Dexamethasone impairs hypoxia-inducible factor-1 function A.E. Wagner 1 , G. Huck, D.P. Stiehl 2 , W. Jelkmann, T. Hellwig-Bürgel * Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany article info Article history: Received 9 May 2008 Available online 21 May 2008 Keywords: Glucocorticoid receptor HIF-1 repression Transcriptional cross-talk Nuclear import Transrepression Heat-shock protein 90 Wound healing abstract Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of a- and b-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory pro- cesses and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1a levels in the cytosol of HepG2 cells, while nuclear HIF-1a levels and HIF-1 DNA- binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced lucif- erase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients. Ó 2008 Elsevier Inc. All rights reserved. The synthetic glucocorticoid (GC) dexamethasone (DEX) is a widely used drug to suppress the development of local heat, red- ness, swelling and tenderness; the symptoms of inflammation. Be- sides these beneficial effects, DEX causes unwanted side effects such as impaired wound healing. Due to the lipophilic nature of GCs they pass through the cell membrane. Once intracellularly lo- cated GCs exert two different modes of action. The first is binding and activating the glucocorticoid receptor (GR), which is in the activated status a dimer of two identical monomers, and subse- quent regulation of several target genes containing glucocorticoid responsive elements (GREs) in their promoter regions. The effect on transcription can be either positive or negative [1,2]. The second mode of action targets several genes containing no GRE in their promoter regions, the so called ‘transcriptional cross-talk’ [1,3,4]. This mode of action is mainly based on the interference with other transcription factors such as NF-jB and AP-1 [3,4]. Hypoxia inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of a- and b-subunits, is of crucial importance for the cellular adaptation to hypoxia [5,6]. While the b-subunit is constitu- tively present within cells, the a-subunit is highly susceptible to oxygen and becomes immediately hydroxylated at specific prolyl- and asparaginyl-residues in the presence of oxygen [7–9]. The pro- lyl-hydroxylated a-subunit is recognized and bound by the von Hip- pel–Lindau protein which is part of an E3 ubiquitin ligase complex. Subsequently, HIF-1a is poly-ubiquitinated and degraded by the proteasome [10–12]. Besides hypoxia, proinflammatory cytokines like TNFa and IL-1b induce HIF-1 activity and it has been demon- strated that HIF-1 itself upregulates IL-1b expression, establishing a positive feedback loop [13–18]. Furthermore, it has been shown that HIF-1a subunits accumulate in cells at the border of artificial incisions in sheep skin during the inflammatory phase of wound healing, indicating that HIF-1 might have an important function dur- ing inflammation and tissue regeneration [19]. Herein, we addressed to the question, as to whether GCs impair HIF-1 function in vitro. Materials and methods. Cell culture. HepG2 (ATCC No. HB 8065) and Hep3B (ATCC No. 8064) cells were maintained in RPMI 1640 medium (Gibco BRL, Kar- lsruhe, Germany), HRG1 (HepG2-derivative) and HRB5 cells (Hep3B- derivative; kindly provided by R. Wenger) were maintained in DMEM high glucose (Gibco). Both media were supplemented with 10% fetal bovine serum (FBS; Sigma, Deisenhofen, or Life Technolo- gies, Karlsruhe, Germany), penicillin (90 U/ml) and streptomycin (90 lg/ml). Medium for HRG1 and HRB5 cells was additionally sup- plemented with G418 (250 lg/ml, Sigma). For experiments cells were grown to 25–50% confluence in tissue culture dishes of 145 mm diameter (Greiner, Frickenhausen, Germany) or to 40–70% confluence in 24-well plates (Nunc, Roskilde, Denmark). All cultures were grown in a humidified atmosphere (5% CO 2 in air) at 37 °C (Heraeus Incubators, Hanau, Germany). For induction of hypoxia 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.061 * Corresponding author. Fax: +49 451 500 4171. E-mail address: hellwig@physio.uni-luebeck.de (T. Hellwig-Bürgel). 1 Present address: Institute of Human Nutrition and Food Science, Department of Food Science, Christian-Albrechts-Universität Kiel, Hermann-Rodewald-Str. 6, D- 24098 Kiel, Germany. 2 Present address: Institute of Physiology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Biochemical and Biophysical Research Communications 372 (2008) 336–340 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc