Doctoral Degrees (Biochemistry)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3909

Browse

Browsing Doctoral Degrees (Biochemistry) by Subject "Acquired glucocorticoid resistance"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    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.