Previous studies in our laboratory have demonstrated that consumption by mammals and birds of a low zinc diet is associated with a diminished output of gene products in response to either endogenous or exogenous estrogen challenge. A recent discovery of the highly conserved zinc finger (or twist) motif in the steroid receptor superfamily suggests that zinc may play a broad role in assuring swift and timely adaptive, regulatory, and developmental responses to this entire class of lipophilic substances. To determine the effect of zinc status at the molecular level, a zinc deficit was created in cell cultures and glucocorticoid regulated and constitutive gene expression monitored. Using a cell impermeable zinc specific chelator, diethylenetriaminepentaacetic acid (DTPA), a zinc deficient state in HeLa cells was created. This deficit was quantified by a fluorescence assay using N-(6-methoxy-8-quinolyl 1)-p-toluenesulfonamide (TSQ) to measure exchangeable zinc and induced coupled plasma analysis to measure total zinc. The 83% reduction in total cellular zinc, corresponding to a 65% decrease in TSQ fluorescence was rapid, nearly maximal within 4 hours, and without significant loss of viability over 72 hours as tested by dye permeability. This was prevented by addition of zinc ions. This cellular system was used to study the effects of zinc deficits separately upon the individual steps in steroid-directed gene expression and constitutive protein synthesis. Differentiation between the effect of zinc supply upon hormone mediated and constitutive gene expression in HeLa cells was accomplished through transient transfections. By examining the expression of a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter gene as compared to CAT activity derived from glucocorticoid-insensitive reporter plasmid, it was determined that glucocorticoid directed expression was reduced to a greater extent than constitutive (88% vs. 15%) when zinc concentrations were reduced 80%. Furthermore, neither the binding affinities of receptors for dexamethasone nor nuclear translocation was altered by zinc concentrations. However, glucocorticoid receptor concentrations were reduced by 50%. This reduction in receptor number is sufficient to produce decreases in gene induction of the magnitude observed. Based on these results in conjunction with previous studies in our laboratory we propose that the most sensitive need for zinc may be in permitting rapid induction of newly synthesized enzymes as seen during hormone responses, rather than serving as a cofactor of metalloenzymes involved in constitutive synthesis. / Ph. D.

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

In zebrafish, transgenic labeling approaches, robust regenerative responses and excellent in vivo imaging conditions enable precise characterization of immune cell behavior in response to injury. Here, we monitored osteoblast-immune cell interactions in bone, a tissue which is particularly difficult to in vivo image in tetrapod species. Ablation of individual osteoblasts leads to recruitment of neutrophils and macrophages in varying numbers, depending on the extent of the initial insult, and initiates generation of cathepsin K+ osteoclasts from macrophages. Osteoblast ablation triggers the production of pro-inflammatory cytokines and reactive oxygen species, which are needed for successful macrophage recruitment. Excess glucocorticoid signaling as it occurs during the stress response inhibits macrophage recruitment, maximum speed and changes the macrophage phenotype. Although osteoblast loss is compensated for within a day by contribution of committed osteoblasts, macrophages continue to populate the region. Their presence is required for osteoblasts to fill the lesion site. Our model enables visualization of bone repair after microlesions at single-cell resolution and demonstrates a pro-osteogenic function of tissue-resident macrophages in non-mammalian vertebrates.

Atherosclerotic and restenotic lesions develop as a result of an excess inflammatory response to vascular injury. Glucocorticoid hormones have widely-recognised anti-inflammatory and anti-proliferative properties which appear to make them ideal candidates for inhibition of vascular lesion development. Indeed, administration of glucocorticoids to experimental animals does inhibit the growth of vascular lesions in some models. In addition, glucocorticoids are currently being trialled clinically as anti-restenotic agents. However, glucocorticoid excess in patients, either as a result of Cushing’s syndrome or chronic steroid therapy, is associated with enhanced CVD risk. Therefore, the effects of glucocorticoids on vascular lesion development remain imperfectly understood. The overall objective of these studies was to explore the influence of endogenous and exogenous glucocorticoids on vascular lesion development using murine models of atherosclerosis (ApoE-/- mice fed a “western” diet) and neointimal hyperplasia (wireinduced femoral artery injury). The work described in this thesis addresses the hypothesis that glucocorticoids are pro-atherogenic, yet anti-restenotic. Mice were bilaterally adrenalectomised to investigate the role of endogenous glucocorticoids on vascular lesion development. Removal of the adrenal glands had no influence on atherogenesis or neointima development. The influence of exogenous glucocorticoids on lesion development was assessed by orally administering dexamethasone (0.1 or 0.8mg/kg/day). Atherosclerotic lesion burden was augmented by dexamethasone administration. Conversely, fibro-proliferative neointimal proliferation was inhibited by dexamethasone. However, this effect was obscured by thrombotic lesion development. It was proposed that this thrombotic effect is attributable to increased plasminogen activator inhibitor-1 (PAI-1), which was tested using PAI-1 deficient mice. Although PAI-1 was found to mediate the systemic pro-thrombotic effect of dexamethasone, it is not required for the enhanced development of thrombotic lesions at the site of intra-luminal injury. These results suggest that physiological levels of endogenous glucocorticoids play a limited role in vascular lesion development. Conversely, although exogenous glucocorticoids inhibit fibro-proliferative intimal hyperplasia, they appear to have significant detrimental influences on lesion development, augmenting atherosclerosis and inducing thrombotic neointimal lesion formation following vascular injury. Further research is therefore required to improve the cardiovascular outcome of patients requiring glucocorticoid therapy and for the use of glucocorticoids as antirestenotic agents.

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, 2013. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)), designed to mimic the actions of the endogenous hormone progesterone (Prog), are extensively used by women as contraceptives and in hormone replacement therapy (HRT). A number of reports have indicated that these synthetic progestins affect immune function in the female genital tract thereby increasing the risk of acquiring sexual transmitted infections. Despite these findings, very little is known about their mechanism of action at the cellular level, in particular their steroid receptor-mediated effects on cytokine gene expression. In the first part of this thesis, the effect of Prog, MPA and NET-A on the expression of the endogenous pro-inflammatory cytokine gene, interleukin (IL)-12p40, and anti-inflammatory cytokine gene, IL-10, was investigated in a human ectocervical epithelial cell line, Ect1/E6E7. Quantitative realtime PCR (qPCR) showed that all three ligands significantly upregulated the tumor necrosis factor alpha (TNF )-induced IL-12p40 gene expression, while IL-10 gene expression was downregulated. Moreover, by reducing the glucocorticoid receptor (GR) levels with siRNA, these effects were shown to be mediated by the GR. A more detailed investigation into the molecular mechanism of the progestogen-induced upregulation of IL-12p40 gene expression, using chromatin immunoprecipitation (ChIP), siRNA, co-immunoprecipitation and re-ChIP analyses, showed that the progestogen-bound GR is recruited to the CCAAT enhancer binding protein (C/EBP)- regulatory element of the IL-12p40 promoter, most likely via an interaction with the transcription factor C/EBP . Similar experiments for the progestogen-induced downregulation of IL-10 gene expression showed that the progestogen-bound GR is recruited to the signal transducer and activator of transcription (STAT)-3 regulatory element of the IL-10 promoter, most likely via an interaction with the transcription factor STAT-3. The second part of this study elucidated the influence of the HIV-1 accessory viral protein R (Vpr) on progestogen-induced regulation of IL-12p40, IL-12p35 and IL-10 in the Ect1/E6E7 cell line. Results showed that in these cells, the overexpression of Vpr significantly modulated the effects of Prog, MPA and NET-A on the mRNA expression of IL- 12p40 and IL-10, while only the NET-A effect was modulated on IL-12p35. Moreover, reducing the GR protein levels by siRNA suggested that the GR is required by Vpr to mediate its effects. Taken together, these results show that Prog, MPA and NET-A promote the pro-inflammatory milieu in the ectocervical environment, and that during HIV-1 infections, this milieu is modulated. Furthermore, the results suggest that the use of MPA or NET in vivo may cause chronic inflammation of the ectocervical environment, which may have important implications for ectocervical immune function, and hence susceptibility to infections such as HIV-1. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), ontwerp om die funksies van die natuurlike hormone progesteroon (Prog) na te boots, word wêreldwyd deur vroue as voorbehoedmiddels sowel as vir hormoon vervangingsterapie (HVT) gebruik. Daar is verskeie aanduidings dat hierdie sintetiese progestiene die immuunfunksie in die vroulike geslagskanaal kan beïnvloed en ook die moontlike vatbaarheid van seksueel oordraagbare infeksies kan verhoog. Ten spyte hiervan, is baie min bekend oor hulle meganisme van werking op ‘n molekulêre vlak, veral in die besonder hul effek op sitokinien geenuitdrukking. Die effek van Prog, MPA en NET-A op die geenuitdrukking van ’n endogene pro-inflammatoriese sitokinien, interleukin (IL)-12, en ’n anti-inflammatoriese sitokinien, IL-10, asook die onderliggend meganisme van werking, in ’n menslike ektoservikale sellyn, Ect1/E6E7, is in die eerste deel van hierdie studie ondersoek. Kwantitatiewe “realtime” polimerisasie ketting reaksie (PKR) het getoon dat al drie die ligande die tumor nekrosis faktor alfa (TNF- )-geïnduseerde IL-12p40 geenuitdrukking opreguleer en IL-10 geenuitdrukking onderdruk. Verder is gevind dat induksie van IL-12p40 en inhibisie van IL-10 deur Prog, MPA en NET-A deur die glukokortikoïed reseptor (GR) gedryf word, aangesien volledige opheffing van die effekte op hierdie sitokinien gene waargeneem is wanneer die GR proteïen vlakke deur middel van kort inmengende ribonukleïensuur (siRNS) verminder is. 'n Meer beskrywende ondersoek in die molekulêre meganisme is uitgevoer deur gebruik te maak van chromatien immunopresipitasie (ChIP), siRNS, mede-immunopresipitasie en her-ChIP analises. Hierdie resultate het voorgestel dat die progestogeen (Prog en die sintetiese progestiene)-gebonde GR tot die CCAAT verbeterende bindings protein (C/EBP)- regulatoriese element van die IL-12p40 promotor betrek word en dat die transkripsie faktor C/EBP benodig word om transkripsie van die IL-12p40 geen te aktiveer. Met betrekking tot IL-10, het die resultate voorgestel dat die progestogeen-gebonde GR tot die sein transduksie en aktiveerder van transkripsie (STAT)-3 regulatoriese element van die IL-10 promotor betrek word en dat die transkripsie faktor STAT-3 benodig word om transkripsie van die IL-10 geen te onderdruk. Die tweede deel van die studie het die invloed van die MIV-1 aksesorale virale proteïen R (Vpr) op sitokinien geenuitdrukking, spesifiek die progestogeen-geïnduseerde regulering van IL-12p40, IL-10 en IL-12p35, in die Ect1/E6E7 sellyn ondersoek. Resultate het getoon dat ooruitdrukking van Vpr in hierdie sellyn die effekte van Prog, MPA en NET-A op die mRNS uitdrukking van IL-12p40 en IL-10, en slegs die NET-A effek op IL-12p35, aansienlik moduleer. Vermindering van die GR proteïen vlakke deur middel van siRNS het getoon dat Vpr die GR benodig om hierdie veranderinge mee te bring. In samevatting, die resultate van hierdie proefskrif stel voor dat Prog, MPA en NET-A die pro-inflammatoriese milieu in die ektoservikale omgewing bevorder, en dat hierdie milieu gedurende MIV-1 infeksies verander. Verder, die resultate van hierdie studie impliseer dat die gebruik van MPA en NET in vivo nadelige lokale immuunonderdrukkende effekte mag hê wat kan lei tot kroniese inflammasie van die ektoservikale omgewing en ‘n moontlike verhoging in die vatbaarheid van infeksies soos MIV-1.

Thesis (PhD (Med))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The increase in the prescription and use of glucocorticoids (GCs) to treat various diseases and resulting decrease in bone density and development of osteoporosis is of growing concern. Glucocorticoid-induced osteoporosis (GCIO) is a relatively under-researched disease with the mechanism by which GCs affect bone metabolism not yet fully delineated. This holds especially true for the early events in bone development. The negative effects of GCs are predominantly seen in osteoblasts, the cells responsible for bone formation, in that GCs diminish both the numbers and function of osteoblastic cells. Osteoblast precursor cell proliferation is crucial to ensure the existence of a healthy pool of osteoblastic cells needed to form new bone after bone resorption by osteoclasts. Previously, it was shown that GCs reduce the proliferation of immortalised osteoblastic cell lines. In addition, early immortalised preosteoblasts were more sensitive to GCs than their mature counterparts. However, these cells have corrupted cell cycles; therefore, primitive primary mesenchymal stromal cells (MSCs) were used in this study to examine the effect of GCs on the mitogen-induced proliferation of early osteoblast precursor cells (naïve MSCs and preosteoblasts) using the synthetic GC, dexamethasone (Dex). Mitogenic conditions established for naïve rat mesenchymal stromal cells (rMSCs) indicated that mild (5% FBS) stimulation is sufficient to induce proliferation, whereas a higher FBS concentration (20% FBS) was mitogenic in primary preosteoblasts. It was also found that pharmacological doses of Dex drastically decreased the mitogen-induced proliferation of both naïve rat MSCs (rMSCs) and preosteoblasts. Mitogen-activated protein kinase (MAPK) signalling pathways, such as ERK1/2, govern cell proliferation. GCs have been shown to decrease the activity of ERK1/2, which is associated with decreased proliferation in osteoblastic cells. In the present study, western blot analysis showed that Dex reduced the proliferation-associated shoulder of the ERK1/2 activity profile in both naïve rMSCs and preosteoblasts. Moreover, the ERK1/2 signalling pathway was shown to be essential for mitogen-stimulated growth of naïve rMSCs and preosteoblasts as the MEK1/2 inhibitor, U0126, inhibited mitogen-induced proliferation. Using western blot analysis, it was shown that, after mitogen administration, ERK1/2 activity exhibited a typical proliferation profile, which was blocked by U0126. Protein tyrosine phosphatases (PTPs) dephosphorylate and inactivate ERK1/2. Utilising sodium vanadate, an inhibitor of PTPs, in vitro phosphatase assays revealed that PTP activity was the predominant phosphatase activity present in naïve rMSCs and preosteoblast lysates after concomitant mitogen and Dex stimulation. The mRNA of the dual specificity phosphatase, MKP-1, was rapidly (within 30 minutes) upregulated after mitogen and Dex administration in both naïve rMSCs and preosteoblasts. However, the protein expression pattern of MKP-1 did not correspond to the mRNA induction, suggesting that the MKP-1 protein could be subjected to rapid degradation. These findings suggest that MKP-1 could possibly be involved in the GC regulation of mitogen-induced proliferation of early osteoblast precursor cells, but closer investigation is needed to fully elucidate this role. In addition, the involvement of other PTPs should not be excluded and warrants further investigation. During the course of the present study, it was found that strong mitogenic stimulation with 20% FBS led to oncogene-induced senescence (OIS). Flow cytometry analysis revealed the presence of two populations in naïve rMSCs preparations and DNA content analysis was consistent with that of cells undergoing OIS. These results indicated that the more primitive osteoblast precursor cells (naïve rMSCs) are more responsive to mitogens than their mature counterparts (preosteoblasts). In addition, it was found that the magnitude of ERK1/2 activation was increased in naïve rMSC after strong mitogenic stimulation, indicating that naïve rMSCs are still highly sensitive to stimulation with strong mitogens. In summary, these findings show that Dex decreased the proliferation of naïve rMSCs and preosteoblasts concomitantly with a decrease in ERK1/2 activity. In addition, Dex upregulated MKP-1 mRNA, but the same effect was not seen on the MKP-1 protein levels. Therefore, this suggests that PTP/s other than MKP-1 could be responsible for the inactivation of ERK1/2 by Dex, leading to decreased proliferation in naïve rMSCs and preosteoblasts. Further identification of PTPs that regulate osteoblast precursor cell numbers and function could lead to the elucidation of the mechanism through which GCs act to negatively influence bone density. This will improve our insights into the pathogenesis of GCIO and aid in the identification of therapeutic targets which can be exploited to develop new agents to treat osteoporosis. / AFRIKAANSE OPSOMMING: Die toename in voorskrifte en gebruik van glukokortikoïede (GKs) om verskillende siektes te behandel en die gevolglike afname in been digtheid, is kommerwekkend. Glukokortikoïed geïnduseerde osteoporosis (GKIO) is 'n relatief min genavorste siekte waarvan die meganisme waardeur GKs been-metabolisme affekteer nog nie ten volle ontrafel is nie. Dit is veral waar ten opsigte van die vroeë stadia in beenontwikkeling. Die negatiewe uitwerking van GK's word oorwegend in osteoblaste, die selle wat verantwoordelik is vir beenformasie, waargeneem, waar GKs beide die getalle en funksie van osteoblaste verminder. Osteoblast voorloper-sel proliferasie is belangrik vir die handhawing van 'n gesonde poel osteoblastiese selle wat benodig word om nuwe been te vorm na beenresorpsie deur osteoklaste. Daar is gevind dat GKs proliferasie van verewigde preosteoblastiese sellyne verminder en dat jong verewigde preosteoblaste meer sensitief is vir GKs as hul meer volwasse ekwivalent. Die selle se selsiklusse is egter gekorrupteer en daarom was primitiewe primêre rot mesenkiem stromaselle (rMSCs) in hierde studie gebruik om die effek van GKs op mitogeen-geïnduseerde proliferasie van vroeë osteoblasvoorloperselle (naïwe MSC en preosteoblaste) deur die sintetiese GK, deksametasoon (Dex), te bestudeer. Mitogeniese kondisies vir naïwe rMSCs het getoon dat matige (5% FBS) stimulasie voldoende is om proliferasie te induseer, terwyl 'n hoë FBS konsentrasie (20% FBS) mitogenies was in primêre preosteoblaste. Daar is ook gevind dat farmokolgiese dosisse Dex die mitogeen-geïnduseerde proliferasie van beide naïwe rMSCs en preosteoblaste verminder. Die mitogeen-geïnduseerde protein kinase (MAPK) pad beheer selproliferasie. Die ekstrasellulêre gereguleerde kinase pad (ERK1/2) is voorheen as die hoofpad wat MBA 15.4 and MG 63 proliferasie beheer geïdentifiseer. Daar is gewys dat GKs die aktiwiteit van ERK1/2 verlaag en proliferasie van die selle verminder. In die huidige studie het western blot analise gewys dat Dex die proliferasie geassosieerde skoueraktiwiteit van die ERK1/2 aktiwiteitsprofiel in beide naïwe rMSCs en preosteoblaste verminder. Die noodsaaklike rol van ERK1/2 pad in mitogeen-gestimuleerde groei van die selle is bevestig deur die MEK1/2 inhibitor, U0126, wat die mitogeen-geïnduseerde proliferasie geïnhibeer het. Western blot analise het gewys dat die ERK1/2 aktiwiteit na mitogeen toediening 'n tipiese proliferasie profile toon wat deur U0126 geblokkeer word. Protein tirosien fosfatases (PTPs) defosforileer and inaktiveer ERK1/2. In vitro fosfatase bepalings met natrium vanadaat, 'n inhibitor van PTPs, het bevestig dat PTP die predominante fosfatase akitiwiteit is in naïwe rMSCs en preosteoblaste lisate is na gelyktydige mitogeen en Dex stimulasie. Die mRNA van die dubbele spesifisiteits fosfatase, MKP-1, is vinnig (binne 30 minute) opgereguleer is na mitogeen en Dex toediening in beide naïwe rMSCs en preosteoblaste. Die proteinekspressie van MKP-1 het egter nie met die mRNA ekspressie ooreengestem nie, wat suggereer dat die MKP-1protein blootgestel is aan vinnige degradasie. Hierdie bevindings stel voor dat MKP-1 moontlik 'n rol speel in die GC-regulering van mitogeen-geïnduseerde proliferasie van vroeë osteoblast voorloperselle maar verdere ondersoek is nodig om die rol ten volle te verklaar. Die betrokkenheid van ander PTPs moet egter nie uitgesluit word nie en regverdig verdere studie. Die huidige studie het bevind dat sterk mitogeniese-stimulasie met 20% FBS tot onkogene- geïnduseerde selgroeistilte (senescence) (OIS) lei. Vloeisitometriese analise het die teenwoordigheid van twee afsonderlike populasies getoon in die naïwe rMSCs preparate en die DNA inhoud was verenigbaar met die van selle wat OIS ondergaan. Die bevindinge stel voor dat die meer primatiewe osteoblast voorloperselle (naïwe rMSCs) is meer vatbaar vir mitogene-stimulasie as hul volwasse ekwivalente (preosteoblaste). Ook is gevind dat die mate van ERK1/2 aktivering hoër was in naïwe rMSCs, selfs na sterk mitogeniese stimulasie wat daarop dui dat naïwe rMSCs steeds hoogs sensitifief is vir stimulasie met sterk mitogene. In opsomming, dui die bevindinge dat Dex die proliferasie van naïwe rMSCs en preosteoblaste onderdruk wat met 'n verlaging van ERK1/2 aktiwiteit gepaard gaan. Verder, het Dex, MKP-1 mRNA opgereguleer maar die effek is nie op die proteinvlak waargeneem nie. Dit suggereer dat PTP/s anders as MKP-1 verantwoordelik kan wees vir die Dex inaktivering van ERK1/2 wat die proliferasie van naïwe rMSCs en preosteoblaste onderdruk.

Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), themineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), influences hippocampalneuron vulnerability to injury. Excessive activation of GR may compromise hippocampalneuron survival after several types of challenge including ischemic, metabolic, and excitotoxicinsults. In contrast, MR prevents adrenalectomy-induced loss of granule neurons in the dentategyrus. The present thesis addresses the respective roles of MR and GR in modulating neuronalsurvival following two forms of neuronal injury, excitotoxicity and traumatic brain injury. MaleSprague-Dawley rats were pretreated with MR antagonist spironolactone or GR antagonistmifepristone (RU486) and subsequently injected with kainic acid, an excitotoxic glutamateanalog, or injured with a controlled cortical impact. Twenty-four hours following injury,hippocampal neuron survival was measured to test the hypotheses that MR blockade wouldendanger and GR blockade would protect hippocampal neurons following injury. MessengerRNA levels of viability-related genes including bcl-2, bax, p53, BDNF, and NT-3 were alsomeasured to test the hypothesis that ACR regulation of these genes wouldcorrelate with neuronal survival. In addition, ACR mRNA levels were measured followingreceptor blockade and injury to test the hypothesis that glucocorticoid signaling is alteredfollowing neuronal injury via regulation of ACR expression.Mineralocorticoid receptor blockade with spironolactone increased neuronal vulnerability toexcitotoxic insult in hippocampal field CA3, and GR blockade with RU486 prevented neuronalloss after traumatic brain injury in field CA1. These results are consistent with the hypothesesthat MR protects and GR endangers hippocampal neurons. Adrenocorticosteroid receptorblockade decreased mRNA levels of the anti-apoptotic gene bcl-2 in select regions of uninjuredhippocampus, yet ACR regulation of bcl-2 did not consistently correspond with measures ofneuronal survival after injury. Kainic acid decreased MR mRNA levels in CA1 and CA3, whileboth kainic acid and controlled cortical impact dramatically decreased GR mRNA levels indentate gyrus. These data suggest that injury modulation of glucocorticoid signaling throughregulation of ACR expression may influence hippocampal neuron viability following injury.