Recent advances in understanding the role of FOXO3

Introduction

The forkhead box O3 (FOXO3, or FKHRL1) protein is one of about 40 forkhead box (FOX) transcription factors encoded by the mammalian genome¹. FOX transcription factors are versatile proteins containing an evolutionarily conserved winged helix DNA-binding motif of about 100 residues at the N-terminal region, the forkhead (FKH) domain^2–4. FOXO3 belongs to the FOXO subclass (made of FOXO1, FOXO3, FOXO4, and FOXO6), which historically is known to regulate insulin signaling (comprehensively reviewed in 5,6). Numerous regulatory processes, including phosphorylation, acetylation, ubiquitination, methylation^7,8, and microRNA (miRNA) binding⁹, can modulate FOXO3 transcriptional activity. Of particular interest for human health, FOXO3 is under the control of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway¹⁰. In its non-phosphorylated form, FOXO3 localizes at the nucleus where it regulates gene transcription. Activation of the PI3K/Akt pathway results in the phosphorylation of FOXO3 at three conserved residues¹¹. This usually causes its exclusion from the nucleus and stops the transcriptional activation of its target genes^1,12. Phosphorylated FOXO3 therefore represents the inactive form of the protein. Through PI3K/Akt, FOXO3 mediates biological processes that are essential for health over the lifespan, including substrate metabolism, protein turnover, cell survival, and cell death^11,13,14.

Our research group investigates skeletal muscle wasting and miRNA-mediated regulation. Although the involvement of FOXO3 in these processes is undeniable, these two topics have not been reviewed in the specific context of FOXO3. In addition, the role and regulation of FOXO3 in the genetics of longevity constitute a very dynamic research field, justifying the need for an updated review encompassing the research articles published over the last 3 years.

FOXO3 and the regulation of skeletal muscle homeostasis

FOXO proteins are expressed across multiple tissues of the body but their expression level, function, and targets are tissue specific. In mice, Foxo3 mRNA is enriched in the heart, brain, spleen, and kidney and, to a certain extent, skeletal muscle¹⁵. FOXO3 is a key player in the control of skeletal muscle protein turnover and a central effector of PI3K/Akt signaling, the main regulator of protein synthesis and degradation in the muscle¹⁶. In anabolic conditions, Akt phosphorylates FOXO3 and suppresses its transcriptional activity. FOXO3 inhibition in turn reduces the expression of the muscle-enriched members of the ubiquitin-proteasome system, atrogin-1 (FBXO32) and muscle RING finger 1 (MURF1)¹⁷, which promote muscle protein degradation. In addition, upon Akt activation, FOXO proteins may play a role in a negative feedback loop that inhibits Akt to maintain the cell homeostatic balance. In non-mammalian cells, FoxO orthologues inhibit the activity of the mechanistic target of rapamycin complex 1 (mTORC1)^18,19, which drives muscle protein synthesis downstream of Akt¹⁶. In mammalian tissue, FOXO proteins reduce mTORC1 activity, thereby activating Akt²⁰. FOXO proteins therefore may play an intricate role in balancing Akt and mTORC1 activities in response to changing metabolic conditions.

In mouse^21–23 and human²⁴ skeletal muscle, FOXO3 mRNA or total protein expression or both are upregulated under artificially induced catabolic conditions such as limb suspension or calorie restriction, suggesting that FOXO3 contributes to muscle wasting in these models. Recent rodent studies using immobilization models point toward myofiber type-specific regulation of FOXO3^23,25. However, a recent study showed no difference in FOXO3 mRNA levels or in the cytoplasmic levels of the inactive phosphorylated FOXO3 protein in overweight young men subjected to energy restriction²⁶, potentially because other factors pertaining to insulin signaling may be at play. The complexity of FOXO protein regulation and the redundancy of FOXO alleles suggest that changes in gene and protein expression levels need to be interpreted with care, as they may not provide direct insights into the mechanistic processes at play.

Disease-induced catabolic states are also characterized by increased FOXO3 expression levels. Foxo3 mRNA levels were elevated in the late symptomatic stage of two mouse models of spinal muscular atrophy²⁷. FOXO3 was also identified in a network-based analysis comparing serum proteomics in patients with Duchenne muscular dystrophy and age-matched controls²⁸, suggesting potential for FOXO3 as a protein biomarker to monitor disease progression in conditions with severe skeletal muscle atrophy. Patients with chronic obstructive pulmonary disease displayed an increased ratio of phosphorylated FOXO3 to total FOXO3 in their muscle when compared with healthy controls with or without sarcopenia²⁹.

Whereas higher levels of FOXO3 are typically observed in pathological catabolic conditions, FOXO3 expression patterns are not upregulated in healthy old muscle. Sarcopenic mice display no change in nuclear or total FOXO3 protein expression despite reduced phosphorylation levels that might be indicative of higher FOXO3 activity³⁰. We and others showed that FOXO3 mRNA^31,32 and FOXO3 nuclear protein levels decreased in old human skeletal muscle³³ whereas total or phosphorylated FOXO3 protein expression did not change³⁴. It is generally accepted that sarcopenia cannot be attributed to an upregulation of the proteolytic system or an induction of FOXO3³⁵. Therefore, in aging muscle, FOXO3 may be similarly or even less active than in younger muscle or in models of artificially or disease-induced atrophy. Overall, these results confirm the idea that a series of upstream regulatory factors that inhibit FOXO3 transcriptional activity, including peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) and PI3K/Akt itself²⁰, protect the muscle from aging-related atrophy^36,37. In addition, the role of FOXO3 in the process of muscle aging might rely on a fine balance between the regulation of protein turnover²⁰ and other, protective anti-aging processes, such as the maintenance of the pool of skeletal muscle stem cells³⁸, which is discussed below.

FOXO3 and the genetics of longevity

FOXO3 is among the few genes associated with human longevity that have been consistently replicated. Genetic variants of FoxO3 are associated with exceptional longevity in worms, flies, and mammals³⁹. In humans, FOXO3 hosts about 40 common, non-coding single-nucleotide polymorphisms (SNPs) that have been consistently associated with longevity in Caucasian^40–44 and Asian^43,45,46 populations. FOXO3 gene and protein expression is associated with age-related phenotypes in multiple tissues⁴⁷. For example, an age-dependent decrease in FOXO3 protein contributes to the loss of anti-inflammatory behavior in macrophages of old mice⁴⁸. Additionally, FOXO3-deficient mice demonstrate signs of pronounced neural activation, apoptosis, and enteric neuronal loss, indicative of premature aging of the enteric nervous system⁴⁸. FOXO3 overexpression also facilitates autophagy, a process of degradation and recycling of cytoplasmic proteins and organelles that is essential for healthy aging in multiple tissues, including skeletal muscle⁴⁹. However, the key role of FOXO3 in aging seems to be via the maintenance of stem cell homeostasis⁵⁰, including in the brain⁵¹, blood⁵², and skeletal muscle³⁸. Whether the modulation of molecular pathways involved in the age-dependent deterioration of stem cell function can reverse aging phenotypes remains controversial⁵³. In skeletal muscle stem cells, termed “satellite cells”, FOXO3 enhances stem cell self-renewal via the activation of Notch signaling, maintaining an available pool of satellite cells that have divided but retain their undifferentiated state³⁸. In fact, FOXO proteins play a dual role, and in situations of cellular damage, they can induce cell cycle arrest and senescence while independently repressing stemness signaling⁵⁴. Yet, in humans, despite consistent associations between FOXO3 genetic variants and exceptional longevity^40–46, a possible link between FOXO3 and healthy aging remains unclear. For example, the G allele of a longevity variant of FOXO3 was associated with a 10% reduction in all-cause mortality in a prospective cohort study of 3,584 older American men⁵⁵. Moreover, in a cross-sectional study including more than 30,000 individuals, the G allele of another longevity variant of FOXO3 was associated with a decrease in concentration of circulating insulin-like growth factor-1 (IGF-1), a marker of insulin resistance and chronic disease⁵⁶. Smaller-scale studies have yielded mixed results, albeit showing consistent trends. In the seminal study on FOXO3 and longevity (n = 615), carriers of FOXO3 longevity variants had lower prevalence of coronary heart disease and insulin resistance⁴³, echoing similar findings on hypertension in Japanese-American women⁵⁷. In two recent studies on older Swedes (n = 1,520)⁵⁸ and Danes (n = 1,088)⁵⁹, carriers of the longevity alleles had better self-rated health even after accounting for cardiovascular disease incidence⁵⁸, higher activity of daily living, and fewer bone fractures⁵⁹. However, these latter findings did not survive adjustment for multiple testing⁵⁹. Similarly, two functional longevity variants of FOXO3 failed to associate with mortality and age-related phenotypes in another sample of 643 long-lived Danes⁶⁰. A recent whole-genome sequencing study also found no differences in genotype distribution at FOXO3 longevity variants between 511 healthy elderly and 686 controls⁶¹. However, these small sample sizes suggest that these negative findings may partly reflect a lack of statistical power.

Two recent studies provide insight into how the longevity variants of FOXO3 may act at the molecular and cellular levels. In carriers of the G allele of a longevity variant of FOXO3, the FOXO3 gene was physically closer to its neighboring genes, and when exposed to stress, FOXO3 mRNA expression in lymphoblastoid cell lines derived from carriers increased more than in cell lines derived from non-carriers⁶². In line with those findings, another study showed that the same genetic variant has enhancer functions and that the G allele allows the creation of a novel transcription factor binding site, which induces FOXO3 mRNA expression in response to diverse stress stimuli⁶³.

Collectively, these results suggest that FOXO3 genetic variants contribute to reaching old age, but there is a paucity of human studies that are sufficiently powered to demonstrate the role of FOXO3 in healthy aging⁶⁰. One mechanism of action of the FOXO3 SNPs was only recently uncovered and involves a complex “interactome” whereby cellular stress causes FOXO3 to move close and physically interacts with no fewer than 46 flanking genes on chromosome 6⁶⁴. Rather than just FOXO3, the strong association of FOXO3 with longevity might rely on the central position of FOXO3 in a chromatin domain containing essential genes involved in cell resilience, including autophagy, stress response, energy/nutrient sensing, cell proliferation, apoptosis, and stem cell maintenance^62–64.

MicroRNA-mediated regulation of FoxO3

MiRNAs are regulatory, small non-coding RNAs. The physiological effect of most miRNAs is based on the post-transcriptional regulation of mRNA expression or the inhibition of protein translation⁶⁵. Whereas correlations are often made between the expression levels of a specific miRNA and its predicted gene and protein targets, miRNA/mRNA direct regulatory relationships can be confirmed only via the means of luciferase reporter experiments in vitro. All of the miRNA/FOXO3 regulatory relationships discussed below were confirmed by luciferase validation.

Conclusions

FOXO3 has versatile functions in human health and disease, and recent research offers new insights into the molecular mechanisms underlying the role and regulation of this essential transcription factor. Over the last 3 years, an important part of FOXO3 research has focused on longevity studies combining population epidemiology and molecular investigations, and the aim has been to pinpoint the mechanisms that underlie the role of FOXO3 in longevity. Simultaneously, numerous new findings highlight the important role of miRNAs in the regulation of FOXO3 and their implication in the regulation of cell cycle-related processes. Overall, despite a strong association of FOXO3 with aging phenotypes, its role in healthy aging remains unclear, especially in skeletal muscle. This may constitute an exciting focus for research in the future.