<p> Proper gene expression programs cellular activities, while aberrant manipulation of transcription factors often leads to devastating consequences, such as cancer or cell death. The transcription factor family activator protein-1 (AP-1) plays an important role in many cellular activities including cell transformation, proliferation and survival (Shaulian and Karin 2002). However, little has been done to obtain a global view of the role of individual AP-1 members and how they cooperate in many cellular activities. We have discovered that blocking the AP-1 pathway by a c-Jun dominant negative mutant, TAM67, induced cell death in RSV-transformed primary chicken embryo fibroblasts (CEF), suggesting that AP-1 activity is vital for cell survival upon v-Src transformation. In addition, accumulation of cytoplasmic vesicles was observed in the cytoplasm of a proportion of RSV-transformed CEF expressing TAM67. Oil-red staining of these vesicles indicated the presence of lipid droplets in these cells, suggesting that the inhibition of AP-1 promotes the adipogenic conversion of v-Src transformed CEF. To understand the role of individual members of the AP-1 family, a retroviral-based shRNA expressing system was designed to stably downregulate individual AP-1 members. This retroviral-based RNAi system provided sustained gene downregulation of AP-1 family members. Reduction of the c-Jun protein level by shRNA induced senescence in normal CEF, while it modestly downregulated AP-1 activity in RSV -transformed CEF indicating that c-Jun is not the main component of the AP-1 complex in RSV-transformed CEF. Inhibition of JunD expression induced apoptosis and was deleterious to both normal and RSV-transformed CEF, suggesting that JunD is crucial for the survival of CEF. Transient express10n reporter-assays also showed that loss-of-function of JunD by shRNA dramatically repressed AP-1 activity. Hence JunD is the main component of the AP-1 complex that regulates the survival of CEF. Furthermore, we determined that loss of JunD expression resulted in an elevated level of tumour suppressor p53. Co-inhibition of p53 and JunD restored the transforming ability of v-Src transformed CEF, as indicated by foci formation in soft agar assays. Hence, repression of p53 induction was able to bypass the death signal released as a result of AP-1 inhibition in v-Src transformed CEF. Downregulation of Fra-2 (Fos-related antigen 2) level by shRNA did not affect the proliferation of normal CEF. However, RSV -transformed CEFs expressing fra -2 shRNA were transformation-defective with the presence of multiple vesicles in cytoplasm. Oil-red staining of these vesicles indicated the presence of lipid droplets, which resembles the effect of T AM67 in RSV -transformed CEF indicating that Fra-2 blocks differentiation. These findings help us to understand the role of individual members of the AP-1 transcription factor family in normal and RSV -transformed CEF. Importantly, global gene profiling of v-Src transformed CEF expressing shRNA for individual AP-1 members will improve our knowledge of the transformation process. Functional characterization of the cascade will rely on the use of retroviral-based shRNA expressing system as described above. </p> / Thesis / Doctor of Philosophy (PhD)

The adrenal cortex is a crucial endocrine gland in the mammalian stress response. In chronic inflammatory stress, cortisol is elevated whereas adrenal androgens are decreased. Furthermore, ACTH levels have poor correlation with the plasma cortisol in these conditions, thus suggesting that other factors are driving the stress response during chronic inflammatory stress. Interleukin-6 (IL-6), a cytokine which is released during chronic inflammatory stress, is assumed to be one such factor. Thus the biochemical pathways by which IL-6 increases cortisol release from the zona fasciculata (ZF), and decreases adrenal androgen release from the zona reticularis (ZR) were investigated. Since IL-6 activates AMP-activated kinase (AMPK) in skeletal muscle, AMPK was investigated for IL-6- induced effects in ZF and ZR tissue. The effects of AMPK activation and IL-6 exposure on the expression of the steroidogenic proteins, steroidogenic acute regulatory protein (StAR) and cholesterol side chain cleavage enzyme (P450scc), and on the steroidogenic nuclear factors steroidogenic factor-1 (SF-1) and adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) were investigated. AMPK activation and IL-6 exposure increased the expression of StAR, P450scc, and SF-1, and decreased DAX-1 in the ZF. Meanwhile, AMPK activation and IL-6 exposure decreased the expression of StAR, P450scc, and SF-1, and increased DAX-1 in the ZR. AMPK inhibition blocked the effects of AMPK activation and IL-6 on the ZF and ZR. Activator Protein-1 (AP-1) was the second biochemical intermediate studied since in other tissues AMPK activation increases the expression and phosphorylation of AP-1 subunits. IL-6 stimulation and AMPK activation increased the expression of the AP-1 subunits cFOS, cJUN, JUN B, and JUN D, while increasing the phosphorylation of cJUN in both the ZF and the ZR. These effects were blocked by AMPK inhibition. Inhibition of AP-1 leads to decreased StAR, P450scc, and SF-1, and increased DAX-1 in the ZF. Meanwhile, AP-1 inhibition leads to increased StAR, P450scc, SF-1, and decreased DAX-1 in the ZR. Therefore the AP-1 complex functions as a biochemical intermediate in the IL-6 and AMPK regulation of steroidogenic enzymes in the ZF and ZR. Overall, the results suggest that IL-6 activates AMPK, which increases the expression and phosphorylation of AP-1 subunits in the ZF and the ZR. However, increased AP-1 activation leads to increased StAR and P450scc in the ZF, but decreased StAR and P450scc in the ZR.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Les récepteurs nucléaires sont des facteurs transcriptionnels impliqués dans la regulation de la croissance cellulaire, en partie dû à leurs effets génomiques. Une voie alternative est l'interference entre les récepteurs nucléaires et le facteur transcriptionnel AP-1 (activator protein-1), composé de protéines induites par plusieurs facteurs de croissance et molécules de signalisation. Dans ce projet, j'ai analysé les mécanismes d'interférence transcriptionnelle entre les récepteurs des oestrogènes (ER), les récepteurs des glucocorticoïdes (GR) et AP-1. / Nuclear receptors constitute a large superfamily of DNA binding transcriptional regulators that can regulate a diversity of important cellular events, such as development, differentiation, and responses to extracellular stimuli. These effects are mediated mostly by the genomic effects of nuclear receptors which bind selectively to DNA (hormone response element), and regulate target gene transcription. An alternative pathway of transcriptional regulation is the crosstalk between nuclear receptors and other transcription factors, such as NPKB, or activator protein-1 (AP-1). Transcription factor AP-1 is composed of nuclear proteins encoded by the Jun family and Fos family of protooncogenes, whose transcription is rapidly induced by a number of growth factors and other signaling molecules. AP-1 is implicated in diverse aspects of cell growth, differentiation and development. In this project, I have focused on the analysis of the mechanisms of transcriptional interference between nuclear receptors and AP-1.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze. / Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock.