres- sion and hypoxia-induced vascular endothelial growth factor expression. Knockdown of enhanced GR gene expression in hypoxia using

specific GR small inhibitory RNA (siRNA) resulted in an attenuation of the enhanced glucocorticoid sensitivity. A role for the hypoxia- Downloaded from inducible transcription factor, HIF-1␣, in the regulation of GR expression and the associated potentiation of glucocorticoid activity in hypoxia was also demonstrated. These results reveal a novel signaling aspect responsible for the incorporation of hypoxic and glu- cocorticoid stimuli, which we hypothesize to be an important co-operative pathway for the control of gene expression observed in complex tissue microenvironments in inflamed states. The Journal of Immunology, 2005, 174: 2250–2257.

lucocorticoids are steroid hormones secreted from the nucleus. The GR can then homodimerize and act directly as a http://www.jimmunol.org/ adrenal cortex as part of the hypothalamic pituitary ad- trans-acting factor binding to two palindromic glucocorticoid re- G renal axis and are critical to physiological function due sponse element (GRE) half sites (5Ј-TGTTCT-3Ј) within the reg- to their regulatory effects on carbohydrate, lipid and protein me- ulatory regions of glucocorticoid responsive genes (1), including tabolism. These hormones are released in response to diverse stim- those involved in endocrine homeostasis and metabolism, such as uli and play essential roles in regulating the stress response, en- TAT and PEPCK, and also inflammation, including the IL-1R an- docrine homeostasis, vascular tone, CNS function, proliferation, tagonist and I␬B (5, 6). As well as transactivation, activated GR and apoptosis (1). They also have major regulatory roles in in- can inhibit gene expression through a mechanism known as trans- flammation and the immune response and are one of the most repression where the GR binds directly to transcription factors widely used pharmacological agents in medicine. They are used such as AP-1 and NF-␬B, thus inhibiting their transactivating po- by guest on September 30, 2021 extensively for their anti-inflammatory properties in the treatment tential for the induction of gene expression (7). Trans-repression of inflammatory and autoimmune disease (2, 3). While much is has been suggested as the main mechanism through, which glu- known regarding the cellular and molecular mechanisms through cocorticoids exert their anti-inflammatory effects (8). which exert their effects, it remains unclear how Hypoxia occurs when the demand for oxygen to maintain nor- they integrate their physiological and pharmacological effects in mal cellular ATP requirements outweighs the vascular supply and complex systemic and tissue environments. has been documented as an integral part of many pathological The actions of glucocorticoids are mediated through an intra- states, including ischemic disease, chronic inflammatory disease, cellular receptor, the glucocorticoid receptor (GR,3 NR3C1), and tumor progression (9, 10). The transcription factor complex which is a member of the nuclear receptor family of ligand de- hypoxia-inducible factor 1 (HIF-1) primarily mediates the adaptive pendent transcription factors (4). Ligand binding in the cytoplasm response to hypoxia. Under low oxygen tension, prolyl hydroxy- causes a dissociation of the GR from it’s chaperone complex, al- lase-dependent degradation of the regulatory subunit of HIF-1, lowing it to become hyperphosphorylated and translocated to the HIF-1␣ is inhibited. This results in nuclear accumulation of HIF-1, it’s binding to the hypoxia response element (HRE) (5Ј-RCGTG- 3Ј), and the subsequent transactivation of genes involved in gly- Conway Institute of Biomolecular and Biomedical Research, University College Dub- lin, Belfield, Dublin, Ireland colysis, angiogenesis, and vasodilatation, including phosphofruc- tokinase, adrenomedullin, and vascular endothelial growth factor Received for publication July 7, 2004. Accepted for publication November 8, 2004. (VEGF) (10). We have also previously reported that prolonged The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance hypoxia may play a role in the resolution of the inflammatory with 18 U.S.C. Section 1734 solely to indicate this fact. response (11) 1 This work was supported by grants from the Health Research Board of Ireland (to The interplay between hypoxia and glucocorticoid-mediated H.R.B. and C.G.), the Wellcome Trust (to H.R.B., C.G., and C.T.T.), and the Science cellular responses in physiology and disease has become increas- Foundation of Ireland (to C.T.T.). ingly appreciated over recent years. Release of glucocorticoids in 2 Address correspondence and reprint requests to Dr. Martin O. Leonard, Department of Medicine and Therapeutics, The Conway Institute of Biomolecular and Biomedical response to atmospheric hypoxia associated with high altitude in Research, University College Dublin, Belfield, Dublin 4, Ireland. E-mail address: humans has been extensively documented and prophylactic treat- [email protected] ment with glucocorticoids has been used to attenuate the associ- 3 Abbreviations used in this paper: GR, glucocorticoid receptor; GRE, glucocorticoid ated mountain sickness (12). Stress-induced erythropoiesis as a response element; HIF-1, hypoxia-inducible factor 1; WT, wild type; DM, double mutant; VEGF, vascular endothelial growth factor; RT, room temperature; 11␤-HSD, result of hypoxia exposure involves glucocorticoid regulation of 11␤-hydroxysteroid dehydrogenase; siRNA, small inhibitory RNA. erythroid progenitor expansion and terminal differentiation arrest

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2251

(13, 14). Mice carrying a dimerization deficient mutation of the Western blot analysis GR exhibit normal erythropoiesis under normoxic conditions but Whole cell extracts were prepared in RIPA lysis buffer (20 mmol/L Tris- fail to respond to hypoxia to increase erythrocyte count and he- HCl, pH 7.4, 50 mmol/L NaCl, 5 mmol/L ethylene diaminetetraacetic acid, matocrit and hemoglobilin content in peripheral blood (13). Glu- 1% Nonidet P-40, 0.1% SDS, 5 mmol/L NaF, 1 mmol/L PMSF, 1 mmol/L ␮ ␮ cocorticoids have been observed to stimulate erythropoiesis indi- Na3VO4,1 mol/L leupeptin, and 0.3 mol/L aprotinin). Protein content rectly through up-regulation of erythropoietin in the (15). was quantified and normalized using the Bradford method (Bio-Rad) and electrophoresed on 10% SDS PAGE gels. Expression levels for the GR and They have also been used in the treatment of many pathological ␤-actin were measured using specific Abs by Western blot analysis as states in which hypoxia plays a major role in the perpetuation of previously described (21). disease, including inflammatory bowel disease and rheumatoid ar- thritis (2, 3) Additionally, it has been observed that glucocorticoids Transient transfection of cells protect against experimental cerebral and hepatic ischemia/reper- Cells were transfected with 2 ␮g of a GRE promoter-luciferase reporter fusion injury (16, 17). construct containing three tandem copies of a GRE enhancer element (BD ␮ Therefore, the integration of hypoxic- and glucocorticoid-medi- Biosciences). Cells were also transfected with 2 g of a GR promoter- reporter construct containing Ϫ1045 bp to ϩ19 bp upstream sequence ated signals is potentially important in various tissue environ- relative to the human GR exon 1C transcription start site (GR1C) (kind gift ments. In the context of the global genomic effects hypoxia and from Dr. W. Vedeckis, Louisiana State University Medical Center, New glucocorticoids exert on many different cell types and the relative Orleans, LA (22). Overexpression of HIF-1␣ was conducted through trans- degree of each stimulus in regulating these genomic responses, it fection with wild-type HIF-1␣ (HIF-1␣ WT) and HIF-1␣ double mutant ␣ is vital to elucidate the precise cross-talk between these pathways (HIF-1 DM), where prolines 564 and 402 were mutated to valine. These residues are responsible for oxygen-dependent degradation of HIF-1␣ in these complex tissue microenvironments. With this in mind, we through a mechanism involving specific hydroxylation. pcDNA3.1 was Downloaded from report for the first time the functional potentiation of glucocorti- used as the vector control. These constructs were kind gifts from Dr. T. coid activity in hypoxia, mediated through an up-regulation of Hagen (University of Nottingham, Nottingham, U.K.). Transfection was the GR. conducted using Fugene 6 transfection reagent (Roche Applied Science) according to the manufacturer’s guidelines. Following transfection for 24 h and subsequent experimental procedures, cells were washed with 2 ml of 1ϫ PBS (ice-cold) and lysed in 200 ␮lof1ϫ luciferase reporter whole cell

Materials and Methods lysis buffer (Promega) as described previously (23). Luciferase activity was http://www.jimmunol.org/ Materials quantified using a luciferin substrate (Promega) and luminometry (Junior LB 9509; Berthold Technologies). All readings were normalized to protein IL-1␤ was purchased from R&D Systems, and the Abs to the GR and using the Bradford method (Bio-Rad). ␤-actin were purchased from Santa Cruz Biotechnology. All other chem- icals were from Sigma-Aldrich unless otherwise specified. Small inhibitory RNA (siRNA) studies Cell culture Cells were transfected with four pooled siRNA duplexes (0–100 nM) di- Human proximal tubular epithelial cells (HK-2; American Tissue Type rected against separate GR mRNA target sequences or with four pooled Culture Collection; Ref. 18) were maintained in DMEM/F-12 containing 5 siRNA duplexes directed against nonspecific mRNA sequences (SMART- ␮ ␮ g/ml insulin, 5 g/ml transferrin, 5 ng/ml selenium, 36 ng/ml hydrocor- Pool) (Dharmacon) for 24 h before experimental protocol. Transfection tisone, 4pg/ml triiodo-L-thyronine, 10 ng/ml epidermal growth factor, 50 was conducted using Lipofectamine 2000 (Invitrogen Life Technologies) by guest on September 30, 2021 ␮ U/ml penicillin, 50 g/ml streptomycin, and 2 mM glutamine (Sigma- and protocols specific for siRNA transfection according to the manufac- Aldrich). Cells were maintained at 37°C in a humidified atmosphere under turer’s instructions. In cotransfection experiments, cells were transfected hypoxic (1% O2, 20 torr) or normoxic/reoxygenation (21% O2, 147 torr) with 2 ␮g of GRE at the same time as siRNA using Lipofectamine 2000 conditions with a balance of 95% N2/5% CO2. Cells were cultured to transfection protocols. confluency in 35-mm diameter petri dishes before culture in hormone- reduced medium (1:25 dilution without ) and subsequent ELISA experimental protocols. Cell culture medium was analyzed for IL-8 and VEGF expression using the DuoSet ELISA system (R&D Systems). Briefly, wells of a 96-well plate RNA isolation, microarray, and RT-PCR analysis (Corning B.V. Life Sciences) were coated with primary capture Ab (2 ␮g/ml) in PBS overnight at RT. Wells were washed three times with 250 After treatment, total cell RNA was isolated using TRI-reagent (Sigma- ␮l of wash buffer (0.05% Tween 20 in PBS; pH 7.4) and 250 ␮l of blocking Aldrich). Briefly, cells were lysed in 1 ml of reagent and left to stand for buffer (PBS containing 1% BSA, 5% sucrose, and 0.05% NaN3) was added 5 min at room temperature (RT) before the addition of 0.2 ml of chloro- for1hatRT.After removal of blocking buffer, 100 ␮l of sample or known form. Samples were then shaken vigorously for 15 s and allowed to stand ϫ standard (0–2000 pg/ml) was added and incubated for2hatRT.Wells for 10 min at RT before centrifugation at 12,000 g for 15 min at 4°C. were then washed three times before addition of 100 ␮l of 1/2000 biotin- The upper aqueous phase was removed to a separate tube, and RNA was ϫ ylated secondary detection Ab made up in reagent diluent (0.05% Tween precipitated using 0.5 ml of isopropanol. After centrifugation at 12,000 20 in TBS (PBS); pH 7.4) and incubated for2hatRT.Wells were washed g for 10 min at 4°C, the pellet was washed with 75% ethanol and resus- ␮ ␮ three times and 100 l of streptavidin-HRP (1/2500 in reagent diluent) was pended in 50 l of TE buffer. RNA was quantified as absorbance at 260 then added to each well and incubated at RT for 20 min. Following three nM. cDNA was synthesized from total RNA using Superscript Choice kit ␮ washes, 100 l of substrate solution containing a 1:1 mixture of H202 and (Invitrogen Life Technologies). Sample preparation and microarray anal- tetramethylbenzidine (Sigma-Aldrich) was added. The plate was allowed to ysis were conducted as previously described (19). Microarray Suite 5.0 develop in the dark for 15 min, and the reaction was stopped with the software (Affymetrix) was used to analyze the relative abundance of each ␮ addition of 50 lof1MH2SO4 (Sigma-Aldrich). OD was determined by gene compared with normoxia control, and results were expressed as fold spectrophotometry at 450 nm with 570 nm as reference filter, and protein change over control. RT-PCR analysis was conducted with primers specific levels were determined by extrapolation from the standard curve. for the GR (forward, 5Ј-CCTAAGGACGGTCTGAAGAGC-3Ј; reverse, 5Ј-GCCAAGTCTTGGCCCTCTAT-3Ј) (Sigma-Genosys) using a standard DNA binding assay reaction mixture for amplification as described previously (20). The am- plification protocol included an initial 2-min denaturation at 94°C, fol- To investigate HIF-1 binding to the consensus HRE sequence we used an lowed by 25 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, with ELISA based approach according to manufacturer’s instructions a final extension of 72°C for 5 min. This protocol was also used for am- (TransAm; Active Motif). Briefly, after the experimental protocol, nuclear plification of 18S RNA (17 cycles) using specific primers (forward, 5Ј- cell extracts were prepared in normoxia or hypoxia and incubated in 96- GTGGAGCGATTTGTCTGGTT-3Ј; reverse, 5Ј-CGCTGAGCCAGTC well plates precoated with an oligonucleotide containing the HRE consen- AGTGTAG-3Ј) as a control for equal sample loading. PCR products were sus motif (5Ј-TACGTGCT-3Ј). Following capture of the transcription fac- analyzed using a 2% agarose gel in 0.5ϫ TAE buffer, stained with ethidium tor by the oligonucleotide, a primary Ab to HIF-1 was added. After bromide and visualized under UV illumination (Uvipro GDS8000; washing, a HRP conjugated secondary Ab was then added followed by UVItech). substrate solution. OD was determined by spectrophotometry at 450 nm 2252 HYPOXIA UP-REGULATES THE GR with 570 nm and the intensity of the absorbance signal was proportional to HIF-1 DNA binding. Statistical analysis All data is presented as mean Ϯ SEM for n independent experiments. Statistical significance was evaluated using one way ANOVA conducted using InStat software package (GraphPad). Results Hypoxia causes an up-regulation of the GR In hypoxic tissue there is a shift in the cellular transcriptional phe- notype toward an adaptive gene expression profile, which aids cell and tissue survival in a lowered oxygen environment (10). We have previously demonstrated that hypoxia causes global tran- scriptomic alterations in gene transcription. Of the genes tightly regulated in an oxygen-dependent manner was the gene for the GR (19). Here we demonstrate that exposure of human proximal tu- bular epithelial (HK-2) cells to hypoxia (1% atmospheric oxygen, 20 torr) caused a time-dependent up-regulation of the GR evalu- ated by microarray analysis (Fig. 1A). This increase in GR expres- Downloaded from sion is oxygen dependent as reoxygenation for 6 h after 36 h of hypoxia attenuated the hypoxia-induced increase. These results were confirmed using semiquantitative RT-PCR analysis (Fig. 1B). Hypoxia also caused a time-dependent increase in GR␣ protein expression up to 48 h, which returned to near normoxic levels after

24 h of reoxygenation (Fig. 1C). We also observed a time-depen- http://www.jimmunol.org/ dent increase in GR promoter reporter luciferase activity (Fig. 1D). Collectively, these data demonstrate a transcriptionally driven in- crease in GR protein expression in response to hypoxia in proximal tubular epithelial cells. Hypoxia causes a potentiation of glucocorticoid-dependent activity Glucocorticoids affect many systems mainly through ligand-de- pendent activation of the GR and subsequent transactivation of by guest on September 30, 2021 genes, which contain regulatory GREs. Because we have observed an increase in GR expression with hypoxic exposure and GR ex- pression levels are tightly correlated with glucocorticoid-depen- dent activity (24), we set out to investigate whether glucocorticoid- dependent GRE transactivating activity is enhanced in hypoxia. Cells were transfected with a GRE-dependent promoter-reporter construct and exposed to hypoxia for 20 h. Treatment with dexa- for a further 24 h in hypoxia resulted in a potentiation of stimulated GRE luciferase activity in hypoxia compared with normoxia with the maximum potentiation observed with the higher dose of dexamethasone used (10 nM; Fig. 2). Glucocorticoids can also act to modulate gene expression through a mechanism termed trans-repression. This mechanism involves direct binding of a ligand activated GR monomer to tran- scription factors such as NF-␬B and AP-1. This prevents these FIGURE 1. Hypoxia stimulates an up-regulation of the GR. HK-2 cells were incubated in hypoxia (hyp) at 1% atmospheric O (20 torr) for various transcription factors’ transactivation potential and inhibits the ac- 2 time points (0–48 h). Cells were also maintained at 21% atmospheric O tivation of genes such as IL-8 in response to the proinflammatory 2 ␤ (147 torr) for normoxic (norm) and reoxygenation (reox) (6 h) treatments. signal IL-1 (25). Pre-exposure of cells to hypoxia caused a po- Affymetrix microarray analysis (A) and RT-PCR analysis (B) revealed an ␤ tentiation of dexamethasone-mediated inhibition of IL-1 -stimu- up-regulation of the GR at the mRNA level. Microarray results were ex- lated IL-8 (Fig. 3A). This was observed to be statistically signifi- pressed as fold over normoxic (0 h) controls (pooled n ϭ 4). RT-PCR cant at all doses of dexamethasone used with an IC50 for results are representative of three individual experiments. Protein expres- dexamethasone in hypoxia of 0.4 nM compared with 7.5 nM in sion for the GR␣ was also increased in hypoxia (C) as assessed using normoxia. To eliminate the possibility of hypoxia-induced alter- SDS-PAGE and Western blot analysis. Again results are representative of ations in the IL-1␤ signaling cascade being responsible for the blots from three individual experiments. Exposure of cells transfected with increased sensitivity to inhibition by dexamethasone, we exposed a GR promoter-luciferase reporter construct to hypoxia revealed a signif- cells in normoxia or hypoxia to varying concentrations of IL-1␤. icant increase in activity (D) compared with empty construct (PxP1). Re- sults are expressed as fold over basal PxP1 relative luciferase unit values Ϯ After 24 h exposure there were no significant alterations in IL-1␤- SEM at normoxia (n ϭ 6), hypoxia at 24 h (n ϭ 3), and hypoxia at 48 h .p Ͻ 0.01 ,ء .(stimulated IL-8 release in hypoxia compared with normoxia (Fig. (n ϭ 6 3B). It is interesting to note that the concentration of dexametha- sone used to inhibit IL-1␤-stimulated IL-8 in hypoxia, 31.6% at The Journal of Immunology 2253

FIGURE 2. Hypoxia pre-exposure results in a potentiation of dexam- ethasone-stimulated GRE promoter-reporter activity. HK-2 cells were transfected with a GRE promoter luciferase-reporter construct for 24 h before incubation under normoxic (21% O2, 147 torr) or hypoxic con- ditions (1% O2, 20 torr) for a further 20 h. Cells were then treated with dexamethasone (Dex) (0–10 nM) for 24 h before cell lysis and analysis Downloaded from of luciferase activity using luminometry. Results were expressed as mean fold over basal nondexamethasone-treated luciferase activity in nor- moxia or hypoxia respectively Ϯ SEM for seven independent experiments .(p Ͻ 0.05 ,ء)

0.1 nM, did not produce a significant induction of GRE activity, http://www.jimmunol.org/ 1.3-fold (Fig. 2). This would indicate that dexamethasone is more potent at inhibiting IL-1␤-stimulated IL-8 than at inducing GRE activity and is consistent with previous reports that glucocorticoid- stimulated GR activity is more potent in transrepression than in transactivation mechanisms (5). Therefore, these results are con- sistent with the hypothesis that enhanced glucocorticoid activity observed in hypoxia is mediated through an increase in the ex- pression of the GR. by guest on September 30, 2021 Knockdown of GR expression results in an attenuation of the FIGURE 3. Hypoxia pre-exposure causes a potentiation of dexametha- ␤ potentiated glucocorticoid activity in hypoxia sone-mediated inhibition of IL-1 -stimulated IL-8 expression. HK-2 cells were incubated under normoxic (21% O2, 147 torr) or hypoxic conditions Having demonstrated that hypoxia causes an increase in the ex- (1% O2, 20 torr) for 20 h. A, Cells were then exposed to various concen- pression of the GR and that hypoxia predisposes to enhanced sen- trations of dexamethasone (Dex) (0–10 ␮M) for 2 h before stimulation sitivity to glucocorticoid-dependent activity, we next set out to with IL-1␤ (2 ng/ml) for a further 18 h. Cell culture supernatant was then determine whether the increase in GR expression is responsible for removed and analyzed for IL-8 release using ELISA. Results are expressed the enhanced activity observed in hypoxia. To address this, cells as mean percentage of nondexamethasone-treated, IL-1␤-stimulated IL-8 Ͻ ء Ϯ were transfected with siRNA (50 and 100 nM) specific to the GR release SEM for four independent experiments ( , p 0.001). B, Cells ␤ before experimental protocols. Western blot analysis revealed an were also treated with various concentrations of IL-1 (0–10 ng/ml) for a ϳ ␣ further 18 h in normoxia or hypoxia. Results are expressed as mean fold 50% reduction in GR expression in hypoxia (Fig. 4A). There ␤ Ϯ ␤ over basal non-IL-1 -treated IL-8 release SEM for three independent were no alterations in control -actin expression. We next went on experiments. to examine the effect of inhibition of GR expression on dexam- ethasone-stimulated glucocorticoid activity. Using GR siRNA (50 nM) we demonstrated a significant reduction of the hypoxia-po- tentiated, dexamethasone-stimulated GRE promoter-reporter ac- glucocorticoid activity observed under hypoxic conditions is due tivity (Fig. 4B). Similarly we observed that treatment with GR to the observed increase in GR expression level. siRNA caused a significant attenuation of the hypoxia-stimulated ␣ potentiation of dexamethasone-mediated inhibition of IL-1␤-stim- Overexpression of HIF-1 causes an up-regulation of the GR ulated IL-8 production (Fig. 4C) compared with nonspecific and potentiates glucocorticoid-dependent activity siRNA control cells. The level to which we observed an attenua- Having established potentiated glucocorticoid activity in hypoxia tion of the potentiated dexamethasone-mediated inhibition of IL- as a functional consequence of increased GR expression, we next 1␤-induced IL-8 production in hypoxia by GR siRNA was not wanted to delineate the precise signaling events responsible for entirely reflective of the levels to which GR protein levels were enhanced GR expression. The transcription factor HIF-1␣ is a reduced, ϳ50%. This may be accounted for by the time point at master regulator of transcriptional responses to hypoxia. We have which the Western blot was conducted, just before treatment with previously demonstrated that overexpression of this transcription dexamethasone and IL-1␤. Analysis of GR protein levels at this factor under normoxic conditions results in an up-regulation of GR later time point, 18 h after treatment with dexamethasone and IL- mRNA (19). In this study, we demonstrate that overexpression of 1␤, may reveal a lesser attenuation by the GR siRNA more com- HIF-1␣ WT or HIF-1␣ DM caused an increase in GR protein parable to the changes observed in IL-8 production. However, al- expression above normoxic and hypoxic vector control levels (Fig. together, these results give further evidence that the potentiated 5A). To investigate whether this increase in GR protein expression 2254 HYPOXIA UP-REGULATES THE GR Downloaded from

FIGURE 5. Overexpression of HIF-1␣ induces the expression of the GR and potentiates GRE activity in hypoxia. HK-2 cells were transfected with HIF-1␣ overexpression constructs, HIF-1␣ WT, HIF-1␣ DM, and the

vector control (pcDNA3.1) for 24 h before incubation under normoxic http://www.jimmunol.org/

(21% O2, 147 torr) or hypoxic conditions (1% O2, 20 torr) for a further 24 h. A, Protein lysates were prepared and probed for GR expression using SDS-PAGE and Western blot analysis and results depicted are represen- tative of three individual experiments. ␤-Actin served as a loading control. B, Cells were also cotransfected with a GRE promoter luciferase-reporter construct. After incubation under normoxic and hypoxic conditions for 24 h, cells were then treated with dexamethasone (Dex) (10 nM) for a further 24 h before cell lysis and analysis of luciferase activity using lu- minometry. Results were expressed as mean fold over basal luciferase ac- by guest on September 30, 2021 ,ء) tivity in normoxia or hypoxia Ϯ SEM for four independent experiments p Ͻ 0.05).

FIGURE 4. Knockdown of the GR using specific siRNA attenuates the compared with vector control (Fig. 5B). These results indicate a hypoxia-induced potentiation of dexamethasone-stimulated glucocorticoid functional role for HIF-1␣ in the regulation of GR expression and activity. HK-2 cells were transfected with siRNA (0–100 nM) specific for the associated increase in glucocorticoid activity under hypoxic the GR before incubation for 24 h under hypoxic conditions. A, Protein lysates were then assessed for GR expression using SDS-PAGE and West- conditions. ern blot analysis and results depicted are representative of three individual Hypoxia preincubation causes a potentiation of dexamethasone- experiments. B, Cells were transfected with a GRE promoter-reporter con- struct together with the GR siRNA (50 nM) before hypoxic incubation for mediated inhibition of VEGF expression but not HIF-1 DNA 24 h. Cells were then treated with dexamethasone (0–0.01 ␮M) for 24 h binding before cell lysis and analysis of luciferase activity using luminometry. Re- VEGF is one of the most potent and specific angiogenic factors sults were expressed as mean fold over basal nondexamethasone-treated acting to promote angiogenesis in hypoxic tissue environments Ͻ ء Ϯ luciferase activity SEM for three independent experiments ( , p 0.05). (26). Glucocorticoids have been demonstrated along with their anti- C, After transfection with GR siRNA (50 nM) and hypoxic exposure, cells inflammatory properties to be angiostatic. One of the mechanisms were treated with dexamethasone (Dex) (0–0.01 ␮M) for 2 h before treat- ment with IL-1␤ (2 ng/ml) for a further 18 h. Cell culture supernatant was suggested for this anti-angiogenic property is through the inhibi- then removed and analyzed for IL-8 release using ELISA. Results are tion of VEGF production (27). Therefore, we investigated whether expressed as mean percentage of nondexamethasone-treated, IL-1␤-stim- hypoxia could alter the sensitivity of glucocorticoids to inhibit (p Ͻ VEGF expression. Cells were exposed to normoxic or short (1-h ,ء) ulated IL-8 release Ϯ SEM for three independent experiments 0.078). and long (24-h) hypoxic preconditions. Cells were then treated with dexamethasone (0–0.1 ␮M) for a further 18 h and maintained under the same conditions. After 18 h, cells preincubated under translates as a potentiation of glucocorticoid activity, we cotrans- short and long hypoxic preconditions demonstrated a 3.37 Ϯ 0.4- fected a GRE promoter-reporter construct with these HIF-1␣ over- and a 4.88 Ϯ 0.46-fold over normoxic VEGF release respectively. expression vectors. Hypoxia alone caused a potentiation of GRE Cells maintained under normoxic or short hypoxic conditions dis- activity compared with the normoxic vector control as previously played a moderate dose-dependent inhibition of VEGF while cells demonstrated. Overexpression of both HIF-1␣ constructs resulted pre-exposed to hypoxia for 24 h showed a significant inhibition of in no significant alteration in GRE activity under normoxic con- VEGF production compared with normoxic or short hypoxic ex- ditions (Fig. 5B). However, under hypoxic conditions HIF-1␣ posure (Fig. 6A). We also assessed HIF-1 DNA binding to a spe- overexpression caused a significant alteration in GRE activity cific HRE oligonucleotide sequence, a measure of HIF-1 activation The Journal of Immunology 2255

sensitivity to GR agonists, which we demonstrate to be mediated through an up-regulation of the GR. It has long been established that the level of GR expression can alter the sensitivity of cells to glucocorticoids (28). Transgenic expression of antisense mRNA specific to the GR in mice resulted in a 50–70% reduction in functional GR expression with a con- comitant 2- to 4-fold decrease in dexamethasone-stimulated glu- cocorticoid-inducible promoter activity (29). We have demon- strated that increased GR levels correlate with a potentiation of glucocorticoid-stimulated activity in hypoxia and that reduction of GR using siRNA attenuates this potentiated activity. Although lev- els of the GR can influence glucocorticoid sensitivity, other factors potentially altered in hypoxia can also alter glucocorticoid-depen- dent activity. The 11␤-hydroxysteroid dehydrogenase (11␤-HSD) are an important prereceptor control mechanism for the activity of glucocorticoids in a tissue-specific manner. They catalyze the in- terconversion of active glucocorticoids with their inert 11-ketone

forms through oxidation and reduction of the 11␤-hydroxyl group. Downloaded from There are two main enzymes: 11␤-HSD type I, which acts mainly to reduce and activate glucocorticoids, and 11␤-HSD type II, which acts to oxidize and inactivate glucocorticoids (30). Levels of these enzymes are tissue specific and confer differential glucocor- ticoid sensitivity. Chemical hypoxia using antimycin A has previ-

ously been observed to reduce 11␤-HSD type II expression at the http://www.jimmunol.org/ transcriptional level (31). However, we have demonstrated that mRNA levels for the 11␤-HSD enzymes are not altered by hyp- oxia up to 36 h revealed using microarray analysis (data not shown). To discount the possibility that activity of these enzymes independent of their expression level could be responsible for the observations in our study we used specific inhibitors of these en- FIGURE 6. Hypoxia pre-exposure potentiates dexamethasone-mediated zymes. Pretreatment of cells with enoxolone and inhibition of hypoxia-induced VEGF expression but not HIF-1 DNA bind- failed to alter the potentiation of glucocorticoid-stimulated GRE ing. HK-2 cells were maintained under normoxic or hypoxic conditions for activity and inhibition of IL-8 production in hypoxia (data not by guest on September 30, 2021 1 and 24 h. Cell culture supernatant was removed and replaced with fresh shown). Together with the fact that dexamethasone does not need medium. A, Cells were then treated with dexamethasone (0–0.1 ␮M) for a to be reduced by these enzymes for activation (32) these data in- further 18 h in normoxia or hypoxia. Cell culture supernatant was removed dicate that the 11␤-HSD enzymes play no role in influencing glu- and analyzed for VEGF release using ELISA. Results are expressed as cocorticoid sensitivity in hypoxia. mean percentage of nondexamethasone-stimulated VEGF release in nor- Glucocorticoids themselves can reduce GR expression level p Ͻ 0.01 ,ء) moxia or hypoxia Ϯ SEM for three independent experiments compared with hypoxia 1 h). B, Cells were also treated with dexametha- through decreased transcription and increased protein turnover sone for2hinnormoxia or hypoxia. Nuclear cell lysates were analyzed for (33). In our present study, however, there is no evidence to suggest binding of HIF-1 to a consensus HIF-1 DNA binding sequence using an that basal glucocorticoid activity in normoxic conditions is higher ELISA based system than that in hypoxia explaining the increased GR expression in hypoxia and thus increased glucocorticoid sensitivity. Quite the contrary, basal levels of GRE activity in hypoxia are higher than those observed under normoxic conditions (data not shown), which as a possible mechanism for the glucocorticoid-mediated inhibi- we hypothesize is due to the hypoxia-induced increase in GR tion of VEGF release. After2hofhypoxic exposure, cells prein- expression. cubated for1hor24hcaused a 4.82 Ϯ 0.53- and a 5.17 Ϯ In the absence of ligand the GR is held in a multiprotein het- 0.61-fold over normoxic HIF-1 DNA binding, respectively. After erocomplex containing hsp90, hsp70, hop, hsp40, and p23. The cell pre-exposure to normoxia or hypoxia for1hor24h,treatment GR must be held in this hsp90 chaperone complex for ligand bind- with dexamethasone for 2 h did not significantly alter HIF-1 DNA ing and activation to occur (34). Disruption of hsp90 function us- binding (Fig. 6B) indicating that the mechanism of potentiated ing the inhibitor geldanamycin prevents GR activation by ligand glucocorticoid inhibition of hypoxia-stimulated VEGF production (35). It is interesting to note that we have observed a hypoxia- is not through alterations in HIF-1 DNA binding. dependent inhibition of hsp90, hsp70, and hsp40 expression (un- published data), suggesting a putative disruption of heterocomplex Discussion formation and inhibition of GR activation. ATP levels, which are It has become increasingly apparent that glucocorticoid and hy- reduced by hypoxia (10) are also necessary for functional complex poxic regulatory responses are closely linked in complex physio- assembly (36). Because the most likely alterations in heterocom- logic and pathophysiologic tissue microenvironments. Although plex formation to occur under hypoxic conditions would interrupt much work has focused on characterization of these responses, complex assembly and GR activation it is unclear whether alter- little work has concentrated on determining the precise molecular ations in complex formation could account for an increased sen- and signaling interplay between these two important stimuli. In sitivity to glucocorticoids under hypoxic conditions. Many other this present study we report for the first time that hypoxia increases factors can contribute to alterations in glucocorticoid sensitivity 2256 HYPOXIA UP-REGULATES THE GR such as protein kinase A activation (37) among others but the hibition of VEGF expression is not likely due to alterations in clearest explanation for increased glucocorticoid sensitivity in HIF-1-mediated VEGF expression in hypoxia. This mechanism hypoxia based on our current study is that hypoxia increase the cannot be totally excluded though, as alterations in transactivation level of GR expression thus increasing the glucocorticoid sensi- potential have been observed without alterations in transcription tivity within the cell. factor DNA binding (42). However, alternate mechanisms for the The GR is classified as a housekeeping gene, whose expression inhibition of VEGF expression by glucocorticoids have been ob- is controlled by a GC-rich promoter lacking a consensus TATA or served through a process involving decreased mRNA stability CAAT box (38). Multiple DNA footprint analysis studies of the (43). Ultimately, we postulate that alterations in GR expression in GR promoter have characterized many transcription factors re- hypoxia not only alter GR-dependent gene expression but also in- sponsible for maintaining basal activity, including SP1, AP-2, fluence hypoxia-dependent gene expression. This cross-talk be- Ku70, Ku80, and Yin Yang 1 (39–41). Therefore, despite the GR tween glucocorticoid and hypoxia-dependent signaling cascades being classed as a housekeeping gene, it is open to alternate reg- has been demonstrated in other studies. One study revealed that ulation through alteration in the activity of these and other tran- ligand-dependent activation of the GR enhances hypoxia-depen- scription factors. We have demonstrated in our study that hypoxia dent gene expression and HRE activity (44). Another study has up-regulates the expression of the GR at the transcriptional level. demonstrated inhibition of HIF-1-dependent activity by glucocor- The most widely described transcription factor complex responsi- ticoids (45). Therefore, we can conclude that the interaction be- ble for driving hypoxia-mediated transcriptional alterations is tween glucocorticoid- and hypoxia-dependent signaling cascades HIF-1. In this present study we have observed that overexpression is significant as well as varied and reflects the complexity and of HIF-1␣ results in the induction of the GR at the protein level context-specific alterations associated with their differential acti- Downloaded from under both normoxic and hypoxic conditions. This observation vation in complex tissue microenvironments. together with previous data demonstrating HIF-1␣-mediated in- Finally, our present data demonstrate an increased sensitivity to duction of GR mRNA (19) indicate that this transcription factor is glucocorticoids after hypoxia exposure, which we postulate to be responsible at least in part for the hypoxic induction of the GR. mediated through a hypoxia- and HIF-1-dependent up-regulation Further evidence for the involvement of HIF-1 in the induction of of the GR. We have previously demonstrated that hypoxia can act

GR expression is revealed by the observation that contained within as a stimulus for resolution of inflammatory conditions through http://www.jimmunol.org/ the 1064 bp of DNA sequence used in the GR promoter-reporter alterations in transcription factor modification (11). In the context construct up-regulated in hypoxia are five putative HIF-1 binding of our current findings we can suggest that hypoxia can also act as HRE sites (5Ј-RCGTG-3Ј). Therefore, we can infer that hypoxia- a stimulus for the resolution of inflammatory disease conditions driven GR up-regulation occurs through a mechanism likely in- through the induction of the GR and the subsequent increased sen- volving HIF-1 binding to one or multiple HRE sites facilitating sitivity to anti-inflammatory properties of glucocorticoids. enhancement of GR transcription. Although HIF-1␣ overexpres- sion was sufficient to induce GR expression in normoxia and hyp- Acknowledgments oxia, a contributory role for other transcription factors including We acknowledge technical assistance from Anne Marie Griffin. SP1/SP3 and AP-2, in the regulation of GR transcription in hyp- by guest on September 30, 2021 oxia cannot be discounted. We also cannot rule out a co-operative References role for nontranscriptional mechanisms such as alterations in 1. Genuth, S. M. 2004. The adrenal glands. In Physiology. R. M. Berne, ed. Mosby, St Louis, pp 883–919. mRNA stability and protein turnover as contributing to the up- 2. Neeck, G., R. Renkawitz, and M. Eggert. 2002. Molecular aspects of glucocor- regulation of the GR in hypoxia. ticoid hormone action in rheumatoid arthritis. Cytokines Cell. Mol. Ther. 7:61. ␣ 3. Nikolaus, S., U. Folscn, and S. Schreiber. 2000. Immunopharmacology of 5-ami- Having observed that overexpression of HIF-1 increases GR nosalicylic acid and of glucocorticoids in the therapy of inflammatory bowel levels, we next went on to investigate the effect of HIF-1␣ on disease. Hepatogastroenterology 47:71. GR-dependent activity. Neither HIF-1␣ WT nor HIF-1␣ DM 4. Schaaf, M. J., and J. A. Cidlowski. 2002. Molecular mechanisms of glucocorti- coid action and resistance. J. Steroid Biochem. Mol. Biol. 83:37. caused any significant alteration in GRE activity in normoxia as 5. Newton, R. 2000. Molecular mechanisms of glucocorticoid action: what is im- compared with vector control indicating that under normoxic con- portant? Thorax 55:603. ditions HIF-1␣-dependent up-regulation of the GR is not sufficient 6. Adcock, I. M., and S. J. Lane. 2003. -insensitive asthma: molecular mechanisms. J. Endocrinol. 178:347. for enhanced GRE activity. However the overexpression of both 7. Webster, J. C., and J. A. Cidlowski. 1999. Mechanisms of glucocorticoid-recep- HIF-1␣ constructs resulted in a significant increase in GRE activ- tor-mediated repression of gene expression. Trends Endocrinol. Metab. 10:396. 8. Barnes, P. J. 1998. Anti-inflammatory actions of glucocorticoids: molecular ity under hypoxic conditions as compared with vector control. mechanisms. Clin. Sci. 94:557. These results reveal that although hypoxia and HIF-1␣ can up- 9. Taylor, C. T., and S. P. Colgan. 1999. Therapeutic targets for hypoxia-elicited regulate the GR, the involvement of other hypoxia-dependent sig- pathways. Pharm. Res. 16:1498. 10. Huang, L. E., and H. F. Bunn. 2003. Hypoxia-inducible factor and its biomedical naling cues necessary to initiate or facilitate GRE-dependent tran- relevance. J. Biol. Chem. 278:19575. scriptional activity are likely involved. 11. Comerford, K. M., M. O. Leonard, J. Karhausen, R. Carey, S. P. Colgan, and The up-regulation of the GR by hypoxia and the subsequent C. T. Taylor. 2003. Small ubiquitin-related modifier-1 modification mediates res- olution of CREB-dependent responses to hypoxia. Proc. Natl. Acad. Sci. USA increased sensitivity to glucocorticoid ligand has major implica- 100:986. tions not only for the regulation of glucocorticoid-dependent re- 12. Basu, M., R. C. Sawhney, S. Kumar, K. Pal, R. Prasad, and W. Selvamurthy. 2002. Glucocorticoids as prophylaxis against acute mountain sickness. Clin. En- sponses but also for the regulation of hypoxia-dependent responses docrinol. 57:761. and gene expression. We have demonstrated a potentiation of glu- 13. Bauer, A., F. Tronche, O. Wessely, C. Kellendonk, H. M. Reichardt, P. Steinlein, cocorticoid-mediated inhibition of hypoxia-induced VEGF expres- G. Schutz, and H. Beug. 1999. The glucocorticoid receptor is required for stress erythropoiesis. Genes Dev. 13:2996. sion when cells were pre-exposed to hypoxia for 24 h compared 14. von Lindern, M., W. Zauner, G. Mellitzer, P. Steinlein, G. Fritsch, K. Huber, with a 1-h control. This result would indicate a potential feedback B. Lowenberg, and H. Beug. 1999. The glucocorticoid receptor cooperates with mechanism for the control of hypoxia-induced VEGF expression the erythropoietin receptor and c-Kit to enhance and sustain proliferation of ery- throid progenitors in vitro. Blood 94:550. through a hypoxia- and HIF-1-mediated up-regulation of the GR. 15. Fisher, J. W. 1998. A quest for erythropoietin over nine decades. Annu. Rev. However we observed no differential regulation of HIF-1 DNA Pharmacol. Toxicol. 38:1. 16. Limbourg, F. P., Z. Huang, J. C. Plumier, T. Simoncini, M. Fujioka, binding by dexamethasone upon hypoxia pre-exposure. This J. Tuckermann, G. Schutz, M. A. Moskowitz, and J. K. Liao. 2002. Rapid non- would indicate that the mechanism of glucocorticoid-mediated in- transcriptional activation of endothelial nitric oxide synthase mediates increased The Journal of Immunology 2257

cerebral blood flow and stroke protection by . J. Clin. Invest. 110: 32. Diederich, S., E. Eigendorff, P. Burkhardt, M. Quinkler, C. Bumke-Vogt, 1729. M. Rochel, D. Seidelmann, P. Esperling, W. Oelkers, and V. Bahr. 2002. 11␤- 17. Glanemann, M., R. Strenziok, R. Kuntze, S. Munchow, N. Dikopoulos, hydroxysteroid dehydrogenase types 1 and 2: an important pharmacokinetic de- F. Lippek, J. M. Langrehr, M. Dietel, P. Neuhaus, and A. K. Nussler. 2004. terminant for the activity of synthetic mineralo- and glucocorticoids. J. Clin. Ischemic preconditioning and both equally reduce hepatic Endocrinol. Metab. 87:5695. ischemia/reperfusion injury. Surgery 135:203. 33. Silva, C. M., F. E. Powell-Oliver, C. M. Jewell, M. Sar, V. E. Allgood, and 18. Ryan, M. J., G. Johnson, J. Kirk, S. M. Fuerstenberg, R. A. Zager, and J. A. Cidlowski. 1994. Regulation of the human glucocorticoid receptor by long- B. Torok-Storb. 1994. HK-2: an immortalized proximal tubule epithelial cell line term and chronic treatment with glucocorticoid. Steroids 59:436. from normal adult human kidney. Kidney Int. 45:48. 34. Kanelakis, K. C., and W. B. Pratt. 2003. Regulation of glucocorticoid receptor 19. Leonard, M. O., D. C. Cottell, C. Godson, H. R. Brady, and C. T. Taylor. 2003. ligand-binding activity by the hsp90/hsp70-based chaperone machinery. Methods The role of HIF-1␣ in transcriptional regulation of the proximal tubular epithelial Enzymol. 364:159. cell response to hypoxia. J. Biol. Chem. 278:40296. 35. Tago, K., F. Tsukahara, M. Naruse, T. Yoshioka, and K. Takano. 2004. Hsp90 20. Taylor, C. T., N. Fueki, A. Agah, R. M. Hershberg, and S. P. Colgan. 1999. inhibitors attenuate effect of dexamethasone on activated NF-␬B and AP-1. Life Critical role of cAMP response element binding protein expression in hypoxia- Sci. 74:1981. elicited induction of epithelial tumor necrosis factor-␣. J. Biol. Chem. 274:19447. 36. Kanelakis, K. C., D. S. Shewach, and W. B. Pratt. 2002. Nucleotide binding states 21. Mitchell, D., K. Rodgers, J. Hanly, B. McMahon, H. R. Brady, F. Martin, and of hsp70 and hsp90 during sequential steps in the process of glucocorticoid re- C. Godson. 2004. Lipoxins inhibit Akt/PKB activation and cell cycle progression ceptor.hsp90 heterocomplex assembly. J. Biol. Chem. 277:33698. in human mesangial cells. Am. J. Pathol. 164:937. 37. Rangarajan, P. N., K. Umesono, and R. M. Evans. 1992. Modulation of glu- 22. Nunez, B. S., and W. V. Vedeckis. 2002. Characterization of promoter 1B in the cocorticoid receptor function by protein kinase A. Mol. Endocrinol. 6:1451. human glucocorticoid receptor gene. Mol. Cell. Endocrinol. 189:191. 38. Zong, J., J. Ashraf, and E. B. Thompson. 1990. The promoter and first, untrans- 23. Hagen, T., C. T. Taylor, F. Lam, and S. Moncada. 2003. Redistribution of in- lated exon of the human glucocorticoid receptor gene are GC rich but lack con- tracellular oxygen in hypoxia by nitric oxide: effect on HIF1␣. Science 302:1975. sensus glucocorticoid receptor element sites. Mol. Cell. Biol. 10:5580. 24. Bamberger, C. M., H. M. Schulte, and G. P. Chrousos. 1996. Molecular deter- 39. Nobukuni, Y., C. L. Smith, G. L. Hager, and S. D. Detera-Wadleigh. 1995. minants of glucocorticoid receptor function and tissue sensitivity to glucocorti- Characterization of the human glucocorticoid receptor promoter. Biochemistry coids. Endocr. Rev. 17:245. 34:8207. 25. Mukaida, N., M. Morita, Y. Ishikawa, N. Rice, S. Okamoto, T. Kasahara, and 40. Warriar, N., N. Page, and M. V. Govindan. 1996. Expression of human glucocor- Downloaded from K. Matsushima. 1994. Novel mechanism of glucocorticoid-mediated gene repres- ticoid receptor gene and interaction of nuclear proteins with the transcriptional sion: nuclear factor-␬B is target for glucocorticoid-mediated interleukin 8 gene control element. J. Biol. Chem. 271:18662. repression. J. Biol. Chem. 269:13289. 41. Breslin, M. B., and W. V. Vedeckis. 1998. The human glucocorticoid receptor 26. Ferrara, N. 2002. VEGF and the quest for tumour angiogenesis factors. Nat. Rev. promoter upstream sequences contain binding sites for the ubiquitous transcrip- Cancer. 2:795. tion factor, Yin Yang 1. J. Steroid Biochem. Mol. Biol. 67:369. 27. Nauck, M., G. Karakiulakis, A. P. Perruchoud, E. Papakonstantinou, and 42. Newton, T. R., N. M. Patel, P. Bhat-Nakshatri, C. R. Stauss, R. J. Goulet, Jr., and M. Roth. 1998. Corticosteroids inhibit the expression of the vascular endothelial H. Nakshatri. 1999. Negative regulation of transactivation function but not DNA ␬ ␬ ␤ growth factor gene in human vascular smooth muscle cells. Eur. J. Pharmacol. binding of NF- B and AP-1 by I B 1 in breast cancer cells. J. Biol. Chem. http://www.jimmunol.org/ 341:309. 274:18827. 28. Vanderbilt, J. N., R. Miesfeld, B. A. Maler, and K. R. Yamamoto. 1987. Intra- 43. Gille, J., K. Reisinger, B. Westphal-Varghese, and R. Kaufmann. 2001. De- cellular receptor concentration limits glucocorticoid-dependent enhancer activity. creased mRNA stability as a mechanism of glucocorticoid-mediated inhibition of Mol. Endocrinol. 1:68. vascular endothelial growth factor gene expression by cultured keratinocytes. 29. Pepin, M. C., and N. Barden. 1991. Decreased glucocorticoid receptor activity J. Invest. Dermatol. 117:1581. following glucocorticoid receptor antisense RNA gene fragment transfection. 44. Kodama, T., N. Shimizu, N. Yoshikawa, Y. Makino, R. Ouchida, K. Okamoto, Mol. Cell. Biol. 11:1647. T. Hisada, H. Nakamura, C. Morimoto, and H. Tanaka. 2003. Role of the glu- 30. Sandeep, T. C., and B. R. Walker. 2001. Pathophysiology of modulation of local cocorticoid receptor for regulation of hypoxia-dependent gene expression. J. Biol. glucocorticoid levels by 11␤-hydroxysteroid dehydrogenases. Trends Endocri- Chem. 278:33384. nol. Metab. 12:446. 45. Hellwig-Buergel, T., A. Wagner, and W. Jelkmann. 2004. Biology of hypoxia: 31. Heiniger, C. D., R. M. Kostadinova, M. K. Rochat, A. Serra, P. Ferrari, B. Dick, the role of oxygen sensing in development, normal function and disease. In Key-

B. M. Frey, and F. J. Frey. 2003. Hypoxia causes down-regulation of 11␤-hy- stone Symposium March 25–30. Abstract 203. R. A. Johnson, A. Giacci, and M. by guest on September 30, 2021 droxysteroid dehydrogenase type 2 by induction of Egr-1. FASEB J. 17:917. C. Simon, ed. Steamboat Springs, Colorado, p. 79.