Repression of transcription factor NF-[kappa]B by glucocorticoids : a molecular mechanism for their anti-inflammatory and immuno-modulatory effects

Abstract: It has been known since the late 1940s that glucocorticoids (GCs) possess anti-inlammatory activity. Their various uses in the clinic include treatment of, rheumatoid arthritis, asthma and AIM skin diseases and also as means for avoiding rejection during transplantation. Despite the wide usage of GCs, it is only during the last five years that we have started to understand the molecular responsible for this anti-inflammatory activity. The anti-inflammatory action of glucocorticoids has been shown to involve repression of NF-[kappa]B activity. Several different molecular mechanisms have been proposed to be involved in this process. These include retention of NF-[kappa]B in the cytoplasm, glucocorticoid receptor (GR) interference with DNA binding of NF-[kappa]B, induction of I[kappa]B levels and GR inhibition of NF-[kappa]B when NF-[kappa]B is still bound to DNA. The GR is a member of the superfamily of nuclear receptors. In its inactive state it is associated with heat shock proteins. This association maintains the receptor in a conformation that has low affinity for DNA and high affinity for its cognate ligands. After the ligand has bound to the receptor the heat shock proteins dissociate and the receptor changes its conformation, acquiring a high affinity for its DNA binding site. The ligand-activated receptors can both activate and repress transcription from target genes. This gene regulatory function is mediated via direct DNA binding of the receptor and/or by physical contact with other transcription factors or signal mediators. In mammalian cells the family of NF-[kappa]B transcription factors consists of Re1A (p65), NFKB1 (p50), c-Re1, p52 and Re1B. ReIA is the most potent transcriptional activator of the Re1 homology protein family and the principal inducible NF-[kappa]B complex is composed of a heterodimer of ReIA and NFKB1. In the non- activated state this heterodimer is sequestered in the cytoplasm through interaction with the NF-[kappa]B -inhibiting protein I[kappa]B[alpha], which masks the nuclear localization sequence of the heterodimer. During the activation process, I[kappa]B[alpha] becomes phosphorylated and degrades. This allows the NF-[kappa]B heterodimer to translocate into the nucleus, where it binds to specific NF-[kappa]B-binding sequences in promoter regions and subsequently activates genes involved in inflammatory and immunoregulatory responses. Published reports included in this thesis describe a mutual transcriptional cross talk between the GR and the NF- KB protein ReIA (I). This cross talk involves repression of transcription regulated by these two factors. Investigation of the region of the GR involved in the repression of NF-[kappa]B revealed that it was mainly localized in the DNA binding domain (DBD), but did not involve DNA binding (I). Usage of in vitro translated proteins in an immunoprecipitation assay demonstrated a physical interaction between -GR and Re1A (I). In order to further map the GR DBD involved in ReIA repression, GR/thyroid hormone receptor JR) chimera constructs were used. TR does not repress ReIA and the results from transfection studies using these chimeras showed the C-terminal zinc finger to harbor the repressive function (II). In further transfection studies using single amino acid mutations within the second zinc finger of GR, two amino acid residues were identified as indispensable for ReIA interference (II). Since it has been a matter of debate whether NF-[kappa]B remains bound to DNA during glucocorticoid repression this question was addressed using an in vivo model. Based on results from this study, evidence was presented supporting a model where NF-[kappa]B is still associated with DNA during glucocorticoid repression of the intercellular adhesion molecule- I promoter, possibly in complex with the GR (III). Organic dust in and around swine confinement buildings promotes strong inflammatory processes in the lungs of healthy humans. Until now little has been known about molecular mechanisms involved in this inflammatory response. This was investigated using a lung epithelial cell line as a model system. By interfering with the NF-[kappa]B pathway at different levels, it was demonstrated that swine dust exposure results in strong NF-[kappa]B activation (IV). Taken together, the data supports a mechanism for glucocorticoid repression of NF-[kappa]B in which GR directly or indirectly masks the transcriptional activation domain of the NF-[kappa]B protein Re1A. Furthermore, results obtained support that the interference between GR and NF-[kappa]B takes place when NF-[kappa]B is bound to the DNA. In addition it was shown that the inflammatory process during exposure to organic dust involves NF-[kappa]B activation and that this activation can be blocked by glucocorticoids.