Glucocorticoid repression of inflammatory gene expression shows differential responsiveness by transactivation- and transrepression-dependent mechanisms
Elizabeth M King, Joanna E Chivers, Christopher F Rider, Anne Minnich, Mark A Giembycz, Robert Newton, Elizabeth M King, Joanna E Chivers, Christopher F Rider, Anne Minnich, Mark A Giembycz, Robert Newton
Abstract
Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the de novo synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent repression by dexamethasone. In conclusion, our data indicate roles for both transrepression and transactivation in the glucocorticoid-dependent repression of inflammatory gene expression. However, transactivation appears to account for the more potent and efficacious mechanism of repression by glucocorticoids on these IL-1β-induced genes.
Conflict of interest statement
Competing Interests: The authors have read the journal’s policy and have the following conflicts: This study was partly supported by funding from AstraZeneca and GlaxoSmithKline. AM is currently employed by Bristol-Myers Squibb and while at Aventis Pharmaceuticals (Bridgewater, NJ) was responsible for conducting the microarray profiling. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Figures
![Figure 1. Effect of IL-1β and dexamethasone…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g001.jpg)
![Figure 2. Effect of dexamethasone on inflammatory…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g002.jpg)
![Figure 3. Effect of ORG34517 and GR-specific…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g003.jpg)
![Figure 4. Effect of IκBαΔN on inflammatory…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g004.jpg)
![Figure 5. Effect of CHX on dexamethasone-dependent…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g005.jpg)
![Figure 6. Relationship between the sensitivity and…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3545719/bin/pone.0053936.g006.jpg)
References
- Barnes PJ (2006) Corticosteroids: the drugs to beat. Eur J Pharmacol 533: 2–14.
- Newton R, Leigh R, Giembycz MA (2010) Pharmacological strategies for improving the efficacy and therapeutic ratio of glucocorticoids in inflammatory lung diseases. Pharmacol Ther 125: 286–327.
- Newton R (2000) Molecular mechanisms of glucocorticoid action: what is important? Thorax 55: 603–613.
- Rhen T, Cidlowski JA (2005) Antiinflammatory action of glucocorticoids–new mechanisms for old drugs. N Engl J Med 353: 1711–1723.
- De Bosscher K, Vanden Berghe W, Haegeman G (2003) The interplay between the glucocorticoid receptor and nuclear factor-kappaB or activator protein-1: molecular mechanisms for gene repression. Endocr Rev 24: 488–522.
- Clark AR, Belvisi MG (2012) Maps and legends: the quest for dissociated ligands of the glucocorticoid receptor. Pharmacol Ther 134: 54–67.
- Ito K, Barnes PJ, Adcock IM (2000) Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol 20: 6891–6903.
- Barnes PJ (2011) Glucocorticosteroids: current and future directions. Br J Pharmacol 163: 29–43.
- Ray A, Prefontaine KE (1994) Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci U S A 91: 752–756.
- Nissen RM, Yamamoto KR (2000) The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev 14: 2314–2329.
- Luecke HF, Yamamoto KR (2005) The glucocorticoid receptor blocks P-TEFb recruitment by NFkappaB to effect promoter-specific transcriptional repression. Genes Dev 19: 1116–1127.
- Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M (1995) Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 270: 286–290.
- Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS Jr (1995) Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science 270: 283–286.
- Brostjan C, Anrather J, Csizmadia V, Stroka D, Soares M, et al. (1996) Glucocorticoid-mediated repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha synthesis. J Biol Chem 271: 19612–19616.
- Ray KP, Farrow S, Daly M, Talabot F, Searle N (1997) Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor alpha, and inhibition by glucocorticoids. Biochem J 328: 707–715.
- Newton R, Hart LA, Stevens DA, Bergmann M, Donnelly LE, et al. (1998) Effect of dexamethasone on interleukin-1beta-(IL-1beta)-induced nuclear factor-kappaB (NF-kappaB) and kappaB-dependent transcription in epithelial cells. Eur J Biochem 254: 81–89.
- Heck S, Bender K, Kullmann M, Gottlicher M, Herrlich P, et al. (1997) I kappaB alpha-independent downregulation of NF-kappaB activity by glucocorticoid receptor. EMBO J 16: 4698–4707.
- Sakai DD, Helms S, Carlstedt-Duke J, Gustafsson JA, Rottman FM, et al. (1988) Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene. Genes Dev 2: 1144–1154.
- Drouin J, Trifiro MA, Plante RK, Nemer M, Eriksson P, et al. (1989) Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription. Mol Cell Biol 9: 5305–5314.
- Bilodeau S, Vallette-Kasic S, Gauthier Y, Figarella-Branger D, Brue T, et al. (2006) Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev 20: 2871–2886.
- Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq C, et al. (2004) Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci U S A 101: 15603–15608.
- So AY, Cooper SB, Feldman BJ, Manuchehri M, Yamamoto KR (2008) Conservation analysis predicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-induced genes. Proc Natl Acad Sci U S A 105: 5745–5749.
- Reddy TE, Pauli F, Sprouse RO, Neff NF, Newberry KM, et al. (2009) Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation. Genome Res 19: 2163–2171.
- Surjit M, Ganti KP, Mukherji A, Ye T, Hua G, et al. (2011) Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor. Cell 145: 224–241.
- Stellato C (2004) Post-transcriptional and nongenomic effects of glucocorticoids. Proc Am Thorac Soc 1: 255–263.
- Clark AR (2007) Anti-inflammatory functions of glucocorticoid-induced genes. Mol Cell Endocrinol 275: 79–97.
- Newton R, Holden NS (2007) Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol Pharmacol 72: 799–809.
- Clark AR, Martins JR, Tchen CR (2008) Role of dual specificity phosphatases in biological responses to glucocorticoids. J Biol Chem 283: 25765–25769.
- Ayroldi E, Riccardi C (2009) Glucocorticoid-induced leucine zipper (GILZ): a new important mediator of glucocorticoid action. FASEB J 23: 3649–3658.
- Kassel O, Sancono A, Kratzschmar J, Kreft B, Stassen M, et al. (2001) Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J 20: 7108–7116.
- Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR (2002) Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol Cell Biol 22: 7802–7811.
- Issa R, Xie S, Khorasani N, Sukkar M, Adcock IM, et al. (2007) Corticosteroid Inhibition of Growth-Related Oncogene Protein-{alpha} via Mitogen-Activated Kinase Phosphatase-1 in Airway Smooth Muscle Cells. J Immunol 178: 7366–7375.
- Kelly M, King E, Rider C, Gwozd C, Holden N, et al... (2011) Corticosteroid-induced gene expression in allergen-challenged asthmatic subjects taking inhaled budesonide. Br J Pharmacol.
- Mittelstadt PR, Ashwell JD (2001) Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 276: 29603–29610.
- Ayroldi E, Migliorati G, Bruscoli S, Marchetti C, Zollo O, et al. (2001) Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB. Blood 98: 743–753.
- Eddleston J, Herschbach J, Wagelie-Steffen AL, Christiansen SC, Zuraw BL (2007) The anti-inflammatory effect of glucocorticoids is mediated by glucocorticoid-induced leucine zipper in epithelial cells. J Allergy Clin Immunol 119: 115–122.
- Ayroldi E, Zollo O, Bastianelli A, Marchetti C, Agostini M, et al. (2007) GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling. J Clin Invest 117: 1605–1615.
- Kwon OJ, Au BT, Collins PD, Baraniuk JN, Adcock IM, et al. (1994) Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells. Immunology 81: 389–394.
- Newton R, Eddleston J, Haddad E, Hawisa S, Mak J, et al. (2002) Regulation of kinin receptors in airway epithelial cells by inflammatory cytokines and dexamethasone. Eur J Pharmacol 441: 193–202.
- Catley MC, Sukkar MB, Chung KF, Jaffee B, Liao SM, et al. (2006) Validation of the anti-inflammatory properties of small-molecule IkappaB Kinase (IKK)-2 inhibitors by comparison with adenoviral-mediated delivery of dominant-negative IKK1 and IKK2 in human airways smooth muscle. Mol Pharmacol 70: 697–705.
- King EM, Holden NS, Gong W, Rider CF, Newton R (2009) Inhibition of NF-kappaB-dependent transcription by MKP-1: transcriptional repression by glucocorticoids occurring via p38 MAPK. J Biol Chem 284: 26803–26815.
- Chivers JE, Gong W, King EM, Seybold J, Mak JC, et al. (2006) Analysis of the dissociated steroid, RU24858, does not exclude a role for inducible genes in the anti-inflammatory actions of glucocorticoids. Mol Pharmacol 70: 2084–2095.
- Kaur M, Chivers JE, Giembycz MA, Newton R (2008) Long-acting beta2-adrenoceptor agonists synergistically enhance glucocorticoid-dependent transcription in human airway epithelial and smooth muscle cells. Mol Pharmacol 73: 203–214.
- Peeters BW, Ruigt GS, Craighead M, Kitchener P (2008) Differential effects of the new glucocorticoid receptor antagonist ORG 34517 and RU486 (mifepristone) on glucocorticoid receptor nuclear translocation in the AtT20 cell line. Ann N Y Acad Sci 1148: 536–541.
- Holden NS, Gong W, King EM, Kaur M, Giembycz MA, et al. (2007) Potentiation of NF-kappaB-dependent transcription and inflammatory mediator release by histamine in human airway epithelial cells. Br J Pharmacol 152: 891–902.
- Catley MC, Chivers JE, Holden NS, Barnes PJ, Newton R (2005) Validation of IKK beta as therapeutic target in airway inflammatory disease by adenoviral-mediated delivery of dominant-negative IKK beta to pulmonary epithelial cells. Br J Pharmacol 145: 114–122.
- Schleimer RP (2004) Glucocorticoids suppress inflammation but spare innate immune responses in airway epithelium. Proc Am Thorac Soc 1: 222–230.
- Cornish AL, Campbell IK, McKenzie BS, Chatfield S, Wicks IP (2009) G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nat Rev Rheumatol 5: 554–559.
- Commins SP, Borish L, Steinke JW (2010) Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol 125: S53–S72.
- Vereecke L, Beyaert R, van Loo G (2011) Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease. Biochem Soc Trans 39: 1086–1091.
- Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15: 1583–1606.
- Honda K, Taniguchi T (2006) IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6: 644–658.
- Taube C, Thurman JM, Takeda K, Joetham A, Miyahara N, et al. (2006) Factor B of the alternative complement pathway regulates development of airway hyperresponsiveness and inflammation. Proc Natl Acad Sci U S A 103: 8084–8089.
- Bartholomew WR, Shanahan TC (1990) Complement components and receptors: deficiencies and disease associations. Immunol Ser 52: 33–51.
- Wang Y, Zhang JJ, Dai W, Lei KY, Pike JW (1997) Dexamethasone potently enhances phorbol ester-induced IL-1beta gene expression and nuclear factor NF-kappaB activation. J Immunol 159: 534–537.
- Hofmann TG, Schmitz ML (2002) The promoter context determines mutual repression or synergism between NF-kappaB and the glucocorticoid receptor. Biol Chem 383: 1947–1951.
- Webster JC, Huber RM, Hanson RL, Collier PM, Haws TF, et al. (2002) Dexamethasone and tumor necrosis factor-alpha act together to induce the cellular inhibitor of apoptosis-2 gene and prevent apoptosis in a variety of cell types. Endocrinology 143: 3866–3874.
- Sukkar MB, Issa R, Xie S, Oltmanns U, Newton R, et al. (2004) Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-gamma and TNF-alpha and regulation by TGF-beta and corticosteroids. Am J Physiol Lung Cell Mol Physiol 287: L1230–L1240.
- King EM, Kaur M, Gong W, Rider CF, Holden NS, et al. (2009) Regulation of tristetraprolin expression by interleukin-1beta and dexamethasone in human pulmonary epithelial cells: roles for nuclear factor-kappaB and p38 mitogen-activated protein kinase. J Pharmacol Exp Ther 330: 575–585.
- Quante T, Ng YC, Ramsay EE, Henness S, Allen JC, et al. (2008) Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1. Am J Respir Cell Mol Biol 39: 208–217.
- Turpeinen T, Nieminen R, Moilanen E, Korhonen R (2010) Mitogen-activated protein kinase phosphatase-1 negatively regulates the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in A549 human lung epithelial cells. J Pharmacol Exp Ther 333: 310–318.
- Newton R, King EM, Gong W, Rider CF, Staples KJ, et al. (2010) Glucocorticoids inhibit IL-1beta-induced GM-CSF expression at multiple levels: roles for the ERK pathway and repression by MKP-1. Biochem J 427: 113–124.
- Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, et al. (2006) Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression. J Exp Med 203: 7–13.
- Diefenbacher M, Sekula S, Heilbock C, Maier JV, Litfin M, et al. (2008) Restriction to Fos family members of Trip6-dependent coactivation and glucocorticoid receptor-dependent trans-repression of activator protein-1. Mol Endocrinol 22: 1767–1780.
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