Homologous down-regulation of the glucocorticoid receptor is influenced by the dimerization state of the receptor

Wilkinson, Legh (2018-03)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

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.

AFRIKAANSE OPSOMMING: Glukokortikoïede (GK's) bly die staatmaker terapeutiese keuse vir die behandeling van inflammasie en oefen hul kragtige anti-inflammatoriese effekte uit via die glukokortikoïede reseptor (GRα). Die chroniese gebruik van GK's, benewens die ontwikkeling van ongewenste newe-effekte (bv. hiperglisemie), lei ook ter tot homoloë afregulering van die GRα. Hierdie afname in GRα proteïenvlakke word gekoppel aan 'n afname in GK-responsiwiteit, in 'n aantal sielkundige en patologiese toestande, wat kan lei tot GK-verworwe weerstand, 'n groot kommer vir chroniese GKgebruikers. Die huidige studie ondersoek of ligand-geïnduseerde afregulering van die GRα beïnvloed word deur die dimerisasietoestand van die reseptor deur menslike wilde tipe GRα (hGRwt) of 'n dimerisasie-defektiewe GRα-mutant (hGRdim) in COS-1-selle te transfekteer. Daarbenewens is Compound A (CpdA), wat GR-dimerisasie ophef, gebruik om die effek van die hGRdim in HepG2-selle wat endogene GRα bevat, na te boots. Verder is die vermoë van 'n endogene mutant, mGRdim, om ligand-geïnduseerde reseptoromset te ondergaan, vergelyk met dié van die wild-tipe GRα, mGRwt, onderskeidelik in MEF-mGRdim en MEF-mGRwt-selle. Heel-selbinding Western klad het aangetoon dat die hGRwt, maar nie die hGRdim nie, homoloë afregulering ondersogaan na behandeling met deksametason (Dex), 'n kragtige sintetiese GK, en kortisol (F), 'n endogene GK. In teenstelling hiermee is ligand-geïnduseerde afregulering van GRα afgeskaf deur CpdA-behandeling of die gebruik van hGRdim, wat 'n splinternuwe rol vir GRadimerisasie in die bemiddeling van reseptoromset voorstel. Hierdie bevindings van die COS-1-selle is ondersteun deur die resultate van die HepG2-selle, en gedeeltelik deur die resultate van die MEFselle. Verder het die dimeriseringstoestand van die GRα die post-translasie-modifisering van die reseptor beïnvloed, wat afbraak deur die proteasoom beïnvloed het. Spesifiek, 'verlies' aan GRα- dimerisasie via CpdA-behandeling of die gebruik van die dimerisasie-defektiewe GRα-mutant, het hiperfosforilering by Ser404, wat gekoppel is aan verhoogde GRα-afbraak, sowel as die interaksie van GRα met die E3-ligase, FBXW7α, beperk wat dus die reseptoromset belemmer het. Laastens is 'n model om verworwe GK-weerstand na te boots daargestel en getoets. Resultate van hierdie eksperimente het getoon dat langdurige GK behandeling van mGRwt (dws 'wins van GRα dimerisasie) lei tot molekulêre GK weerstand (dws GILZ) en kliniese GK weerstand (dws FKBP51), terwyl die opregulering van 'n metaboliese geen (dws TAT ) behoue bly. In teenstelling hiermee verminder 'verlies' van GRα-dimerisasie die verworwe weerstand, op molekulêre en kliniese vlak, terwyl 'n verbeterde newe-effekprofiel vertoon word in terme van die beperking van die uitdrukking van 'n metaboliese geen (dws TAT). Hierdie resultate brei ons begrip uit van faktore wat bydra tot GK-weerstand en kan klinies ontgin word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103838
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