Peter L.J. de Keizer

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.

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.

Mdm2 induces mono-ubiquitination of FOXO4.

Background The Forkhead box O (FOXO) class of transcription factors are involved in the regulation of several cellular responses including cell cycle progression and apoptosis. Furthermore, in model organisms FOXOs act as tumor suppressors and affect aging. Previously, we noted that FOXOs and p53 are remarkably similar within their spectrum of regulatory proteins [1]. For example, the de-ubiquitinating enzyme USP7 removes ubiquitin from both FOXO and p53. However, Skp2 has been identified as E3 ligase for FOXO1, whereas Mdm2 is the prime E3 ligase for p53. Principal Findings/Methodology Here we provide evidence that Mdm2 acts as an E3 ligase for FOXO as well. In vitro incubation of Mdm2 and FOXO results in ATP-dependent (multi)mono-ubiquitination of FOXO similar to p53. Furthermore, in vivo co-expression of Mdm2 and FOXO induces FOXO mono-ubiquitination and consistent with this result, siRNA-mediated depletion of Mdm2 inhibits mono-ubiquitination of FOXO induced by hydrogen peroxide. Regulation of FOXO ubiquitination by Mdm2 is likely to be direct since Mdm2 and FOXO co-immunoprecipitate. In addition, Mdm2-mediated ubiquitination regulates FOXO transcriptional activity. Conclusions/Significance These data identify Mdm2 as a novel E3 ligase for FOXOs and extend the analogous mode of regulation between FOXO and p53.

The Peptidyl-Isomerase Pin1 Regulates p27kip1 Expression through Inhibition of Forkhead Box O Tumor Suppressors

The Forkhead box O (FOXO) protein family is an evolutionarily conserved subclass of transcription factors recently identified as bona fide tumor suppressors. Preventing the accumulation of cellular damage due to oxidative stress is thought to underlie its tumor-suppressive role. Oxidative stress, in turn, also feedback controls FOXO4 function. Regulation of this process, however, is poorly understood but may be relevant to the ability of FOXO to control tumor suppression. Here, we characterize novel FOXO4 phosphorylation sites after increased cellular oxidative stress and identify the isomerase Pin1, a protein frequently found to be overexpressed in cancer, as a critical regulator of p27(kip1) through FOXO4 inhibition. We show that Pin1 requires these phosphorylation events to act negatively on FOXO4 transcriptional activity. Consistent with this, oxidative stress induces binding of Pin1 to FOXO, thereby attenuating its monoubiquitination, a yet uncharacterized mode of substrate modulation by Pin1. We have previously shown that monoubiquitination is involved in controlling nuclear translocation in response to cellular stress, and indeed, Pin1 prevents nuclear FOXO4 accumulation. Interestingly, Pin1 acts on FOXO through stimulation of the activity of the deubiquitinating enzyme HAUSP/USP7. Ultimately, this results in decreased transcriptional activity towards target genes, including the cell cycle arrest gene p27(kip1). Notably, in a primary human breast cancer panel, low p27(kip1) levels inversely correlated with Pin1 expression. Thus, Pin1 is identified as a novel negative FOXO regulator, interconnecting FOXO phosphorylation and monoubiquitination in response to cellular stress to regulate p27(kip1).

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence.

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAF(V600E) oncogene, which arises commonly in melanoma. BRAF(V600E) signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH(2) terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr(223), Ser(226), Thr(447), and Thr(451). BRAF(V600E)-induced FOXO4 phosphorylation resulted in p21(cip1)-mediated cell senescence independent of p16(ink4a) or p27(kip1). Importantly, melanocyte-specific activation of BRAF(V600E) in vivo resulted in the formation of skin nevi expressing Thr(223)/Ser(226)-phosphorylated FOXO4 and elevated p21(cip1). Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response.

In this study, we searched for proteins regulating the tumor suppressor and life‐span regulator FOXO4. Through an unbiased tandem‐affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double‐strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor‐suppressive function in part through transcriptional regulation of the cell cycle arrest p27kip1 gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4‐mediated p27kip1 transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27kip1 transcription. In addition, in contrast to wild‐type mouse embryonic stem (ES) cells, Ku70−/− ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70‐FOXO4 interaction stoichiometry followed a nonlinear dose‐response curve by hydrogen peroxide‐generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 µM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.—Brenkman, A. B., van den Broek, N. J. F., de Keizer, P. L. J., van Gent, D. C., Burgering, B. M. T. The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response. FASEB J. 24, 4271–4280 (2010). www.fasebj.org

Senescent Cells Drive Frailty through Systemic Signals

Senescent cells drive ageing and the associated loss in health and lifespan. Whether this is mediated by systemic signalling remained unclear. Recently, Xu et al. [1] (Nat. Med. 2018;24:1246-1256) answered this question by injecting senescent cells into young mice and observing a long-lasting increase in frailty and mortality.

Musculoskeletal senescence: a moving target ready to be eliminated

Aging is the prime risk factor for the broad-based development of diseases. Frailty is a phenotypical hallmark of aging and is often used to assess whether the predicted benefits of a therapy outweigh the risks for older patients. Senescent cells form as a consequence of unresolved molecular damage and persistently secrete molecules that can impair tissue function. Recent evidence shows senescent cells can chronically interfere with stem cell function and drive aging of the musculoskeletal system. In addition, targeted apoptosis of senescent cells can restore tissue homeostasis in aged animals. Thus, targeting cellular senescence provides new therapeutic opportunities for the intervention of frailty-associated pathologies and could have pleiotropic health benefits.