Browsing by Author "Louw, Ann"

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    Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model
    (Public Library of Science, 2013) Robertson, Steven; Rohwer, Johann M; Hapgood, Janet P; Louw, Ann
    Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.
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    Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ
    (Public Library of Science, 2014) Verhoog, Nicolette; Allie-Reid, Fatima; Berghe, Wim Vanden; Smith, Carine; Haegeman, Guy; Hapgood, Janet; Louw, Ann
    Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg . Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ’s involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ.