Browsing by Author "Wilkinson, Legh"

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    Disease- and treatment-associated acquired glucocorticoid resistance
    (BioScientifica, 2018-10-11) Wilkinson, Legh; Verhoog, Nicolette J. D.; Louw, Ann
    ENGLISH ABSTRACT: The development of resistance to glucocorticoids (GCs) in therapeutic regimens poses a major threat. Generally, GC resistance is congenital or acquired over time as a result of disease progression, prolonged GC treatment or, in some cases, both. Essentially, disruptions in the function and/or pool of the glucocorticoid receptor α (GRα) underlie this resistance. Many studies have detailed how alterations in GRα function lead to diminished GC sensitivity; however, the current review highlights the wealth of data concerning reductions in the GRα pool, mediated by disease-associated and treatment-associated effects, which contribute to a significant decrease in GC sensitivity. Additionally, the current understanding of the molecular mechanisms involved in driving reductions in the GRα pool is discussed. After highlighting the importance of maintaining the level of the GRα pool to combat GC resistance, we present current strategies and argue that future strategies to prevent GC resistance should involve biased ligands with a predisposition for reduced GR dimerization, a strategy originally proposed as the SEMOGRAM–SEDIGRAM concept to reduce the side-effect profile of GCs.
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    Homologous down-regulation of the glucocorticoid receptor is influenced by the dimerization state of the receptor
    (Stellenbosch : Stellenbosch University, 2018-03) Wilkinson, Legh; Louw, Ann; Verhoog, Nicolette J. D.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Glucocorticoids (GCs) remain the mainstay therapeutic choice for the treatment of inflammation, and exert their potent anti-inflammatory effects via the glucocorticoid receptor (GRα). However, the chronic use of GCs, in addition to generating undesirable side-effects (e.g. hyperglycemia), results in homologous down-regulation of the GRα. This reduction in GRα protein levels has been coupled to a decrease in GC-responsiveness, in a number of psychological and pathological conditions, which may culminate in GC-acquired resistance, a major concern for chronic GC users. The current study investigated whether ligand-induced down-regulation of the GRα is influenced by the dimerization state of the receptor by transfecting human wild type GRα (hGRwt) or a dimerization deficient GRα mutant (hGRdim) into COS-1 cells. In addition, Compound A (CpdA), which abrogates GR dimerization, was used to mimic the effect of the hGRdim in HepG2 cells containing endogenous GRα. Furthermore, the ability of an endogenous mutant, mGRdim, to undergo ligand-induced receptor turnover was compared to that of the wild-type GRα, mGRwt, in MEF-mGRdim and MEF-mGRwt cells, respectively. Whole-cell-binding and Western blotting revealed that the hGRwt, but not the hGRdim, underwent homologous down-regulation following dexamethasone (Dex), a potent synthetic GC, and cortisol (F), an endogenous GC, treatment. In contrast, ligand-induced down-regulation of GRα was abolished by CpdA treatment or the use of hGRdim, suggesting a novel role for GRα dimerization in mediating receptor turnover. These findings from the COS-1 cells were supported by results from the HepG2 cells, and, in part, by results from the MEF cells. Moreover, the dimerization state of the GRα influenced the posttranslational processing of the receptor, impacting its degradation via the proteasome. Specifically, ‘loss’ of GRα dimerization via CpdA treatment or the use of the dimerization deficient GRα mutant, restricted hyper-phosphorylation at Ser404, which has been coupled to increased GRα degradation, as well as restricted the interaction of GRα with the E3 ligase, FBXW7α, thus hampering receptor turnover. Lastly, a model to mimic acquired GC resistance was established and tested. Results from these experiments demonstrated that prolonged GC treatment of mGRwt (i.e. ‘gain’ of GRα dimerization) leads to molecular GC resistance (i.e. GILZ) and clinical GC resistance (FKBP51), whilst maintaining the up-regulation of a metabolic gene (i.e. TAT). In contrast, ‘loss’ of GRα dimerization partially restricts acquired resistance, at a molecular and clinical level, whilst displaying an improved side-effect profile in terms of restricting the expression of a metabolic gene (i.e. TAT). These results expand our understanding of factors that contribute to GC-resistance and may be exploited clinically.
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    Novel role for receptor dimerization in post-translational processing and turnover of the GRα
    (Nature, 2018) Louw, Ann; Wilkinson, Legh; Verhoog, Nicolette
    Glucocorticoids (GCs), acting via the glucocorticoid receptor (GRα), remain the mainstay therapeutic choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable side-effects, results in GRα down-regulation, often coupled to a decrease in GC-responsiveness, which may culminate in acquired GC resistance. The current study presents evidence for a novel role of the dimerization state of the GRα in mediating GC-mediated GRα turnover. Through comparing the effects of dimerization promoting GCs on down-regulation of a transfected human wild type GRα (hGRwt) or a dimerization deficient GRα mutant (hGRdim), we established that a loss of receptor dimerization restricts GRα turnover, which was supported by the use of the dimerization abrogating Compound A (CpdA), in cells containing endogenous GRα. Moreover, we showed that the dimerization state of the GRα influenced the post-translational processing of the receptor, specifically hyper-phosphorylation at Ser404, which influenced the interaction of GRα with the E3 ligase, FBXW7α, thus hampering receptor turnover via the proteasome. Lastly, the restorative effects of CpdA on the GRα pool, in the presence of Dex, were demonstrated in a combinatorial treatment protocol. These results expand our understanding of factors that contribute to GC-resistance and may be exploited clinically.