Forkhead box O and the control of cellular oxidative stress

Abstract

FOXO Forkhead transcription factors are a subfamily of the large superfamily of Forkhead transcription factors, which consists of over 100 different members, expressed in species ranging from yeast to humans. Members of this family of transcription factors have been demonstrated to play important roles in cell proliferation and differentiation, both during development and in the adult organism. In addition to these roles in normal development, several Forkhead transcription factors have also been suggested to be involved in neoplasia. FOXO transcription factors, for example, have been shown to be part of chromosomal translocations associated with several forms of human cancer. In recent years, a lot of research is performed to investigate the regulation and the role of the FOXO Forkhead transcription factors. Members of this subfamily have been identified in several organisms including in Caenorhabditis elegans, Drosophila Melanogaster, mice, and humans. FOXOs are mainly regulated by the phosphoinositide 3-kinase (PI-3K)/protein kinase B (PKB) pathway. Upon growth factor stimulation FOXOs are directly phosphorylated by PKB, resulting in nuclear export of FOXO and inhibition of the transcriptional activity. Several other kinases, which regulate phosphorylation of FOXO, have been identified as well. Also other processes like acetylation and ubiquitination may play a role in the regulation of FOXO activity. FOXO transcription factors have been implicated in to play a role in the regulation of a multitude of biological processes including cell cycle, apoptosis, DNA repair, differentiation, metabolism, and protection from oxidative stress. These data indicate a role for FOXOs as a meeting point within the cell, integrating several signalling pathways and regulating cell fate. In C. elegans, the FOXO orthologue DAF-16 is involved in the regulation of longevity, dauer formation and stress resistance. Overexpression of DAF-16 in the worm will lead to an increased lifespan, correlated with increased resistance to oxidative stress. Recent data in mammalian systems show that FOXOs are involved in inducing a cell cycle arrest and quiescence. On the other side, FOXOs can protect cells from oxidative stress by regulating the expression of MnSOD and catalase, two anti-oxidant enzymes important in the defense to oxygen radicals. Thus, these data indicate, that, like in C. elegans, also in mammalian systems FOXOs can regulate both quiescence and stress resistance. In this thesis we tried to further understand the role of mammalian FOXOs in the control of cellular oxidative stress. We focus on the possibility that FOXOs not only repress cellular oxidative stress by increasing anti-oxidants expression, but that cellular oxidative stress itself also signals to FOXOs, creating a feedback mechanism. FOXO transcription factors can directly bind to regions within promoters of their target genes, thereby regulating the transcription of these genes. However, in recent years, a variety of cofactors for FOXO-induced effects on transcription have been described, for example p300 and several nuclear receptors. A yeast-two-hybrid screen for new interactors for -catenin revealed FOXOs as potential binding partners. -catenin is a multifunctional protein that regulates gene transcription within the Wnt signalling pathway by direct binding to members of the Lef-1/TCF family of transcription factors. The results described in Chapter 2 show that the binding of FOXO to -catenin also occurs in cells, and that this binding enhances the transcriptional activity of FOXO. The binding between FOXO and -catenin is induced under conditions of increased cellular oxidative stress, further increasing FOXO transcriptional activity under these conditions. In addition to the biochemical data, our genetic analysis in C. elegans demonstrate that this interaction is evolutionary conserved. The -catenin homologue BAR-1 is required for DAF-16 dependent dauer formation, lifespan regulation, oxidative stress resistance and the expression of the DAF-16 target gene SOD-3 following oxidative stress. These data implicate -catenin binding to FOXO as part of the mechanism by which cellular oxidative stress signals to FOXO. The binding of -catenin to FOXOs not only influences the function of FOXOs; this interaction also results in the inhibition of -catenin/TCF transcriptional activity (Chapter 3). The -catenin/TCF complex is activated upon Wnt signalling and it regulates transcription of several downstream target genes. -catenin levels in a cell are tightly controlled by the APC/GSK3/Axin complex. In the absence of a Wnt signal, this complex targets -catenin for degradation by inducing phosphorylation of -catenin. The inhibition of -catenin/TCF transcriptional activity is probably due to competition between TCF and FOXO for binding to -catenin. The FOXO mediated inhibition also correlated with the ability of FOXOs to shuttle -catenin out of the nucleus. Mutants of FOXO that are predominantly localized within the nucleus are impaired in their ability to inhibit -catenin/TCF transcriptional activity and do not increase cytosolic levels of -catenin, whereas mutants of FOXO that do inhibit -catenin/TCF activity are predominantly localized in the cytosol and also increase cytosolic -catenin levels. Finally, the increased binding of FOXO to -catenin upon cellular oxidative stress also results in a further enhancement of the ability of FOXOs to inhibit -catenin/TCF activity. These results further demonstrate a cross talk between FOXO and TCF signalling in which -catenin is the central player. Phosphorylation of FOXOs is regulated by several kinases, including PKB. Activation of the Ras/Ral pathway also results in phosphorylation of FOXO, as was described for FOXO4. Activation of Ral results in phosphorylation of FOXO4 on two residues in the C-terminal part of the protein, Thr447 and Thr451. In Chapter 4 we show that phosphorylation of these sites by the Ral pathway is increased under conditions of cellular oxidative stress. Upon treatment of cells with H2O2, Ral is activated and this results in JNK dependent phosphorylation of Thr447 and Thr451. Oxidative stress induces nuclear localization of FOXO4 and an increase in the transcriptional activity of the protein. In addition, we show that this signalling pathway is also employed by TNFa to activate FOXO4 transcriptional activity. Taken together, these data provide evidence for a feedback mechanism from oxidative stress to FOXO transcription factors, involving the small GTPase Ral and the stress kinase JNK. In the Addendum of Chapter 4 we compare different forms of stress and the ability of FOXOs to reduce JNK activation in cells in response to these stresses. Inhibition of JNK activation by FOXOs in response to oxidative stress is dependent on MnSOD, since FOXOs can no longer inhibit JNK activity after H2O2 treatment in SOD-/- MEFs. However, FOXOs can still reduce JNK activation in response to UV treatment in these cells. Experiments in TKO MEFs, which lack all members of the RB family and thus are incapable of going into cell cycle arrest, indicate an involvement of a cell cycle arrest in stress induced by H2O2, whereas UV induced stress is likely to be independent of the ability to go into cell cycle arrest. In conclusion, this thesis describes the role of FOXOs in oxidative stress signalling in more detail. We show that FOXOs are part of a feedback mechanism, in which an increase in ROS levels in a cell will signal towards FOXO, leading to Ral-mediated, JNK-dependent phosphorylation and activation of FOXO. This results in activation of antioxidant mechanisms in a cell, and thus protection of the cell. Furthermore, we have identified a new binding partner for FOXOs, -catenin. Interaction between these two proteins is increased under conditions of oxidative stress and affects both FOXO and -catenin/TCF signalling in a cell. These findings indicate an important role for FOXO in the defense against oxidative stress. Together with its role in cell cycle regulation this further indicates a link between the protection against oxidative stress and the regulation of the cell cycle and quiescence via the regulation of one subfamily of transcription factors, FOXOs.