Abstract
Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol11:1245-1255, 1997) repress interleukin (IL)-1β-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor κB (NF-κB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-κB-dependent transrepression (∼40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1β-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory “dissociated” GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.
Footnotes
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J.E.C. was a collaborative Biotechnology and Biological Sciences Research Council student supported by Aventis Pharmaceuticals (now Sanofi-Aventis). R.N. is a Canadian Institutes of Health Research (CIHR) New Investigator and Alberta Heritage Foundation for Medical Research (AHFMR) scholar. This work was supported by both CIHR and the AHFMR. R.N. also received an unrestricted grant from GlaxoSmithKline.
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J.E.C., W.G., and E.M.K. contributed equally to this work and are listed alphabetically.
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ABBREVIATIONS: GR, glucocorticoid receptor; AMP N, aminopeptidase N; AP-1, activator protein 1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GILZ, glucocorticoid-inducible leucine zipper protein; GRE, glucocorticoid response element; IL, interleukin; MET 1X, metallothionein 1X; MAPK, mitogen-activated protein kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; NF-κB, nuclear factor κB; PG, prostaglandin; PGES prostaglandin E synthase; PCR, polymerase chain reaction; RGS2, regulator of G-protein signaling 2; RT-PCR, reverse transcriptase-polymerase chain reaction; SFM, serum-free medium; ELISA, enzyme-linked immunosorbent assay; RU486, mifepristone.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received April 19, 2006.
- Accepted September 19, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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