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Potentiation of Glucocorticoid Activity in Hypoxia through Induction of the Glucocorticoid Receptor 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 References This article cites 43 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/174/4/2250.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 glucocorticoid receptor (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 dexamethasone-induced glucocorticoid response element promoter activity and enhanced dexamethasone-mediated inhibition of IL-1␤-stimulated IL-8 expression 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 glucocorticoid 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 glucocorticoids 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 kidney (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

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