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The effect of glucocorticoid receptor alpha dimerization state on receptor turnover
Thesis (MSc)--Stellenbosch University, 2021.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867613933878116352 |
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| access_status_str | Open Access |
| author | Spies, Lee-Maine Lorin |
| author2 | Louw, Ann |
| author_browse | Louw, Ann Spies, Lee-Maine Lorin |
| author_facet | Louw, Ann Spies, Lee-Maine Lorin |
| author_sort | Spies, Lee-Maine Lorin |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/124298 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:01Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/124298 The effect of glucocorticoid receptor alpha dimerization state on receptor turnover Spies, Lee-Maine Lorin Louw, Ann Verhoog, Nicolette Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Glucocorticoids -- Receptors Dimerization Glucocorticoid receptor alpha Glucocorticoids UCTD Thesis (MSc)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Due to the unique anti-inflammatory properties of glucocorticoids (GCs) these steroid hormones, which mediate their effects via the ligand-activated transcription factor, the glucocorticoid receptor alpha (GRα), have been used as therapeutics in the treatment of various autoimmune and inflammatory-linked diseases, for over 70 years. However, aside from the onset of severe side-effects, chronic GC therapy often leads to the ligand-mediated downregulation of the GRα, which in turn, leads to a decrease in GC sensitivity, and effectively, the development of an acquired GC resistance. Although the ligand-mediated downregulation of GRα is well documented, the precise factors which influence this process is not well understood and thus, the development of an acquired GC resistance presents an ever-increasing challenge to the pharmaceutical industry. Following the recently demonstrated dimerization-dependent downregulation of GRα, through the primary use of transiently transfected receptors, the current study sought to determine the effect of GRα dimerization state on the ligand-mediated downregulation of the receptor, within an endogenous cellular environment. The current study, through the use of mouse embryonic fibroblast (MEF) cells containing endogenous levels of either wild type GRα (GRwt) or the dimerization deficient GRα mutant (GRdim), confirms the dimerization dependent downregulation of both GRα protein and mRNA levels. Specifically, treatment with the dimerization promoting GRα agonist, dexamethasone (Dex) led to both the time- and dose-dependent downregulation of GRwt protein, as well as the downregulation of GRwt mRNA levels. In contrast, Dex was unable to affect either the mRNA or protein levels of the receptor upon the restriction of receptor dimerization, through the use of GRdim. Similarly, neither GRwt nor GRdim protein underwent downregulation following both the time- and dose-dependent treatment with the dimerization abrogating selective GRα modulator, compound A (CpdA). Furthermore, through the use of the proteasomal inhibitor, MG132, results in the current study strongly implicated the ubiquitin- proteasome system (UPS) as the predominant molecular mechanism for the downregulation of GRwt protein upon its Dex-mediated dimerization, while transcriptional inhibition, through the use of amanitin, indicated a modest contribution of the miRNA-mediated destabilization of GRwt mRNA to the cumulative Dex- mediated downregulation of the GRwt levels. In contrast, the restriction of receptor dimerization via GRdim, inhibited the Dex-mediated downregulation of the receptor by the UPS. However, upon transcriptional inhibition via amanitin, a significant increase in both the efficacy and potency of Dex to mediate the downregulation of GRdim protein was seen, compared to Dex treatment alone. Due to the recently discovered association of GRα with non-coding RNAs via the DNA-binding domain of the receptor, the current study postulates that the increase in GRdim protein downregulation upon transcriptional inhibition occurs due to the inhibition of non-coding RNA transcription, and thus, suggests a novel link between the stability of Dex-bound GRdim and non- coding RNAs. Collectively, the findings of the current study contribute to the broad understanding of how and where ligand-mediated downregulation of GRα occurs and provides pharmacological targets that may be used to develop more beneficial GCs with a reduced potential for developing resistance. AFRIKAANSE OPSOMMING: As gevolg van die unieke anti-inflammatoriese eienskappe van glukokortikoïede (GKs) word hierdie steroïedhormone, wat hul effekte deur die ligand-geaktiveerde transkripsie faktor die glukokortikoïed reseptor alfa (GRα) bemiddel, vir al meer as 70 jaar as terapeutiese middels gebruik in die behandeling van verskeie outo-immuun en inflammatoriese siektes. Afgesien van die aanvang van ernstige newe-effekte, lei chroniese GK-terapie egter dikwels tot die ligand-bemiddelde af-regulering van die GRα, wat weer lei tot 'n afname in GK-sensitiwiteit, en uiteindelik die ontwikkeling van verworwe GK-weerstandigheid. Alhoewel die ligand-bemiddelde af-regulering van GRα goed gedokumenteer is, word die presiese faktore wat hierdie proses beïnvloed nie goed verstaan nie, en die ontwikkeling van 'n verworwe GK-weerstandigheid bied dus toenemend 'n uitdaging vir die farmaseutiese industrie. Na die onlangs gedemonstreerde dimerisering-afhanklike af-regulering van GRα, deur die primêre gebruik van kortstondig getransfekteerde reseptore, het die huidige studie beoog om die effek van die GRα-dimeriserings toestand op die ligand-bemiddelde af-regulering van die reseptor binne 'n endogene sellulêre omgewing te bepaal. Die huidige studie, deur die gebruik van muis-embrionale fibroblast (MEF) selle wat endogene vlakke van wilde tipe GRα (GRwt) of die dimerisering-gebrekkige GRα mutant (GRdim) bevat, bevestig die dimerisering-afhanklike af-regulering van beide GRα proteïen- en mRNA- vlakke. Spesifiek, lei behandeling met die dimerisering-bevorderende GRα-agonis, deksametasoon (Dex) tot die tyd- en dosisafhanklike af-regulering van GRwt proteïen, sowel as die af-regulering van GRwt mRNA vlakke. Daarteenoor kon Dex nie die mRNA- of proteïenvlakke van die reseptor beïnvloed indien reseptor-dimerisering beperk is deur die gebruik van GRdim nie. Net so het nie GRwt- of GRdim-proteïen af-regulering ondergaan na die tyd- en dosisafhanklike behandeling met die dimerisering voorkomende selektiewe GRα-modulator, compound A (CpdA). Verder, deur die gebruik van die proteasomale inhibitor, MG132, het resultate in die huidige studie die ubiquitin-proteasoom-stelsel (UPS) sterk geïmpliseer as die oorheersende molekulêre meganisme vir die af-regulering van GRwt-proteïene na afloop van Dex- bemiddelde dimerisering van die reseptor, terwyl transkripsie-inhibisie deur die gebruik van amanitien, op 'n beskeie bydrae van die miRNA-bemiddelde destabilisering van GRwt mRNA tot die kumulatiewe Dex-bemiddelde af-regulering van die GRwt-vlakke dui. Daarenteen het die beperking van reseptor-dimerisering via GRdim die Dex-bemiddelde af-regulering van die reseptor deur die UPS geïnhibeer. By transkripsie-inhibisie via amanitien is 'n beduidende toename in die effektiwiteit en die sterkte van Dex om die af-regulering van GRdim-proteïen te bemiddel egter gesien in vergelyking met Dex-behandeling alleen. As gevolg van die pas ontdekte assosiasie van GRα met nie-koderende RNAs via die DNA-bindings domein van die reseptor, postuleer die huidige studie dat die toename in GRdim-proteïen af-regulering wat tydens transkripsie-inhibisie plaasvind as gevolg van die inhibisie van nie-koderende RNA-transkripsie is, en stel dus 'n nuwe verband voor tussen die stabiliteit van Dex- gebonde GRdim en nie-koderende RNAs. Gesamentlik dra die bevindings van die huidige studie by tot die breë begrip van hoe en waar ligand-bemiddelde af-regulering van GRα voorkom en bied dus farmakologiese teikens wat gebruik kan word om voordeliger GKs te ontwikkel met 'n verminderde potensiaal om weerstand te ontwikkel. Masters 2021-07-16T15:04:29Z 2022-02-22T10:23:46Z 2021-07-16T15:04:29Z 2022-02-22T10:23:46Z 2021-12 Thesis http://hdl.handle.net/10019.1/124298 en_ZA Stellenbosch University xii, 137 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Glucocorticoids -- Receptors Dimerization Glucocorticoid receptor alpha Glucocorticoids UCTD Spies, Lee-Maine Lorin The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title | The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title_full | The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title_fullStr | The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title_full_unstemmed | The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title_short | The effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| title_sort | effect of glucocorticoid receptor alpha dimerization state on receptor turnover |
| topic | Glucocorticoids -- Receptors Dimerization Glucocorticoid receptor alpha Glucocorticoids UCTD |
| url | http://hdl.handle.net/10019.1/124298 |
| work_keys_str_mv | AT spiesleemainelorin theeffectofglucocorticoidreceptoralphadimerizationstateonreceptorturnover AT spiesleemainelorin effectofglucocorticoidreceptoralphadimerizationstateonreceptorturnover |