Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study (2 x 2 factor design) describes an investigation into the physiological interaction between the peripheral endocrine and cytokine systems after the organism has been exposed to psychological stress. An in vivo rodent model with two interventions was used: (1) mild psychological stress (immobilisation for 2 hours per day, for 4 days); (2) an antiinterleukin (IL)-6-antibody injection. Thirty-nine male Wi star rats were divided into 4 groups and given either the antibody (CA, control antibody) or stress (IP, immobilisation placebo), or both (IA, immobilisation antibody), or neither (CP, control placebo). Antibody and placebo (saline) were injected intraperitoneally. Differences between groups for the following parameters were determined in blood or metabolic tissues, viz. skeletal muscle and liver: 1) corticosterone concentrations, 2) glucocorticoid receptor (GR) binding capacity and 3) activities of metabolic enzymes, tyrosine aminotransferase (TAT) and glutamine synthetase (GS). Groups lP and lA showed a significant loss in body mass (CP vs. lP, p<O.01; CA vs. lA, p<O.001), indicating a main effect of stress. The corticosterone concentrations of only group lP were significantly elevated compared to that of group CP (CP vs. lP, p<O.01), again indicating a main effect of stress. All three intervention groups (CA, lP, lA) had decreased GR binding capacity, with group lA showing a statistically greater decrease (CP vs. CA, p<O.05; IP vs. IA, p<O.01; CP vs. IP, p<O.001; CA vs. IA, p<O.001), indicating main effects of stress and antibody treatment. In groups IP and IA increased activities of both enzymes (TAT and GS) were measured (main effect of stress), with IA again showing the greatest statistically significant increase for both enzymes. The liver tissue displayed greater sensitivity to the stress and antibody regimes. This study provides the first conclusive in vivo evidence for IL-6 modulation of glucocorticoid action in peripheral tissues in response to mild psychological stress. / AFRIKAANSE OPSOMMING: Hierdie studie (met 'n 2 X 2 faktorontwerp) beskryf 'n ondersoek oor die fisiologiese interaksie tussen die perifere endokrien- en sitokiensisteme in organismes blootgestel aan psigologiese stres. Daar word gebruik gemaak van 'n in vivo-rotmodel met twee intervensies: (1) matige psigologiese stres (immobilisering vir 2 uur per dag vir 4 dae); (2) 'n anti-interleukin (IL)-6-antiliggaam inspuiting. Nege-en-dertig manlike Wistar rotte is in vier groepe verdeel en het óf antiliggaam (CA, antiliggaam kontrole), óf stres (IP, immobilisasie placebo), óf beide stres en antiliggaam (lA, immobilisasie antiliggaam) of geen behandeling ontvang (CP, placebo kontrole). Die antiliggaam- en placebo (soutoplossing)- inspuitings is intraperitoneaal toegedien. Verskille tussen die groepe van die volgende parameters, in metaboliese weefsels (skeletspier en lewer), was bepaal: 1) kortikosteroon konsentrasies, 2) glukokortikoïed reseptor (GR) bindingskapasiteit en 3) aktiwiteite van die metaboliese ensieme, tirosien aminotransferase (TAT) en glutamien sintetase (GS). Groepe IP en IA het 'n beduidende afname in gewig getoon (CP vs. IP, p<O.01;CA vs. IA, p<O.001), wat 'n hoof-effek van stres aandui. Die kortikosteroon konsentrasies van slegs IP het beduidend toegeneem in vergelyking met CP (CP vs. IP, p<O.01),wat weereens 'n hoof-effek van stres aandui. AI drie intervensiegroepe (CA, IP, IA) het verlaagte GR bindingskapasiteit getoon, met lA wat 'n groot statistiese afname getoon het (CP vs. CA, p<O.05; IP vs. IA, p<O.01;CP vs. IP, p<O.001;CA vs. IA, p<O.001),wat hoof-effekte van beide stres en antiliggaam-behandeling aandui. In groepe IP and IA is toenames in beide ensiemaktiwiteitvlakke (TAT en GS ensieme) getoon (hoof-effek van stres), met IA wat weereens die grootste toename gewys het. Die lewer het ook verhoogde sensitiwiteit tot die stres- en antiliggaamregimente. Hierdie studie lewer die eerste daadwerklike in vivo bewyse vir IL-6 modulering van glukokortikoïedaksie in perifere weefsels na reaksie op psigologiese stres.

Thesis (PhD)--Stellenbosch University, 2011. / See also the post-print version of the article that was published from the PhD - http://hdl.handle.net/10019.1/19557 / ENGLISH ABSTRACT: Glucocorticoid receptor (GR) levels, which modulate the response to glucocorticoids (GCs), vary between tissues and individuals and are altered by physiological and pharmacological effectors. In this study we set out to investigate the effects and implications of differences in GR concentration. Firstly, we established conditions that resulted in three statistically different GR populations in transiently transfected COS-1 cells. We demonstrated, using whole cell saturation ligand binding experiments, that high levels of wild type GR, but not of dimerization deficient GR, exhibited positive cooperative ligand binding with a concomitant increased ligand binding affinity. Furthermore, we established, through co-immunoprecipitation and fluorescent resonance energy transfer, that ligand independent dimerization correlates with positive cooperative ligand binding. This is the first time that positive cooperative ligand binding and increased ligand binding affinity have been explicitly correlated and linked to increased ligand independent dimerization of the GR. The downstream consequences of variation in GR concentration and dimerization included modulation of GR import and export rates, as investigated through live cell as well as immunofluorescent analysis. Furthermore, the nuclear distribution of GR was also influenced by GR dimerization. The major function of the GR is as a transcription factor, which mediates the response to GCs via activation or repression of genes. We have revealed direct influences of GR concentration and dimerization in a number of promoter reporter assays as well as in the transactivation of an endogenous gene. Specifically, cooperative ligand binding was found to be responsible for the GR level dependent potency shift in transrepression of an NF B containing promoter reporter construct via dexamethasone and the shift in the bio-character of Compound A, a dissociative GR agonist. Transactivation potency of dexamethasone as well as the partial agonist bio-character of medroxyprogesterone and mifepristone via a multiple GRE containing promoter reporter construct were influenced directly by cooperative ligand binding. Dimerization of the GR was shown to be crucial for ligand dependent transactivation of a single GRE containing promoter reporter construct, while ligand independent transactivation of both single and multiple GRE containing constructs was significantly increased due to an increase in GR concentration. The endogenous GC responsive glucocorticoid induced leucine zipper (GILZ) gene demonstrated significant ligand independent transactivation at GR levels, which displayed ligand independent dimerization. An increase in GR concentration resulted in an increase in efficacy through all promoter reporter constructs as well as the endogenous GILZ gene. Positive cooperative binding and the concomitant increase in ligand binding affinity to the GR at high levels may be a crucial factor in determining both the efficacy and potency of the GC response. Considering the significant differences in GR concentrations expressed by different tissues and by individuals within the same tissue, our findings may explain the interindividual as well as tissue specific responses to GC treatment and suggest an important mechanism of action through which the GR is primed to responsed to subsaturating GC concentrations and displays a significant level of ligand independent activity. / AFRIKAANSE OPSOMMING: Glukokortikoïed reseptor (GR) vlakke, wat die gedrag van glukokortikoïede (GCs) moduleer, wissel tussen weefsels en onder individue en word verander deur fisiologiese en farmakologiese effektore. In hierdie studie ondersoek ons die gevolge en implikasies van verskille in GR konsentrasie. Eerstens het ons die kondisies vasgestel wat benodig word om drie statisties verskillende GR populasies te vestig in kortstondige getransfekteerde COS-1 selle. Ons het getoon, met behulp van die heel sel versadigings ligand bindings eksperimente, dat hoë vlakke van wilde-tipe GR, maar nie van dimeriserings gebrekkige GR, positiewe koöperatiewe ligand binding, met 'n gepaardgaande toename in ligand bindings affiniteit, toon. Verder het ons bevestig, deur ko-immunopresipitasie en fluoressente resonansie energie-oordrag, dat ligand onafhanklike dimerisering korreleer met positiewe koöperatiewe ligand binding. Dit is die eerste keer dat positiewe koöperatiewe ligand binding en verhoogde ligand bindings affiniteit uitdruklik gekorreleer en gekoppel word aan verhoogde ligand onafhanklike dimerisering van die GR. Die daarop nagevolge van variasie in GR konsentrasie en dimerisering sluit in modulasie van die invoer en uitvoer tempo van die GR, soos ondersoek deur lewendige sel sowel as immunofluorescente analise. Verder is die verspreiding van die GR in die kern ook beïnvloed deur GR dimerisering. Die belangrikste funksie van die GR is as 'n transkripsie faktor, wat die respons van GCS bemiddel via aktivering of onderdrukking van gene. Ons het die direkte invloed van GR konsentrasie en dimerisering in 'n aantal promotor verslaggewer essais sowel as in die transaktivering van endogene gene onthul. Spesifiek, is gevind dat koöperatiewe ligand binding verantwoordelik is vir die GR vlak afhanklike verskuiwing in transrepressie potensie van 'n NF B bevattende promotor verslaggewer konstruk via deksametasoon en die verskuiwing van die biokarakter van verbinding A,' dissosiatiewe GR agonis. Transaktiverings potensie van deksametasoon, asook die gedeeltelike agonis bio-karakter van medroksie-progesteroon en mifepristoon, via 'n veelvoudige GRE bevattende promotor verslaggewer konstruk is direk beïnvloed deur koöperatiewe ligand binding. Dimerisering van die GR is getoon om deurslaggewend vir ligand afhanklike transaktivering van 'n enkele GRE bevattende promotor verslaggewer konstruk te wees, terwyl ligand onafhanklike transaktivering van beide enkel-en veelvoudige GRE bevattende konstrukte aansienlik toegeneem het as gevolg van toename in GR konsentrasie. Die endogene GC responsiewe glukokortikoïed geïnduseerde leusien rits (GILZ) gene het beduidende ligand onafhanklike transaktivering gedemonstreer op GR vlakke wat ligand onafhanklike dimerisering toon. 'n toename in GR konsentrasie het gelei tot toename in die effektiwiteit van al die promotor verslaggewer konstrukte, sowel as die endogene GILZ gene. Positiewe koöperatiewe ligand binding en die gepaardgaande toename in ligand bindings affiniteit van die GR by hoë vlakke kan 'n belangrike faktor wees in die bepaling van sowel die effektiwiteit as die potensie van die GC respons. As die aansienlike verskille in GR konsentrasies van verskillende weefsels en tussen verskillende individue in dieselfde weefsel in ag geneem word, kan ons bevindings die inter-individuele sowel as weefsel spesifieke response op GC behandeling verduidelik en stel dit 'n belangrike meganisme van aksie voor waardeur die GR voorberei word om op sub-versadigings konsentrasies van GC te reageer deur 'n beduidende vlak van ligand onafhanklike aktiwiteit te toon.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • the influence of a methanolic extract of unfermented Rooibos and five major Rooibos flavonoids, aspalathin, nothofagin, rutin, orientin and vitexin, on the activities of key adrenal steroidogenic enzymes - cytochrome P450 17β- hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase • the development of a novel UPLC-MS/MS method for the separation and quantification of 21 adrenal steroid metabolites; • the influence of Rooibos and aforementioned flavonoids on adrenal steroid hormone production in H295R cells - a human adrenal carcinoma cell line expressing the enzymes catalysing the production of mineralocorticoids, glucocorticoids and adrenal androgens, assayed under both basal (normal) and forskolin-stimulated (stressed) conditions; • the influence of Rooibos on the inter-conversion between cortisol and cortisone by 11βHSD1 and 11βHSD2 expressed in CHO-K1 cells; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male Wistar rats; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male and female human test subjects at risk for developing cardiovascular disease. (3βHSD2), cytochrome P450 21-hydroxylase (CYP21A2) and cytochrome P450 11β-hydroxylase (CYP11B1), expressed in non-steroidogenic COS-1 cells; / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: • die invloed van metanoliese ekstrakte van ongefermenteerde Rooibos en vyf van die hoof flavonoïedverbindings in Rooibos, aspalatien, notofagien, rutien, oriëntien en viteksien, op die aktiwiteite van ensieme wat steroïedbiosintese in die bynier kataliseer – sitochroom P450 17α-hidroksilase/17,20-liase (CYP17A1), 3β-hidroksisteroïed dehidrogenase (3βHSD2), sitochroom P450 21-hidroksilase (CYP21A2) en sitochroom P450 11β-hidroksilase (CYP11B1), uitgedruk in nie-steroïed produserende COS-1 selle; • die ontwikkeling van ‘n geskikte UPLC-MS/MS metode vir die skeiding en kwantifisering van 21 steroïedmetaboliete in die bynier; • die invloed van Rooibos en die bg. flavonoïede op steroïedproduksie in H295R selle – ‘n menslike bynier kanker sellyn gekenmerk deur die ekspressie van die steroidogeniese ensieme wat die produksie van mineralokortikoïede, glukokortikoïede en bynierandrogene kataliseer, geanaliseer onder beide basale (normale) en forskoliengestimuleerde (gestresde) kondisies; • die invloed van Rooibos op die omeenskakeling tussen kortisol en kortisoon deur 11βHSD1 and 11βHSD2 in CHO-K1 selle; • die invloed van Rooibosinname op vlakke van sirkulerende steroïed hormone en relatiewe verhoudings in die bloed van manlike Wistarrotte; • die invloed van Rooibosinname op sirkulerende steroïed hormoon vlakke en relatiewe verhoudings in die bloed van mans en vrouens met ‘n hoë risiko vir die ontwikkeling van kardiovaskulêre siektes.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells. / AFRIKAANSE OPSOMMING: Die gonadotropienvrystellings hormoon (GnRH) reseptor is ‘n G-proteïen-gekoppelde reseptor in die pituitêre gonadotrope en is ’n belangrike beheerpunt vir reproduksie. GnRH bind aan die GnRH reseptor (GnRHR) met die gevolg dat follikel stimulerende hormoon (FSH) en luteïeniserende (LH) gesintetiseer en vrygestel word. Die sensitiwiteit van die pituitêre klier vir GnRH kan direk met GnRHR vlakke gekorreleer word. Die muis GnRHR promotor bevat drie cis elemente met bindingssetels vir steroïedogeniese faktor 1 (SF1), naamlik setel 1 (-15/-7), setel 2 (-244/-236) en setel 3 (-304/-296) sowel as ’n aktiveerder proteïen 1 (AP-1) tipe konsensus sekwens (TGAGTCA) in posisie -336/-330. Terwyl setels 1 en 2 en die AP-1 setel voorheen getoon is om by die regulering van transkripsie van die muis GnRHR (mGnRHR) promotor in party sellyne betrokke te wees, is die rol van setel 3 nog nie vantevore bestudeer nie. In hierdie studie is ondersoek of die transkripsie van die mGnRHR geen deur GnRH en glukokortikoïede in die LβT2 gonadotroop pituitêre sellyn gereguleer word, en die rol van setel 3 en die AP-1 setel en hulle binders, deur gebruik te maak van in vitro proteïen-DNA bindings studies en promotor-verslaggewer essais. Die rol wat setel 3 en die AP-1 setel in basale transkripsie van die mGnRHR gene in LβT2 selle gespeel het, was die eerste onderwerp wat in hierdie studie bestudeer is. Lusiferase verslaggewer plasmiede wat die eerste 600 bp van die mGnRHR promotor bevat het en waarin setel 3 en die AP-1 setels óf wilde tipe óf gemuteer was, is gebruik. Two konstrukte is vanaf die wilde tipe konstruk berei, naamlik wilde tipe (LG), ’n setel 3 mutant (m3) en ’n AP-1 mutant (mAP-1). Transfeksie van LG, m3 en mAP-1 plasmiede in LβT2 selle is deurgevoer om te bepaal wat die effek van hierdie mutasies op die basale ekspressie van die mGnRHR gene was. Mutasie van setel 3 het ’n 1.5-voudige toename in die transkripsionele aktiwiteit van die mGnRHR promotor tot gevolg gehad. Dit suggereer dat setel 3 ’n rol in die inhibisie van die basale transkripsievlakke van die mGnRHR promotor in LβT2 selle speel. Mutasie van die AP-1 setel het tot ‘n 50% verlaging in basale transkripsievlakke van die mGnRHR promotor in LβT2 selle gelei. Dit suggereer dat die AP-1 setel betrokke is in die positiewe bemiddeling van die basale transkriptionele respons van die GnRHR promotor in LβT2 selle. Eksperimente wat gemik was om die meganisme van die cis-elemente (setel 3 en die AP-1 setel) op die mGnRHR promotor te verklaar, asook om die rol van proteïen kinase A (PKA) paaie, proteïene daarby betrokke en die effek van varieende dosisse vir verskillende tye van GnRH, sowel as die oorekspressie van PKA en die SF-1 proteïen, is deurgevoer. Dit is gevind dat setel 3 en die AP-1 setel nie betrokke by die GnRH respons is nie. Die resultate suggereer dat setel 3 gedeeltelik betrokke is by die PKA respons van LβT2 selle. Setel 3 kan SF-1 proteïen bind soos getoon deur kompeterence elektroforetiese mobiliteits verskuiwings essais (EMSA). As EMSA’s deurgevoer is op die AP-1 setel is bevind dat die c-Fos proteïen nie betrokke is in die aktivering van die AP-1 setel nie. ’n Faktor is gevind om aan die AP-1 setel te bind wat nie ’n intakte AP-1 setel vereis het nie, wat gesuggereer het dat dit die c-Jun proteïen kan wees wat aan die AP-1 setel onder basale omstandighede bind. ’n Ander area wat ondersoek is, is of die GnRHR promotor gereguleer kan word deur deksametasoon (dex) óf via die AP-1 setel óf via setel 3. ’n Dosis en tyds-afhanklike toename in promotor aktiwiteit is waargeneem met dex. ’n Vereiste vir hierdie effek blyk om die teenwoordigheid van setel 3 en die AP-1 setel te wees, soos aangetoon deur die totale verlies aan response as hierdie twee setels individueel gemuteer is, en wat weereens in ooreenstemming met die funksionele interaksie tussen setel 3 en die AP-1 setel in LβT2 selle is.

published_or_final_version / Medicine / Master / Master of Philosophy

Die akute Graft-versus-Host Erkrankung (acute Graft-versus-Host disease, aGvHD) ist eine pathogene Folgeerscheinung der hämatopoetischen Stammzell-transplantation. Zur Therapie werden zumeist Glukokortikoide eingesetzt, die sich durch ihre anti-inflammatorische und immunsuppressive Wirkung, insbesondere auf die an der aGvHD beteiligten alloreaktiven T Zellen und Antigenpräsentierenden Zellen, auszeichnen. Die chronische Verabreichung hochdosierter Glukokortikoide ist jedoch mit zahlreichen Nebenwirkungen verbunden, und kann zum Auftreten Glukokortikoid-resistenter Verlaufsformen führen. Aus diesem Grund wurden in der vorliegenden Arbeit zwei Ansätze verfolgt, die als Grundlage für die Entwicklung verbesserter Verfahren zur Therapie der aGvHD dienen könnten. Zuerst wurde die Möglichkeit eines adoptiven Transfers ex vivo aus dem Knochenmark generierter und mittels Glukokortikoiden und IL-4 zum M2-Phänotyp polarisierter Makrophagen analysiert. Die Kombinationsbehandlung zeigte in vitro teils ausgeprägte synergistische Effekte in Hinblick auf die Polarisation der Zellen, insbesondere auf Ebene der Genexpression. Nichtsdestotrotz konnte nach Transfer der anti-inflammatorischen Makrophagen kein therapeutischer Effekt im Mausmodell der aGvHD beobachtet werden. Als mögliche Erklärung hierfür kommt insbesondere der Verlust der M2-Polarisation in vivo in Frage. Als alternative Strategie wurden daraufhin verschiedene zelltypische Wirkungen der Glukokortikoide in der aGvHD untersucht, um auf diesem Weg Angriffspunkte für eine zielgerichtetere Therapie zu identifizieren. Hierbei zeigte sich, dass eine überschießende Sekretion von IL-6 durch Zellen des Empfängers einen fulminanten Krankheitsverlauf zur Folge hat. Diese Wirkung konnte durch Applikation eines neutralisierenden Antikörpers verhindert werden, was durch einen geringeren Abfall der Körpertemperatur und Blutglukose begleitet war. Durch Verwendung konditionaler knock-out Mäuse konnten weiterhin die Makrophagen als einer der zentralen Zelltypen definiert werden, welche für die Glukokortikoid-Wirkung im Empfänger verantwortlich sind. Zuletzt ergaben Untersuchungen, dass Glukokortikoide auch wesentlich auf die transplantierten allogenen T-Zellen einwirken. Hier zeigte sich, dass sie vor allem die Zytotoxizität der CD8+-T-Zellen vermindern und auf diese Weise einem fulminanten Verlauf der aGvHD entgegen wirken. Zusammengefasst zeigen die Ergebnisse der Arbeit, dass eine Therapie der aGvHD mittels M2-Makrophagen nur dann möglich sein wird, wenn es gelingt, die Stabilität deren Phänotyps in vivo zu erhalten. Alternativ erscheinen jedoch auch Strategien vielversprechend, die auf einer gezielten Beeinflussung einzelner Zelltypen durch hoch-spezifische Glukokortikoide beruhen, da sich auf diese Weise möglicherweise Nebenwirkungen und Resistenzen umgehen lassen.

No description available.

Glucocorticoids (GC) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic BCL-2 family-regulated pathway. It has been shown that BIM (BCL-2 interacting mediator of cell death), a BH3-only pro-apoptotic protein, is up-regulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia (ALL) cells. Furthermore, BIM is inactivated by extracellular signal-regulated kinase (ERK)-mediated phosphorylation. We therefore hypothesized co-treatment with Dex and MEK/ERK inhibitors would promote apoptosis in ALL cells through BIM up-regulation and activation. We show here that a MEK inhibitor, PD184352 synergistically enhances Dex lethality in CCRF-CEM (T-ALL) cells. Co-treatment with Dex and PD184352 results in BIM accumulation. Down-regulation of BIM by short-hairpin RNA in CCRF-CEM cells suppressed apoptosis by Dex/PD184352 co-treatment. In contrast, another BH3-only protein, BAD is dispensable. Thus, BIM is a critical molecule in this regimen, and targeting BIM by drugs combination could be effective on ALL and possibly other malignancies.

11-hydroxysteroid dehydrogenase (11HSD1) is an oxidoreductase which catalyzes conversion of inactive 11-oxo steroid derivatives into active 11-hydroxy forms. 11HSD1 elevates intracellular level of active glucocorticoid (GC) hormones: cortisol in human tissues and corticosterone in rodents, therefore local level of active GCs can be set independently from systemic secretion driven by hypothalamo-pituitary-adrenal axis (HPA axis). Chronic systemic excess of GCs results in development of Cushing's syndrome which is characterised by central obesity and other metabolic disturbances. Despite normal serum levels of GCs, the patients with idiopathic obesity also develop metabolic syndrome. It was suggested that GCs could be elevated locally in target tissues due to enhanced 11HSD1 activity. This hypothesis was confirmed in transgenic rodent models. Prague hereditary hypertriglyceridemic (HHTg) rats represent a non-obese model of metabolic syndrome without genetic manipulations or specific mutations. The strain was bred by cross-mating of Wistar rat individuals with elevated serum levels of triglycerides (TGs). The strain exhibit hypertriglyceridemia and hypertension. When kept on high carbohydrate diet HHTg rats exhibit alterations in glucose homeostasis. Since there are no data that would describe...

L’action physiologique des glucocorticoïdes (GC) est de mobiliser certaines ressources de l’organisme pour s’adapter à des changements d’origine endogène ou exogène susceptibles de perturber l’homéostasie de l’organisme. Des facteurs métaboliques/nutritionnels modifient l’intensité d’action des GC. Ils agissent au niveau i) de l’activation de l’axe corticotrope, ii) de leur biodisponibilité des GC (régulation « pré-récepteur »), iii) de l’activation transcriptionnelle des récepteurs des GC (régulation « post-récepteur »). L’objectif général de ce travail repose sur l’exploration du rôle de certains facteurs métaboliques/nutritionnels dans la modulation pré- et post-récepteur de l’action des GC sur l’organisme. Dans une approche clinique, notre attention s’est tout d’abord focalisée sur le rôle de l’équilibre diabétique et/ou l’état inflammatoire des patients atteints de diabète de type I et le métabolisme pré-récepteur du cortisol. Par l’étude en spectrométrie de masse des métabolites du cortisol, nous montrons une augmentation significative de l’activité de la hydroxystéroïde-déshydrogenase 1, principale enzyme de régénération intracellulaire du cortisol. Cette augmentation est corrélée à des marqueurs de l’inflammation chez les enfants diabétiques. Ces résultats suggèrent un lien entre le diabète et l’existence d’une inflammation à bas bruit et l’augmentation de l’exposition cellulaire aux GC. Dans une approche expérimentale, nous nous sommes ensuite intéressés à l’action de l’acide rétinoïque all trans (atAR), métabolite actif de la vitamine A, sur l’activité transcriptionnelle des GC. Nous avons choisi un modèle in vitro de cellules hippocampiques en raison d’effets contrastés de l’atAR et des GC sur les fonctions mnésiques in vivo. Nous observons une interaction entre les voies de signalisation transcriptionnelle de l’atAR et des GC sur leurs propres récepteurs et sur des protéines de la plasticité synaptique. Par ailleurs, l’atAR est responsable de modifications de la phosphorylation du récepteur aux GC altérant ainsi ses fonctions transcriptionnelles. Enfin atAR et GC modifient différemment l’organisation du cytosquelette d’actine sans modification transcriptionnelle ou traductionnelle. La compréhension du rôle de certains facteurs environnementaux dans la signalisation des GC pourrait permettre de réduire certains des effets délétères du stress. L’utilisation de certains nutriments, vitamine A par exemple, pourrait atténuer certaines conséquences d’un tonus glucocorticoïde excessivement prolongé. / Rôles to mobilize body resources and to adapt to endogenous or exogenous changes that might disrupt the homeostasis of the body. Nutritional & metabolic factors may modify the intensity of GC action in: i) the activation of the corticotrope axis and their secretion by the adrenals, ii) their bioavailability ("pre-receptor" regulation), iii) the transcriptional activation of their receptors ("post-receptor" regulation). The main target of this work is to explore the role of some metabolic/nutritional endogenous or exogenous factors in modulating pre- and post-receptor action of GC. Our attention first focused on the role of diabetes and the related inflammation in patients with type I diabetes, and pre-receptor metabolism of cortisol. We showed that a significant increase in the activity of hydroxysteroid dehydrogenase 1, the main enzyme of intracellular cortisol regeneration, is correlated with markers of inflammation in diabetic children. This suggests a link between diabetes and the low-level chronic inflammation and increased cellular exposure to GC . Then, we focused on the action of all-trans retinoic acid (atRA), the active metabolite of vitamin A on the transcriptional activity of GC. We used an in vitro model of hippocampal cells as GC and atRA have contrasted effects on mnesic processes in vivo. We observed a transcriptional interaction between the GC and retinoic pathways targeting their receptors and genes involved in neuronal plasticity. atRA also affects the phosphorylation of the GC receptor and modifies its transcriptional activity. Lastly, both atRA and GC affect cellular organisation of actin cytoskeleton. The knowledge acquired by studying the action of nutritional molecules on GC action could be used to easily reduce the deleterious effects of GC in chronic stress. Clinical studies have started in this direction.