Dynamic Regulation of Metabolic Syndromes by Posttranslational modifications of Forkhead transcription factor FOXO1

Monica Rivera-Torres1

1 University of Puerto Rico, Cayey, PRDepartment of Biology

Abstract Proteins such as FOXO1, follow a systematic pathway regulated by

phosphorylation. In this pathway many proteins, kinases, and phosphatases are activated and inactivated, leading to sometimes very dramatic results such as excessive amounts of hormones, or their deficiency. The purpose of this review article is to find the molecular mechanisms by which insulin signaling in FOXO1 is related to metabolic syndromes. The nuclear activity of the transcription factor FOXO1 is dependent on reversible phosphorylation. Therefore, it is crucial to understand how posttranslational modifications such as phosphorylation regulate the activity of certain genes.

Introduction

Forkhead box O (FOXO) transcription factors are involved in the regulation of the cell cycle, apoptosis and metabolism. The activity of Forkhead box transcription factors is tightly controlled by posttranslational modifications including phosphorylation, acetylation, and ubiquitination. Insulin/growth factor signaling has been shown to negatively regulate FOXOs through phosphorylation by AKT, also known as Protein Kinase B, resulting in their active nuclear export and inhibition of their transcriptional activities. Phosphorylation of FOXOs by other kinases, in response to stress, results in their translocation to the nucleus. FOXOs are the mammalian orthologs of DAF-16. Orthologs are

genes in different species that evolved from a common ancestral gene. DAF-16 is a receptor in the cell membrane that regulates longevity, metabolism, and fertility in the nematode Caenorhabditis elegans.

The FOXO family is composed of four members including FOXO1, FOXO3, FOXO4, and FOXO6. FOXOs regulate diverse cell functions, such as cell cycle progression, apoptosis, stress resistance, and metabolism. Because disruption of the FOXO1 gene in mice results in embryonic lethality, the role of FOXO1 in the regulation of reproduction is unknown. Although disruption of FOXO4 has no overt phenotype, FOXO3 null mice have an age-dependent reduction in fertility

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caused by defective follicular growth in the ovary similar to premature ovarian failure in women. In addition to genetic analyses, mechanistic studies have established a role for FOXOs in reproductive tissues, such as the ovary and uterus, but the function of FOXOs in the neuroendocrine control of reproduction remains to be elucidated. Other studies have established another role for FOXOs in patients with hyperinsulinemia, where it may be a regulator of chronic liver disease.

Given that FOXO1 has been shown to regulate Luteinizing Hormone (LH) and Folicule Stimulating Hormone (FSH), one of the purposes of this study was to investigate what regulates FOXO1. Since Gonadotropin Releasing Hormone (GnRH) controls LH and FSH production, and along with insulin it is pulsated in the blood stream, we are investigating the effect of GnRH in FOXO1 phosphorylation. We found that FOXO1 has been expressed in adult mouse pituitaries and mouse gonadotrope cells given the name of LBT2-PS cells and that insulin signaling can regulate FOXO1 phosphorylation and cellular localization in an immortalized gonadotrope derived cell line. (Arriola, 2012) We also found, through previous Western blot analyses, that GnRH decreases insulin-induced phosphorylation; and okadaic acid stops GnRH induced dephosphorylation at FOXO1 phospatase Thr24 site. (Unpublished data from study entitled FOXO1 Transcription Factor Inhibits Follicle Stimulating Hormone β Gene Expression in Pituitary Gonadotrope Cells, University of California San

Diego, 2013)

Insulin has also been shown to increase the activation of plasminogen activator inhibitor-1 (PAI-1). Elevated levels of (PAI-1) in patients with hyperinsulinemia, has been shown to be risky for chronic liver disease. Since insulin inactivates FOXO1 but activates PAI-1, it is critical to understand if this activation has anything to do with the inactivation of FOXO1.

Posttranslational modifications

Post-translational modification of proteins is critical for cellular function. Reversible phosphorylation is a post-translational modification carried out by kinases and phosphatases. Kinases phosphorylate proteins by transferring a phosphate from an ATP molecule to an amino acid such as Serine or Threonine, and phosphatases catalyze the removal of the phosphate. The nuclear activity of the transcription factor FOXO1 is dependent on reversible phosphorylation.

Reversible Phosphorylation

Most proteins in eukaryotic cells undergo reversible phosphorylation. This is a posttranslational mechanism that involves a protein kinase and phosphatases in order to modulate the biological activity of the cell. Protein phosphatases are divided in two major functional groups, protein tyrosine phosphatases (PTPs) and protein serine/threonine phosphatases (PPs). Ser/Thr phosphatases are then divided into

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two families, PPP and PPM. The PPM family compromises phosphatases dependent on Magnesium. The PPP family includes PP1, PP2A, PP4, PP6, PP2B, PP5, and PP7. The most important PPs are represented by PP1 and PP2A.

FOXO1 Transcription Factor

The Forkhead box family is composed of four members including FOXO1, FOXO3, FOXO4, and FOXO6. FOXOs regulate diverse cell functions, such as cell cycle progression, apoptosis, stress resistance, and metabolism. Because disruption of the FOXO1 gene in mice results in embryonic lethality, the role of FOXO1 in the regulation of reproduction is unknown. Although disruption of FOXO4 has no overt phenotype, FOXO3 null mice have an age-dependent reduction in fertility caused by defective follicular growth in the ovary similar to premature ovarian failure in women. In addition to genetic analyses, mechanistic studies have established a role for FOXOs in reproductive tissues, such as the ovary and uterus, but the function of FOXOs in the neuroendocrine control of reproduction remains to be elucidated.

Plasminogen Activator 1

Previous researchers have been studying the role of FOX01 in terms of activation of the (PAI-1). Through a series of Northern Blot analyses of the expression of PAI-1

in insulin stimulation, scientists found that activation of FOXO1 prevents activation of PAI-1 gene expression. This also demonstrates that inactivation of FOXO1 by the insulin induced phosphorylation increases PAI-1 gene expression. Excess of Plasminogen Activator Inhibitor 1 in patients with hyperinsulinemia is a risk factor for chronic liver disease.

Studies using C. elegans

C. elegans are becoming a very popular model for the study of reproduction. They synthesize this model without prostaglandin G/H synthase homologs. Studies in this nematode model have shown that insulin signaling is required for multiple reproductive processes. Some of these are oogenesis, ovulation, oocyte maturation, and sperm guidance. Prostaglandins (PGs) are a lipid hormone that is critical for fertilization, ovulation and other reproductive processes, but its role in sperm guidance is still being debated. Previous sperm guidance RNAi screen implicated two genes with similarity to PG synthases. The results showed that insulin signaling initiated the conversion of a polyunsaturated fatty acid to a type of Prostaglandins. Scientist are still trying to determine weather the increase in velocity of the sperm is due to the sperm’s recognition of the PG cyclopentane ring. These prostaglandins could function to guide sperm to the fertilization site, but this has not been proven, however, it has been shown that they are critical for ovulation and fertilization. The inhibition of these prostaglandins in various mammals

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is associated with reversible infertility in human females.

Infertility studies using mouse pituitary gonadotrope cells

FOXO1 has been identified as a negative regulator of luteinizing hormone (LH) production, a hormone critical for human reproduction. Studies in mice pituitary gonadotrope cells (LBT2 cells) demonstrate that FOXO1 might regulate fertility through modulation of Lhb transcription, the rate-limiting step in the production of the mature hormone. Therefore it is interesting to study the effects of Gonadotropin Releasing Hormone (GnRH) in FOXO1 phosphorylation. Since GnRH decreases the insulin induced phosphorylation signal at two sites of the insulin/FOXO1 pathway, FOXO1 and the protein kinase B(AKT), the pharmacological phosphatase inhibitor okadaic acid has been used because of its specificity to PP2A, PP4, and PP6. It is expected that okadaic acid inhibits the phosphorylation effect on the specific phosphatases PP2A, PP4, and PP6. In a previous Western Blot analysis, Okadaic acid did not affect phosphorylation at the AKT sites, therefore it could be that GnRH is not affecting the Kinase. On the other hand Okadaic acid has been shown to inhibit the GnRH induced dephosphorylation at the FOXO1 pThr24 site. This leads to a hypothesis that GnRH may be affecting a phosphatase of the PP2A, PP4, and PP6 family. (Unpublished data from study entitled FOXO1 Transcription Factor Inhibits Follicle Stimulating Hormone β Gene Expression in Pituitary

Gonadotrope Cells, University of California, San Diego, 2013)

Conclusion

Forkhead transcription factors are found in a vast number of species. These transcription factors are in nucleus, active transcriptionally, and increase hepatic glucose production, decrease insulin secretion, increase food intake and cause degradation of skeletal muscle for supplying substrates for glucose production. They are the major regulators of metabolic homeostasis and insulin signaling regulates them. Reversible phosphorylation of FOX01s is the key point to many processes inside the cell. Understanding the functions of FOXO1s in their active and inactive states may shed the light to treatments for diseases like infertility, chronic liver disease, type 2 diabetes and other metabolic syndromes.

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