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ORIGINAL ARTICLE

Identification of Endogenous Glucocorticoid Repressed Genes Differentially Regulated by a Glucocorticoid Receptor Mutant Able to Separate between Nuclear Factor-B and Activator Protein-1 Repression

Departments of Medical Nutrition (L.-G.B., S.O.) and Biosciences (J.L., K.D-W., M.R.), Karolinska Institutet and Karo Bio AB, Novum, Huddinge, Sweden (S.N.)

Abstract

Glucocorticoids are commonly used in the clinic, but long-term treatment is often associated with severe side effects. One way to reduce unwanted effects is to restrict glucocorticoid receptor (GR) signaling through defined pathways. In this study, we examine endogenous target genes regulated by a GR mutant that in contrast to the wild-type GR is unable to repress stimulated nuclear factor-B (NF-B) activity, whereas repression of activator protein-1 (AP-1) activity is maintained. This GR mutant (GR_R488Q) harbors a point mutation in the second zinc finger of the DNA binding domain. Its ability to distinguish between NF-B and AP-1 repression is defined using reporter genes regulated by both simple and natural promoters. The inability of GR_R488Q to repress NF-B was not related to its inability to activate target genes through a glucocorticoid response element. Furthermore, the discriminating property was observed in three different cell lines, suggesting that this is not a cell-specific effect. These results show that different receptor surfaces or mechanisms are involved in repression of NF-B and AP-1, respectively. It is interesting that the GR_R488Q still interacted physically with NF-B. Gene expression profiling of human embryonic kidney 293 cells, which express the wild-type GR and the GR_R488Q mutant allowed identification of endogenous genes preferentially repressed by GR interference with NF-B activity. The genes differentially regulated by GR_R488Q mutant versus the wild-type GR after 2 h of treatment seem mainly to be involved in control of transcription and cell growth. At 8 h, no such distinction could be seen.

Address correspondence to: Dr. Sam Okret, Department of Medical Nutrition, Karolinska Institutet, Karolinska University Hospital in Huddinge, Novum, SE-141 86 Huddinge, Sweden. E-mail: sam.okret{at}mednut.ki.se

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