1. Dynamic Regulation of Metabolic Syndromes by
Posttranslational modifications of Forkhead
transcription factor FOXO1
Monica Rivera-Torres1
1University of Puerto Rico, Cayey, PR
Department 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
Caenorhabditis elegans.
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
modificationsincludingphosphorylation,
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 orthologsof 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
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 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
1

2. hyperinsulinemia, where it may be a
regulator of chronic liver disease.
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.
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
GonadotropinReleasing 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 LBT2PS 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 Hormoneβ Gene
Expression in Pituitary Gonadotrope Cells,
University of California San Diego, 2013)
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 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.
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.
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
Posttranslational modifications
Post-translational modification of
proteins is critical for cellular function.
Reversible phosphorylation is a posttranslational modification carried out by
2

3. 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.
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 is
associated with reversible infertility in
human females.
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 1in patients with
hyperinsulinemia is a risk factor for
chronic liver disease.
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
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
3

4. 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 Hormoneβ
Gene Expression in Pituitary Gonadotrope
Cells, University of California, San Diego,
2013)
Regulates Ovarian Prostaglandins
Critical for Reproduction. J. Dev.
Cell. 19, 858-871.
2. Yun-A J, Kyeong-Min L, Mi-Kyung
K, 2009, Fofkhead Transccription
factor Fox01 inhibits insulin- and
transformin
growth
factor-Bstimulated plasminogen activator
inhibitor-1 expression, Biochem
and Biophy R.C. 386 (2009) 757–
761
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.
3. Arriola, D. J., Mayo, S. L., Skarra,
D. V. 2012 FOXO1 Transcription
Factor Inhibits Luteinizing Hormone
β Gene Expression in Pituitary
Gonadotrope Cells. J. Biol. Chem.
287:33424-33435
4. Nakae J, Oki M, Cao Y, The FoxO
transcription factors and metabolic
regulation, 2007, FEBS Letters 582
(2008) 54-67
5. Kovacs K, Lengyel F, Wilhelm F,
2010, Involvement of FKHR
(FOXO1) transcription factor in
human uterus leiomyoma growth,
J. Fert and Stert vol. 94, No. 4
1491-1495
References:
1. Edmonds J W, Prasain J K, Dorand
D. 2010 Insulin/FOXO Signaling
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