FOXO Signaling Pathways
February 17, 2021
The above diagram represents the FOXO gene pathways. FOX proteins are named for a gene found in fruit flies that cause the insects to have forked structures on their heads (supplying the “F”) and a particular group, known as “box”, of specialized genes (supplying the “OX”). They’re named alphabetically, from FOXA to FOXS. There are over 100 subclasses of FOX proteins in humans, such as FOXA, FOXR, FOXE, etc. and they have many functions. An important group of FOX proteins is the class “O”. This class is regulated by the insulin/Akt/mTOR signaling pathway. Invertebrates have a single FOXO gene, whereas mammals have four: FOXO1, FOXO3, FOXO4, and FOXO6. FOXO proteins regulate stress resistance, cellular turnover, apoptosis, glucose and lipid metabolism, and inflammation. FOXO factors are evolutionarily conserved mediators of insulin and growth factor signaling. FOXO proteins act as transcription factors by binding to specific regions on DNA, thereby controlling the transmission of genetic information and influencing the chemical “blueprint” for proteins. Of all the different groups the FOXO group may be the most important. The following is a summary of these ideas:
As stated, there is accumulating evidence that FOXO factors play an important role in stem cell biology and tissue homeostasis. There is also a great deal of research on the FOXO pathway and its relationship with osteoarthritis and osteoporosis both of which consume a large portion of our health care dollars. During aging, the balance of removal and regeneration of cells in tissues becomes disturbed mainly due to a decrease in the regenerative potential of adult stem cells. The FOXO family of transcription factors (proteins that can bind to DNA and “switch on” other genes) regulate the expression of genes in cellular physiological events including apoptosis (cellular programmed death), cell-cycle control, glucose metabolism, oxidative stress resistance, and longevity. These six pillars can be the blueprint for significant anti-aging strategies in addition to allowing for greater stem cell success in both the lab and the real world.
Many transcription factors play a key role in cellular differentiation and the delineation of cell phenotype (the physical appearance from the expression of one or more genes). Transcription factors are regulated by phosphorylation, ubiquitination, acetylation/deacetylation and interactions between two or more proteins controlling multiple signaling pathways. The regulation of these various processes typically involves the addition or removal of certain chemical compounds to a protein. These pathways regulate different physiological processes and pathological events, such as cancer and other diseases.
The forkhead transcription factors have four members: FOXO1, FOXO3, FOXO4, and FOXO6. FOXO1 and FOXO3 are expressed in nearly all tissues. FOXO4 is highly expressed in muscle, kidney, and colorectal tissue while FOXO6 is primarily expressed in the brain and liver. The following illustration shows the various Forkhead transcription factors. It shows the far-ranging influences that these transcription factors have:
Over the last decade, studies have demonstrated that FOXOs play critical roles in a wide variety of cellular processes. FOXOs transcriptionally activate or inhibit downstream target genes, thereby playing an important role in proliferation, apoptosis, autophagy, metabolism, inflammation, differentiation, and stress resistance. Remember when we are dealing with anti-aging we want to influence downstream events from an upstream process. Deletion of FOXOs has given insight into their function. For instance, deletion of FOXO1 is lethal; it causes embryonic cell death due to incomplete vascular development. Deletion of FOXO3 is not lethal but affects lymph proliferation, widespread organ inflammation, age-dependent infertility, and decline in the neural stem cell pool. Deletion of FOXO4 exacerbates colitis in response to inflammatory stimuli. Deletion of FOXO6 displays normal learning but impaired memory consolidation.
The process of aging is accompanied by a decline in physiological function and cellular maintenance. It is known that aging dramatically alters gene expression and transcription factor activity. FOXO functions downstream of insulin/insulin-like growth factor (insulin/IGF). Studies have found that lifespan extension effects of insulin/IGF deficiency depend on FOXO activity, probably through the transcriptional regulation of key longevity assurance pathways. However, how FOXO elicits this response remains to be fully elucidated.
FOXO proteins are tightly regulated to ensure that transcription (first step in protein synthesis) of specific target genes is responsive to environmental conditions. A major form of regulation is Akt-mediated phosphorylation of FOXO in response to insulin or growth factors. This can be seen on the following diagram:
In the absence of insulin or growth factors, FOXO transcription factors are located in the nucleus, where they specify target gene expression. We are able to see the various tasks accomplished by the FOXO genes including DNA repair, Cell Cycle arrest, help eliminate reactive oxygen species, and have some effects on glucose metabolism. When insulin and other growth factors are present they result in phosphorylation which subsequently results in the export of the FOXO proteins from the nucleus to the cytoplasm thereby decreasing expression of FOXO target genes. This is regulated by the Akt-pathway. The opposite happens when the FOXO genes are stimulated. FOXO proteins are phosphorylated by other protein kinases which phosphorylate FOXO under conditions of oxidative stress. This phosphorylation causes the movement of FOXO from the cytoplasm to the nucleus, thus opposing Akt’s action. Once in the nucleus the FOXO genes can do their work.
WHAT ABOUT THE DIFFERENT CELLULAR PROCESSES?
AUTOPHAGY is a key player in the aging process. Autophagy involves the disassembly and recycling of unnecessary or dysfunctional cellular components. It allows the orderly degradation and recycling of cellular components. Premature aging and age-related disorders have been related to defects in autophagy. FOXO proteins regulate many genes responsible for autophagy.
Autophagy has important effects that occur both within the cell and outside of the cell. Within the cell, autophagy helps to decrease oxidative stress, increase genomic stability (which aids in the prevention of cancer), increase bioenergetic metabolism, and increase the elimination of waste. Outside of the cell, autophagy helps to decrease inflammatory responses, increase neuroendocrine homeostasis, increase surveillance of cancer by the immune system, and increase the elimination of aging cells.
CELL CYCLE ARREST
Cells constantly monitor their cell cycle status at various checkpoints. These checkpoints help ensure the accuracy of DNA replication and division and provide time for DNA repair. In some scenarios, FOXO blocks the cell cycle by either switching on cell cycle inhibitors or by switching off cell cycle activators. But FOXO is highly sensitive to physiological context and needs, and under conditions of cellular stress, it mediates cell cycle arrest to allow time for repair of damaged DNA and cellular detoxification.
When dealing with the cell cycle it might appear strange that FOXO could induce both stress resistance and cell death? The regulation of stress-resistance genes and pro-apoptotic genes by FOXO is not necessarily a paradox. FOXO factors may orchestrate different patterns of gene expression based on the intensity of the stimulus, perhaps activating stress-resistance genes under mild conditions but pro-apoptotic genes when the intensity of stress stimuli increases beyond a certain threshold. It is also possible that FOXO factors regulate different genes in different cell types, causing apoptosis in some cells (e.g. neurons, lymphocytes) while promoting survival in others. Importantly, the induction of apoptosis by FOXO may cause the death of damaged or abnormal cells, therefore benefiting the longevity of the entire organism.
FOXO PATHWAYS AND ENERGY HOMEOSTASIS
The FOXO pathway has been called the Transcriptional Chief of Staff of Energy Metabolism. FoxO1 is highly expressed in insulin-responsive tissues, including pancreas, liver, skeletal muscle and adipose tissue, as well as in the skeleton. In all these tissues FoxO1 orchestrates the transcriptional cascades regulating glucose metabolism. Indeed, FoxO1 is a major target of insulin which inhibits its transcriptional activity via nuclear exclusion. In skeletal muscle FoxO1 maintains energy homeostasis during fasting and provides energy supply through breakdown of carbohydrates, a process that leads to atrophy and underlies glycemic control in insulin resistance. In a dual function, FoxO1 regulates energy and nutrient homeostasis through energy storage in white adipose tissue, but promotes energy expenditure in brown adipose tissue. In its most recently discovered novel role, FoxO1 acts as a transcriptional link between the skeleton and pancreas as well as other insulin target tissues to regulate energy homeostasis. We can see the importance of these concepts in the following:
FoxO1 is a unifying regulator of energy metabolism through the skeleton and peripheral organs
FOXO PATHWAYS AND OSTEOARTHRITIS
FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. Observations suggest that FoxO transcription factors play a key role in cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage. FoxO transcription factors control the expression of genes that are essential for maintaining joint health. The following illustration shows what the lack of FOXO protein transcription and subsequent oxidative stress contribute to in the joint:
The next illustration shows this more succinctly:
FOXO PATHWAYS AND OSTEOPOROSIS
Just like in the joint, FOXO pathways have significant effects on osteoporosis. The effects can sometimes be confusing. How the FOXO proteins function in bone metabolism is a bit more complicated than in the joint. The proper stimulation of the FOXO pathways will encourage the formation of new bone. The cells which make new bone, namely the osteoblasts will have increased survival by FOXO stimulation. At the same time the FOXO pathway will diminish activity of cells which cause bone resorption. Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas FoxO transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, 3 and 4 in three-month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 gene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis, and increased osteoblast number, bone formation rate and vertebral bone mass. FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis. In the future, research will become devoted to the study of supplements and medication which stimulate the FOXO pathway which may become a viable alternation for Osteoporosis treatment. The following diagram shows some of the relationships between the FOXO proteins and the various cells in bone metabolism. There is still much we need to learn concerning this topic.
How to Increase FOXO Proteins
The enzyme SIRT1 increases FOXO DNA binding by deacetylating FOXO in response to oxidative stress. So, what happens is that the FOXO leaves the cytoplasm and enters the nucleus ultimately affecting the DNA. FOXO proteins get increased in response to cellular stress and increased energy depletion. Taking it one step further we find that many things which stimulate the Sirtuin genes will stimulate the FOXO genes. Calorie restriction increases sirtuins as well as FOXO factors. For instance, fasting for forty-eight hours elevates FOXO1,3, and 4 by 1.5-fold and but when one eats it will drop back to baseline. FOXO1 is also critical for adapting to fasting by activating gluconeogenesis in the liver, which can make the liver produce glucose whether from amino acids or fatty acids. This can be important in someone who is following a Keto diet. Another method of increasing FOXO is high intensity exercise. FOXO factors are important for regulating muscle energy homeostasis.
In response to heat stress, FOXO contributes to increased heat shock protein levels. Heat shock proteins will protect DNA from damage and maintains cellular resistance. One way they do this is to make sure that proteins fold properly in the cell. Taking this to a more practical level, taking a sauna or exercising and sweating can promote FOXO activation and subsequent heat shock protein. Exposure to cold stress production. Hypoxia will also activate FOXO3. The general trend for increasing FOXO follows the same pattern as the other longevity pathways such as AMPK and Sirtuins. Energy deprivation and adaptation to stress can lead to more resilient and longer life. It forces the body to continue producing energy and survive in situations of low nutrients and thus become really efficient at its own metabolic processes. FOXO3 is activated by dietary components, such as EGCG, which is found in green tea, and by quercetin, which is found in onions and apples.
WHY STIMULATE THE FOXO PROTEINS?
Why would you want to activate FOXO proteins? FOXO proteins activate genes that maintain healthy joints and bone structure. People with osteoarthritis have significantly lower FOXO proteins. FOXO transcription factors modulate autophagy, which promotes cellular turnover and maintenance. Defects in autophagy are associated with age-related diseases. FOXO factors are important for stem cell production and DNA repair. FOXO1 and FOXO3 promote mitophagy which is mitochondrial autophagy FOXO proteins suppress tumorigenesis in cancer. FOXO factors increase the antioxidant capacity of cells, which influence aging and promote longevity. Reactive oxygen species and oxidative stress activate FOXO pathway to adapt to the stress. Inactivity of FOXO factors accelerates atherosclerosis and compromises stem cell proliferation.
HOW ABOUT THE FUTURE OF FOXO PROTEINS?
Is there a connection between FOXO and cancer? FOXO proteins were originally identified in human tumors. They play an important role in cell-cycle arrest, DNA repair, and apoptosis cell functions that go awry in cancer the FOXO family is thought to coordinate the balance between longevity and tumor suppression. An example of this is found in certain breast cancers. In these cancers, FOXO3 is sequestered in the cytoplasm and inactivated. Expression of active forms of FOXO in tumor cells prevents tumor growth in vivo. Additionally, protein partners of FOXO, such as p53 and SMAD transcription factors, are tumor suppressors. Investigating the ensemble of FOXO protein partners will provide insight into the connection between aging and cancer. The following illustration best defines this relationship:
The above entities show the far-reaching hands of the FOXO proteins. These hands all have a direct effect on aging and disease prevention.
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