Tobias B. Dansen

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

Unravelling the tumor-suppressive functions of FOXO proteins.

Members of the forkhead box O (FOXO) family of transcription factors have been postulated to be tumor suppressors because of their established roles in cell-cycle arrest, apoptosis, DNA-damage repair and scavenging of reactive oxygen species. Recently, several animal model studies have shown that the FOXO proteins are indeed tumor suppressors. Furthermore, FOXO proteins have recently been implicated in the negative regulation of signaling by the hypoxia-inducible factor 1 during vascular development, raising the possibility that the FOXO proteins suppress not only tumor formation but also tumor angiogenesis and, possibly, metastasis. Here, we discuss recent advances in the understanding of the roles of FOXO family members in tumor suppression.

Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy

The PI(3)K–PKB–FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K–PKB–FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.

Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and FoxO4 activity

Cellular damage invoked by reactive oxygen species plays a key role in the pathobiology of cancer and aging. Forkhead box class O (FoxO) transcription factors are involved in various cellular processes including cell cycle regulation, apoptosis and resistance to reactive oxygen species, and studies in animal models have shown that these transcription factors are of vital importance in tumor suppression, stem cell maintenance and lifespan extension. Here we report that the activity of FoxO in human cells is directly regulated by the cellular redox state through a unique mechanism in signal transduction. We show that reactive oxygen species induce the formation of cysteine-thiol disulfide-dependent complexes of FoxO and the p300/CBP acetyltransferase, and that modulation of FoxO biological activity by p300/CBP-mediated acetylation is fully dependent on the formation of this redox-dependent complex. These findings directly link cellular redox status to the activity of the longevity protein FoxO.

Peroxisomes in human fibroblasts have a basic pH.

eroxisomes are single-membrane-bound organelles found in nearly all eukaryotic cells. These organelles have a central function in lipid metabolism, including the beta-oxidation of verylong-chain and branched-chain fatty acids and the biosynthesis of ether phospholipids and cholesterol. Another characteristic of peroxisomes is their ability to degrade hydrogen peroxide by catalase. A deficiency in one or more peroxisomal enzymes has been linked to at least 20 (often lethal) disorders, showing the key role of this organelle in normal functioning of the human body. Peroxisomes are fragile structures that easily lose their integrity upon isolation. This poses a serious problem for studying these organelles in vitro and explains why our knowledge about the properties of the peroxisomal membrane, including the change in pH across it, is limited. In vivo, peroxisomes have been shown to be closed structures that are impermeable to NADH and NADPH, implying the existence of NADP redox shuttles. Here we study the pH in peroxisomes by targeting a pH-sensitive fluorescent reporter group to these organelles in living fibroblasts. We attained specific targeting by conjugating the fluorophore to a membrane-permeable peptide that contains a type-I peroxisome-targeting sequence (PTS1; amino-acid sequence AKL). Using this peptide probe, we establish that peroxisomes of human fibroblasts have a pH of 8.2 ± 0.3. Fibroblasts from RCDP (rhizomelic form of chondrodysplasia punctata) type 1 patients with severe mutations in the PEX7 protein, which result in an isolated defect in peroxisomal import of proteins with a PTS2 sequence, are still capable of importing the probe into peroxisomes, but have a pH of 6.5 ± 0.3. We covalently linked the pH-sensitive (5and 6-)carboxySNAFL-2 moiety to the PTS1-containing heptapeptide acetylCKGGAKL-COOH at the lysine near the amino terminus. This peptide probe (SNAFL-2–PTS1) was rapidly taken up into the cells and a punctate pattern of fluorescence was found, indicative of a peroxisomal localization. To confirm that these structures are indeed peroxisomes, we used a fixable analogue (BODIPY–PTS1) in co-localization studies with Cy5-labelled antibodies against different peroxisomal proteins (Fig. 1a–c). The probe was targeted only towards the peroxisomes (Fig. 1b). Further evidence that the probe was incorporated into peroxisomes came from studies of human fibroblasts with defects in peroxisomal import of PTS1bearing proteins. This probe was not incorporated into peroxiP

Glucose Withdrawal Induces Oxidative Stress followed by Apoptosis in Glioblastoma Cells but not in Normal Human Astrocytes

Tumor cells rely preferentially on anaerobic glycolysis rather than on respiration for ATP generation, a phenomenon known as the Warburg effect. We explored the effects of glucose withdrawal on glioblastoma multiforme–derived cell lines and their nontransformed counterparts, normal human astrocytes. We found that glucose withdrawal induces extensive apoptosis in glioblastoma multiforme cells but not in normal astrocytes. In all cells examined, ATP levels are sustained on glucose withdrawal due to elevation of fatty acid oxidation and ensuing respiration; however, we show that oxidative stress generated in the mitochondrial respiratory chain is the direct cause of cell death in glioblastoma multiforme cells. Oxidative stress that only occurs in glioblastoma multiforme cells underlies the selective susceptibility to glucose withdrawal–induced apoptosis documented in the malignant cells. This study implicates glycolysis as a potentially efficient and selective target for glioblastoma multiforme treatment. (Mol Cancer Res 2006;4(5):319–30)

Forkhead Box O as a Sensor, Mediator, and Regulator of Redox Signaling

The forkhead box O (FOXO) family of transcription factors regulates a variety of cellular programs, including cell cycle arrest, reactive oxygen species (ROS) scavenging, and apoptosis, and are of key importance in the decision over cell fate. In animal model systems it has been shown that FOXO is involved in the regulation of long lifespan. FOXO activity is tightly controlled by the insulin signaling pathway and by a multitude of ROS-induced posttranslational modifications. In the cell, ROS levels can be sensed by virtue of stimulatory and inhibitory oxidative modification of cysteine residues within proteins that control various signaling cascades. Recently, it was shown that cysteines in FOXO can also act as sensors of the local redox state. In this review we have outlined the cysteine-dependent redox switches that regulate both the insulin and ROS signaling pathways upstream of FOXO. Further, we describe how FOXO controls ROS levels by transcriptional regulation of a multilayered antioxidant system. Finally, we will discuss how cysteine-based redox signaling to FOXO could play a role in fine-tuning the optimal cellular response to ROS to control organismal lifespan.

Release of Mps1 from kinetochores is crucial for timely anaphase onset

Mps1 regulates its own turnover at kinetochores to ensure mitotic checkpoint silencing in metaphase.

Redox-dependent control of FOXO/DAF-16 by transportin-1

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of insulin signaling, reactive oxygen species (ROS) can activate FOXO/DAF-16 through nuclear accumulation. How ROS regulate the nuclear translocation of FOXO/DAF-16 is largely unknown. Cysteine oxidation can stabilize protein-protein interactions through the formation of disulfide-bridges when cells encounter ROS. Using a proteome-wide screen that identifies ROS-induced mixed disulfide-dependent complexes, we discovered several interaction partners of FOXO4, one of which is the nuclear import receptor transportin-1. We show that disulfide formation with transportin-1 is required for nuclear localization and the activation of FOXO4/DAF-16 induced by ROS, but not by the loss of insulin signaling. This molecular mechanism for nuclear shuttling is conserved in C. elegans and directly connects redox signaling to the longevity protein FOXO/DAF-16.

Specific Requirement for Bax, Not Bak, in Myc-induced Apoptosis and Tumor Suppression in Vivo

Bax and Bak comprise the mitochondrial gateway for apoptosis induced by diverse stimuli. Loss of both bax and bak is necessary to block cell death induced by such stimuli, indicating a great degree of functional overlap between Bax and Bak. Apoptosis is the major intrinsic pathway that limits the oncogenic potential of Myc. Using a switchable mouse model of Myc-induced apoptosis in pancreatic β cells, we have shown that Myc induces apoptosis in vivo exclusively through Bax but not Bak. Furthermore, blockade of Myc-induced apoptosis by the inactivation of Bax, but not Bak, eliminates all restraints to the oncogenic potential of Myc, allowing the rapid and synchronous progression of invasive, angiogenic tumors.