FoxO3a is a transcription factor that regulates various cellular functions such as cell cycle or cell death. However, its role in the innate immune response is not clear. I investigated the impact of FoxO3a signaling on the immune response during infection with Salmonella Typhimurium (ST). My results revealed that FoxO3a regulated the homeostasis of myeloid cells in the spleen and blood of mice during steady-state. Following infection of macrophages with ST, FoxO3a signaling promoted the expression of pro-inflammatory cytokines such as IL12 and TNFα, but inhibited the expression of the anti-inflammatory cytokine IL10. Phenotypic analysis revealed that FoxO3a signaling had no effect on classical macrophage polarization into M1 vs M2 phenotypes, although it appeared to regulate mitochondrial function during infection with ST. Inflammatory responses are critical during infection with virulent intracellular pathogens, and these results provide new insights into the role of FoxO3a signaling in inflammatory responses.

FOXO3a is a pro-apoptotic transcription factor which shows increased activation in vascular smooth muscle cells (VSMCs) of advanced atherosclerotic plaques, specifically within the intimal layer. Since VSMC apoptosis plays a crucial role in the pathophysiology of atherosclerosis, we investigated the mechanisms underlying FOXO3a-mediated cell death in this particular cell type. We aimed to characterise a novel VSMC system (FOXO3aA3ERTM) and use these cells to validate MMP-13 and TIMP3 as new FOXO3a target genes. Also, we sought to determine the mechanisms of FOXO3aA3ERTM-mediated VSMC apoptosis, particularly regarding MMP-13 and TIMP3, potential MMP-13 substrates in the extracellular matrix and the precise apoptotic signalling involved. Furthermore, we aimed to investigate whether VSMC-specific activation of FOXO3aA3ERTM in mouse affects vascular remodelling during injury and whether this is reliant on MMP-13. Lastly, we aimed to address if endogenous FOXO3a upregulates MMP-13 in mouse and human VSMCs. Our laboratory has created a transgenic rat VSMC line which stably expresses an inducible FOXO3a mutant allele known as FOXO3aA3ERTM and previous microarray experiments identified matrix metalloproteinase 13 (MMP-13) as a potential novel FOXO3a target gene. Initially, we described several key features of the FOXO3aA3ERTM VSMCs used throughout this thesis, and subsequently demonstrated that MMP-13 is a bona fide target whose expression is rapidly upregulated upon FOXO3a activation, leading to markedly higher levels of protein, cleavage and proteolytic capacity. This induction of MMP-13 was responsible for the vast majority of FOXO3a-mediated apoptosis which was accompanied by prominent degradation of fibronectin, a glycoprotein found in the extracellular matrix. However, we could not identify a terminal apoptotic pathway. FOXO3a also downregulated the endogenous MMP inhibitor TIMP3, the recombinant protein of which reduced both MMP-13 proteolysis and FOXO3a-mediated apoptosis. Activation of FOXO3aA3ERTM in the VSMCs of medium and large arteries in mice resulted in heightened expression of MMP-13 in the vessel wall, which contributed to enhanced neointimal formation during carotid ligation. Finally, endogenous FOXO3a activation leads to increased MMP-13 expression in human VSMCs, but not mouse. Overall, we have shown that FOXO3a promotes VSMC apoptosis through MMP-13 both in vitro and in vivo, a novel pathway that has important implications for the pathogenesis and treatment of vascular disease.

Recent research suggests that [beta]-hydroxy-[beta]-methylbutyrate (HMB), a metabolite of leucine (Leu), increases muscle mass and attenuates muscle damage during resistance training. Although Leu acts as a potent stimulator of protein synthesis, HMB, but not Leu, has been reported to be effective in suppressing proteolysis in skeletal muscle. However, mechanisms for the effects of HMB on cell signaling pathways controlling muscle protein turnover during rest and after endurance exercise are still poorly understood. Furthermore, the effects of HMB on cell signaling pathways controlling protein synthesis and degradation under normal in vivo conditions warrant further investigation. For optimal gains in muscle mass, the appropriate type and amount of protein (PRO) is required for positive protein balance to occur in skeletal muscle. Therefore, this dissertation was designed to determine the effect of HMB, PRO and Leu, individually and in combination, on the regulation of cellular signaling pathways controlling muscle protein turnover during sedentary and post-exercise conditions. Study 1 demonstrated that, compared with HMB and PRO alone, the combination of HMB and PRO was more effective in activating the mTOR signaling pathway, which controls protein synthesis, and inhibiting FOXO3A, a major regulator of the ubiquitin-proteasome proteolytic signaling pathway. Study 2 demonstrated that, compared with its individual components, a novel HMB/PRO/Leu supplement better activated protein-synthetic signals and inhibited proteolytic signals in skeletal muscle, and these effects were better sustained. Finally, Study 3 demonstrated that adding Leu to PRO-enriched mixtures after exercise additively activated protein-synthetic signals in a fiber type-specific manner, and adding HMB clearly inhibited proteolytic signaling proteins. Furthermore, provision of an HMB/PRO/Leu supplement after exercise was found to favorably modulate signaling pathways controlling both protein synthesis and degradation. Taken together, the results of these studies suggest that a novel nutrient supplement, composed of HMB, Leu and PRO, additively enhances the intracellular signaling proteins controlling protein synthesis and attenuates signaling proteins controlling proteolysis in skeletal muscle during sedentary and post-exercise recovery. Therefore, such a supplement may be beneficial for both athletic and therapeutic purposes. / text

The innate immune response mediates immediate control of the pathogen and is followed by the acquired immune response which is slower but ensures comprehensive elimination of the pathogen. Dendritic cells are unique innate immune cells that can phagocytose the pathogen and generate pathogen-associated antigenic peptides for presentation to T cells in order to initiate the acquired immune response. Dendritic cells also express cytokines which facilitate pathogen control and development of acquired immune responses, thus acting as a bridge between innate and acquired immune responses. CD8+ T cells are important cells of the adaptive immune system that play a key role in mediating clearance and protection against intracellular pathogens. Upon engagement by antigen-presenting cells, CD8+ T cells undergo massive expansion followed by a swift, extensive contraction to restore homeostasis. The mechanisms behind the expansion and contraction of CD8+ T cells are yet to be completely elucidated. FoxO3a is a transcription factor that is involved in the regulation of various vital cellular processes ranging from cell proliferation and cell metabolism to stress resistance and cell death. I have, therefore, investigated the role of FoxO3a signaling in the activation of dendritic cells and CD8+ T cells. My initial experiments indicated that FoxO3a regulates the homeostasis of various immune cells including CD8+ T cells and dendritic cells. CD8+ T cells lacking FoxO3a displayed enhanced proliferation, as evaluated by cell imaging, CFSE dilution and Ki67 staining, upon polyclonal stimulation in vitro. The modulation of cell proliferation by FoxO3a seemed to be p27kip-independent, as evaluated by western blotting. At later stages of stimulation, FoxO3a-deficient CD8+ T cells underwent reduced cell death, as assessed by cell counting and 7-AAD staining, and this seemed to be independent of Bim, Caspase 8 or Caspase 3 activation. In addition, FoxO3a regulated cytokine expression by CD8+ T cells while displaying similar NFκB activation in comparison to WT CD8+ T cells. Similar results were observed in dendritic cells upon LPS stimulation in vitro, wherein cytokine expression was higher in the FoxO3a-deficient dendritic cells and they also displayed enhanced antigen presentation to CD8+ T cells, as evaluated by CFSE dilution. Taken together, these results indicate that FoxO3a acts as a negative regulator of CD8+ T cell and dendritic cell activation.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

Accumulated evidence indicates that, by the phosphorylation of its physiological substrates, Akt promotes cell survival, proliferation and angiogenesis. While a number of Akt targets have been identified, the mechanism by which Akt regulates cell survival and growth and induces malignant transformation still remains elusive. During the last 5 years, I have shown that AKT1 cross-talks with Src/Stat3 pathway. AKT1 is a direct target gene of Stat3. Protein/mRNA levels and promoter activity of AKT1 are significantly induced by constitutively active Src and Stat3. Knockdown of Stat3 or dominant-negative Stat3 reduced AKT1 expression induced by constitutively active Src. Blockage of AKT1 expression largely reduced Stat3 function in cell survival and angiogenesis. Furthermore, I have shown that proapoptotic protein 24p3 is a major target of Akt to mediate IL3 signaling in hematopoietic cells. Forkhead transcription factor FOXO3a directly binds to and activates 24p3 promoter leading to expression of 24p3 in response to IL3 withdrawal. Akt phosphorylates FOXO3a and inhibits its action toward 24p3. Finally, I have identified a novel transcription factor TZP that interacts with Akt and p53. Expression of TZP inhibits cell growth and survival and induces both G1 and G2/M cell cycle arrest. TZP directly binds to the p53 promoter and induces p53 transcription. In addition, TZP interacts with p53 and prevents p53 from Mdm2-mediated degradation. In response to genotoxic stress, both TZP and p53 were upregulated and knockdown of TZP reduced p53 expression. Akt phosphorylated TZP resulting in its translocation from the nucleus into the cytoplasm, and thus inhibits TZP function. These data indicate that Akt induced by STAT3 confers oncogenesis through inhibition of the transcription factors.

No description available.

L’acide docosahexaénoïque (DHA) est un acide gras polyinsaturé oméga-3 avec des propriétés anti-inflammatoires et anti-tumorales. L’effet du DHA dans le cadre du cancer colorectal pourrait être la conséquence d'une action anti-proliférative directe sur les cellules cancéreuses et de sa capacité à réduire l’inflammation propice au développement de la tumeur. Le Tumor Necrosis Factor-alpha (TNFa) est une cytokine pro-inflammatoire et présente des effets paradoxaux. En fonction du contexte cellulaire, le TNFa activera une voie de signalisation dépendante de la kinase RIP1 engageant la cellule cancéreuse vers la prolifération ou la mort cellulaire. Notre objectif fut d'évaluer le rôle du TNFa dans l'effet anti-prolifératif du DHA sur des cellules cancéreuses coliques et de préciser les mécanismes moléculaires régulant l'expression de cette cytokine. Le DHA induit l'expression de TNFa et sa sécrétion par les cellules cancéreuses. Nous avons montré que des anticorps neutralisant l'action autocrine du TNFa sur les cellules cancéreuses prévenait l'effet pro-apoptotique du DHA et abolissait l'effet anti-cancéreux observé dans des souris nude avec tumeurs HCT-116 sous régime DHA. L’induction de l'expression de TNFa par le DHA prend son origine à un niveau post-transcriptionnel par la répression du microARN miR-21 perdant sa capacité à dégrader l'ARNm TNFa. Le DHA par l'activation des kinases AMPKa et RIP1 déclenche la translocation nucléaire du facteur de transcription FOXO3a se fixant sur le promoteur miR-21 et diminuant l’expression de ce microARN. Nos travaux mettent en évidence un nouveau mécanisme moléculaire soutenant l'action anti-tumorale du DHA. / Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with anti-inflammatory and anti-tumoral properties. The anti-tumor effect of DHA in colorectal cancer might be attributed to direct anti-proliferative action on cancer cells and to its ability to reduce inflammatory status involved in tumor growth. Tumor Necrosis Factor-alpha (TNFa) is an inflammatory cytokine with paradoxical effect in cancer biology. According to the cellular context, TNFa activates RIP1 kinase dependent signaling pathway leading to proliferation or cell death. Our aim was to evaluate the role of TNFa in anti-proliferative effect of DHA in colon cancer cells and to precise the molecular mechanisms regulating TNFa expression.DHA treatment increased TNFa expression and secretion by cancer cells. We have shown that neutralization of autocrine TNFa action prevented the pro-apoptotic effect of DHA colon cancer cells and abolished anti-cancer effect in tumor HCT-116 bearing nude mice fed a DHA-enriched diet. Induction of TNFa expression by DHA occured at post-transcriptional level through microRNA miR-21 repression reducing its ability to induce TNFa mRNA degradation. DHA activates AMPKa and RIP1 kinases triggering nuclear translocation of the transcription factor Foxo3a which bound to miR-21 promoter and repressed the microRNA expression. Our works highlight a new molecular mechanism supporting the anti-cancer action of DHA.

The extension of life span by caloric restriction has been studied across species from yeast and Caenorhabditis elegans to primates. No generally accepted theory has been proposed to explain these observations. Here, we propose that the life span extension produced by caloric restriction can be duplicated by the metabolic changes induced by ketosis. From nematodes to mice, extension of life span results from decreased signaling through the insulin/insulin-like growth factor receptor signaling (IIS) pathway. Decreased IIS diminishes phosphatidylinositol (3,4,5) triphosphate (PIP3) production, leading to reduced PI3K and AKT kinase activity and decreased forkhead box O transcription factor (FOXO) phosphorylation, allowing FOXO proteins to remain in the nucleus. In the nucleus, FOXO proteins increase the transcription of genes encoding antioxidant enzymes, including superoxide dismutase 2, catalase, glutathione peroxidase, and hundreds of other genes. An effective method for combating free radical damage occurs through the metabolism of ketone bodies, ketosis being the characteristic physiological change brought about by caloric restriction from fruit flies to primates. A dietary ketone ester also decreases circulating glucose and insulin leading to decreased IIS. The ketone body, d-β-hydroxybutyrate (d-βHB), is a natural inhibitor of class I and IIa histone deacetylases that repress transcription of the FOXO3a gene. Therefore, ketosis results in transcription of the enzymes of the antioxidant pathways. In addition, the metabolism of ketone bodies results in a more negative redox potential of the NADP antioxidant system, which is a terminal destructor of oxygen free radicals. Addition of d-βHB to cultures of C. elegans extends life span. We hypothesize that increasing the levels of ketone bodies will also extend the life span of humans and that calorie restriction extends life span at least in part through increasing the levels of ketone bodies. An exogenous ketone ester provides a new tool for mimicking the effects of caloric restriction that can be used in future research. The ability to power mitochondria in aged individuals that have limited ability to oxidize glucose metabolites due to pyruvate dehydrogenase inhibition suggests new lines of research for preventative measures and treatments for aging and aging-related disorders.