Glucocorticoid receptor cross-talk with NF-kappaB and AP-1 : functional role and mechanisms

Abstract: The protein investigated in this thesis is the glucocorticoid receptor (GR), which is a ligand activated transcription factor that belongs to the superfamily of nuclear hormone receptors. The GR contains three functional independent domains, the N-terminal domain containing a major transactivation region, the central DNA binding domain (DBD) and the C-terminal domain that binds the hormone/ligand. Glucocorticoid hormones, which bind to and activate the intracellular located GR, are steroid hormones that are involved in several important physiological processes such as metabolism, inflammatory and immune responses and cell proliferation. The GR exerts its effects by activating and repressing transcriptional activity of target genes. Repression of target genes in many cases involves interference with the activity of the transcription factors NFkappaB and AP-1, a process usually referred to as cross-talk. Results presented in studies I-II show that GR cross-talk with NF-kappaB and AP-1 signaling pathways can be separated. Using a model system devoid of endogenous functional GR (HEK293 cells), it was possible to study effects of different GR mutants. It was demonstrated that a point mutation in the C-terminal zinc-finger of the DBD impaired the receptor's ability to repress NFkappaB but not AP-1. Using this 'loss-of-function' mutant it was revealed that inhibition of activated extracellular signal-regulated kinase (ERK), is an important determinant for functional GR mediated repression of NF-kappaB. In wild type GR, but not GR mutant containing cells, induction of the endogenous ERK inhibitor MKP-1 was observed, highlightening this glucocorticoid mediated mechanism to be important for NF-kappaB repression in contexts were ERK is activated. In conclusion, GR cross-talk with NF-kappaB and AP-1 can be separated. This allowed us using gene expression profiling to identify endogenous genes preferentially repressed by GR interference with NF-kappaB. Glucocorticoids exert an antiproliferative effect on most cells. However, the molecular mechanisms are still largely unclear. In study III, the antiproliferative mechanism by glucocorticoids is further investigated using GR mutants that discriminate between cross-talk with NF-kappaB and AP-1, transactivation and transrepression and antiproliferative versus nonantiproliferative responses. Gene expression profiling revealed that the NF-kappaB inhibitor, IkappaBalpha, was upregulated in cells displaying an antiproliferative response to glucocorticoids. Unexpectedly, the induction of IkappaBalpha was shown to be a glucocorticoid response element (GRE) independent mechanism. Selective expression of IkappaBalpha demonstrated that IkappaBalpha upregulation was sufficient for the antiproliferative effect. In summary, this demonstrates that glucocorticoid inhibition of NF-kappaB is an important mechanism in the antiproliferative response to glucocorticoids. In HeLa cells the antiproliferative mechanism by glucocorticoids has been demonstrated to involve a direct transcriptional induction of the cyclin dependent kinase inhibitor p57Kip2 . In study IV the promoter region of p57Kip2 was further investigated. These studies identified a functional GRE in the human p57Kip2 promoter, located 5 kb upstream of the transcriptional start site. The GRE was shown to be well conserved in the mouse, both regarding sequence similarity and function, indicating a high biological relevance for this element.