INTERACTIONS OF INSULIN-LIKE GROWTH FACTOR-I AND ESTROGEN IN THE BRAIN
Pablo Mendez\ Gloria Patricia Cardona-Gomez^ and Luis Miguel Garcia-Segura^
^Instituto Cajal, C.S.LC, E-28002 Madrid, Spain; ^Group of Neuroscience, School of Medicine, University ofAntioquia, PO Box 1226 Medellin, Colombia
Key words: Estradiol; gonadotrophins; IGF-I; neuronal development; neuroprotection;
synaptic plasticity.
1. INTRODUCTION
Insulin-like growth factor exerts different effects on the development and
plasticity of the nervous system and is a potent neuroprotectant (see Chapters
8 and 10). As in other organs, the actions of IGF-I in the nervous system
may be affected by the interaction with other molecules. In the brain, these
include neurotransmitters, neuromodulators, hormones and other growth
factors. For instance, growth hormone regulates IGF-I expression in the
brain in an anatomically specific manner^ and leukemia inhibitory factor has
been shown to regulate IGF-I levels in peripheral nerves^. The cross-talk
between IGF-I intracellular signaling with the signaling pathways of other
factors may also play an important role in the actions of IGF-I in the nervous
system. This is the case of the interaction of IGF-I and nerve growth factor
(NGF) in peripheral nerves, where the phosphatidylinositol-3 kinase (PI3K)-
Akt-glycogen synthase kinase 3 (GSK3) pathway seems to underlie the
synergism of these two factors on axonal growth^. Neurotransmitters, such as
glutamate, may affect the actions of IGF-I in the brain by interfering with
IGF-I receptor signaling. Recently it has been shown that glutamate at high
concentrations induces loss of sensitivity to IGF-I by phosphorylating the IGF-I receptor docking protein insulin-receptor-substrate (IRS)-l in Ser(307) through a pathway involving activation of protein kinase (PK) A and PKC"^.
One of the best characterized factors that affect IGF-I's actions in the nervous system is the ovarian hormone estradiol. Evidence has accumulated over the past years indicating a close interdependence between the actions of IGF-I and estradiol in the brain. Both factors have in common the duality of being hormones, as well as locally produced neuromodulators, and they exert similar pleiotropic actions in the developing and adult brain. During the development of the nervous system, IGF-I and estradiol promote the differentiation and survival of specific neuronal populations. In the adult brain, IGF-I and estradiol act as neuromodulators, regulate synaptic plasticity, are involved in the response of neural tissue to injury, regulate neurogenesis, and protect neurons against different neurodegenerative stimuli^.
IGF-I and its receptors are expressed at high levels by neurons and glia in the developing rodent brain. However, the expression of IGF-I is dramatically reduced in many brain areas, excluding the hypothalamus, by the time of puberty. In contrast, expression of the IGF-I receptor is maintained in neurons and glia in the adult brain^, suggesting that under physiological conditions the actions of peripheral IGF-I are predominant in adult life^'l
Estradiol is mainly produced in the ovary and reaches the brain from the circulation. In addition, estradiol is formed in the brain from androgen precursors present in plasma and probably also from androgen precursors locally synthesized from cholesterol. Local production of estradiol is important in the developing brain, as inferred from the high level of expression of aromatase, the enzyme that converts testosterone into estradiol^. In the adult rodent brain, the level of aromatase expression is substantially reduced, suggesting a decrease in the local formation of estradiol. Interestingly, both IGF-I and estradiol synthesis are induced in damaged brain regions following brain injury, suggesting a possible role for the local production of these factors in brain recovery^^'^^
In this chapter we focus on the functional implications and the molecular
mechanisms involved in the interaction of IGF-I and estradiol in the brain.
2. INTERACTION OF IGF-I AND ESTROGEN IN NEUROENDOCRINE AND REPRODUCTIVE EVENTS IN THE BRAIN
The nervous system is a target for the ovarian hormone estradiol. This hormone regulates brain development and function, acting on neurons, synapses, and glial cells^^. For many years, the actions of estradiol in the brain were thought to be restricted to areas involved in neuroendocrine regulation and the control of sexual behavior. Today it is evident that estradiol exerts a broad spectrum of actions in many brain areas that are not directly related to reproduction^^'^^.
One of the main mechanisms of action of estradiol in the brain, as in other organs, is the activation of nuclear estrogen receptors. These receptors belong to the steroid/thyroid nuclear receptor superfamily, and once activated they homo- or heterodimerize, interact with DNA, and recruit a cohort of transcriptional cofactors to the regulatory regions of target genes.
The two mammalian estrogen receptors cloned to date, denominated a and ß, are widely distributed throughout the central nervous system (Table 1). In addition, estradiol exerts rapid membrane effects on neural cells, modulating ion channels, neurotransmitter transporters, intracellular calcium levels, and phosphorylation of different kinases^'^^'^^.
As an endocrine signal, IGF-I represents a link between the growth and reproductive axes, and the interaction between IGF-I and estradiol in the brain is of particular physiological relevance for the regulation of growth, sexual maturation, and adult neuroendocrine function. IGF-I regulates gonadotrophins by acting at the level of both the hypothalamus and pituitary^^'^^ IGF-I from peripheral origin reaches the rat hypothalamus, where it appears to be a signal related with the initiation of puberty^^.
Coincident with the onset of puberty, IGF-I mRNA and protein levels increase dramatically in the rat mediobasal hypothalamus and median eminence^^'^^. Decreased IGF-I plasma levels in growth hormone deficient mice, as well as in growth hormone receptor deficient mice, are associated with reproductive deficits, a delay in female puberty, and alterations in the functioning of the hypothalamic-pituitary-gonadal axis. In addition, deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/IGF-I receptor signaling cascade, causes infertility in females^^.
Interactions between estrogen and IGF-I have been reported in brain
areas involved in neuroendocrine control. One such area is the hypothalamic
arcuate nucleus, a key center for neuroendocrine regulation of the growth
and reproductive axes. This nucleus shows estrogen-induced and estrous
cycle-related plastic changes in synaptic connectivity and glial-neuronal
interactions in female rodents^^. During the preovulatory and ovulatory
phases of the estrous cycle, there is a transient disconnection of axo-somatic
y-aminobutyric acid-(GABA)ergic synapses on the arcuate somas of adult female rats. This synaptic remodeling is induced by estradiol and is linked to plastic modifications in astroglial processes^^.
During the estrous cycle, estradiol increases IGF-I accumulation in the arcuate nucleus^^ IGF-I levels increase in the afternoon of proestrus and remain high in the morning of estrus. This effect may be in mediated in part by the increased expression of insulin-like growth factor binding protein-2 by hypothalamic tanycytes, specialized glial cells that express IGF-I receptors and accumulate IGF-I from the cerebrospinal fluid^^. Furthermore, results from in vivo experiments using intracerebroventricular infusion of specific receptor antagonists have shown that both estrogen receptors and IGF-I receptors are involved in the induction of synaptic and glial plastic modifications in the arcuate nucleus that take place during the estrous cycle and are linked to luteinizing hormone release^^. Under normal conditions, the number of synaptic inputs on arcuate neuronal somas of cycling female rats decreases between the morning of proestrus and the morning of estrus. This phasic synaptic and glial remodeling is blocked when the selective and specific IGF-I receptor antagonist JBl is administered in the lateral cerebral ventricle to neutralize the local actions of IGF-I. In contrast, JBl did not affect the number of synapses in proestrus rats, suggesting that IGF-I receptor activation is necessary for the estrogen-dependent synaptic plastic changes, but not for the normal maintenance of synaptic inputs^"^.
Arcuate neurons express IGF-I receptors^ and may, therefore, be a direct target for the actions of JBl. This antagonist may affect pre- and/or postsynaptic mechanisms, since ultrastructural studies have shown that IGF-I receptors are present both in axo-somatic presynaptic terminals, as well as in neuronal somas of the rat arcuate nucleus^. Arcuate astrocytes are another possible cellular target for the IGF-I receptor antagonist, since they also express IGF-I receptors^. Arcuate astrocytes appear to be directly involved in the regulation of synaptic inputs to arcuate neurons^^. Synaptic disconnection of arcuate neurons in estrous females is accompanied by increased expression of glial fibrillary acidic protein (GFAP) and by an increase in the ensheathment of neuronal surfaces by astrocytic processes^^. Interestingly, studies on hypothalamic tissue fragments from ovariectomized rats have shown that IGF-I receptor activation is needed for the induction of GFAP changes by estrogen in the arcuate nucleus^^.
Another alternative mechanism involved in the blocking effect of JB1 on
arcuate nucleus synaptic plasticity may be the regulation of local IGF-I
levels as a consequence of the antagonistic effect of JBl on IGF-I receptors
in tanycytes. As mentioned previously, tanycytes, a specialized form of glial
cells present in the walls of the third ventricle and in the median eminence,
may play an important role in the regulation of local IGF-I levels in the
arcuate nucleus. Tanycytes express IGF-I receptors, are able to accumulate IGF-I from extrahypothalamic sources, and show changes during the estrous cycle in IGF-I immunoreactivity and IGF-I accumulation. Therefore, tanycytes could represent a possible site of action for JB 1 in the inhibition of synaptic plasticity. Indeed, administration of JBl in the rat lateral cerebral ventricle is able to block the accumulation of IGF-I by arcuate nucleus tanycytes^^.
Quesada and Etgen^^'^^ have reported several functional interactions between estrogen and IGF-I receptor in neuroendocrine brain areas. These authors have shown that estradiol treatment induces alpha(lB)-adrenoceptor expression in the hypothalamus and preoptic area and that this hormonal effect is blocked by intracerebroventricular infusion of the IGF-I receptor antagonist JBl. Furthermore, estradiol potentiates the effects of IGF-I on alpha(l)-adrenergic receptor activation in the preoptic area and hypothalamus. Blockade of the IGF-I receptor during estrogen priming blocks estrogen-induced luteinizing hormone release and partially inhibits hormone-dependent reproductive behavior. Quesada and Etgen have proposed that the interaction of estrogen with IGF-I receptor may help coordinate the timing of ovulation with the expression of sexual receptivity .
3. INTERACTION OF IGF-I AND ESTRADIOL ON NEURONAL DEVELOPMENT
The interactions of estrogen and IGF-I are also relevant for brain development. The interaction between these two factors contributes to the development of structural sex differences in the brain through regulation of the survival and differentiation of developing neurons in brain areas involved in the regulation of neuroendocrine events and reproduction^^.
Several studies have demonstrated an interdependence of estrogen receptors
and the IGF-I receptor in promoting the survival and differentiation of
developing hypothalamic neurons^^'^\ Both estradiol and IGF-I promote
neuronal survival and differentiation in primary neuronal cultures grown in a
defined medium deprived of serum and hormones. In these cultures, the
induction of neuronal survival and differentiation by IGF-I is prevented by
inhibiting the synthesis of estrogen receptor a by the addition of a specific
antisense oligonucleotide^^ The effect of IGF-I can also be prevented by the
estrogen receptor antagonist ICI 182,780^\ In turn, the promotion of
neuronal survival and differentiation by estradiol is prevented by blocking
the synthesis of IGF-I in the cultures by using a specific IGF-I antisense
oligonucleotide^\ as well as by the pharmacological blockade of the MAPK and the PI3K signaling pathways, both used by the IGF-I receptor^^.
4. INTERACTION OF IGF-I AND ESTRADIOL ON THE RESPONSE TO BRAIN INJURY AND ADULT NEUROGENESIS
Estradiol and IGF-I may also interact in areas of the brain that are not directly involved in the control of neuroendocrine events and reproduction, as has been shown in studies assessing the neuroprotective effects of these two factors. IGF-I and estradiol have neuroprotective properties and prevent neuronal cell death in different experimental models of neurodegenerative diseases^^'^"^. The interaction of IGF-I and estradiol in neuroprotection has been assessed in ovariectomized rats, using systemic administration of kainic acid to induce degeneration of hippocampal hilar neurons^^, an experimental model of excitotoxic cell death. Both systemic administration of estradiol and intracerebroventricular infusion of IGF-I prevent hilar neuronal loss induced by kainic acid. The neuroprotective effect of estradiol is blocked by intracerebroventricular infusion of the IGF-I receptor antagonist JBl, while the neuroprotective effect of IGF-I is blocked by the intracerebroventricular infusion of the estrogen receptor antagonist ICI 182,780^^. Similar results have been obtained after the unilateral infusion of 6-hydroxdopamine into the medial forebrain bundle to lesion the nigrostriatal dopaminergic pathway^^, a model of Parkinson's disease. Pretreatment with estrogen or IGF-I significantly prevents the loss of substantia nigra compacta neurons and the related motor disturbances. Blockage of IGF-I receptors by intracerebroventricular JB-1 attenuates the neuroprotective effects of both estrogen and IGF-I. These findings suggest that the neuroprotective actions of estradiol and IGF-I after brain injury depend on the coactivation of both estrogen and IGF-I receptors.
Another functional outcome of the interaction of IGF-I with estrogen in
the brain is the regulation of adult neurogenesis. Neural precursors located in
the subgranular zone of the dentate gyrus of the hippocampus proliferate in
adult rodents. The newly generated neurons are functional, integrate in
hippocampal circuits and may be involved in certain forms of hippocampus-
dependent learning. IGF-I and estradiol are among the molecules that have
been identified as modulators of adult neurogenesis^'^^. A recent study has
shown that intracerebroventricular administration of the estrogen receptor
antagonist ICI 182,780 blocks IGF-I-induced neurogenesis in adult
ovariectomized rats^^ suggesting that estrogen receptors are involved in the
action of IGF-I on adult hippocampal neurogenesis (Fig. 1). This interaction of IGF-I with estrogen receptors may occur directly in the proliferating cells, since they express receptors for both IGF-I and estrogen^^.
5. MECHANISMS OF INTERACTION BETWEEN IGF-I AND ESTRADIOL IN THE BRAIN
Analysis of the distribution of the IGF-I receptor and of the two known forms of estrogen receptors (a and ß) in the rat brain reveals that most neural cells expressing IGF-I receptor also express estrogen receptors^^, Colocalization of estrogen receptor a and IGF-I receptor immunoreactivity is observed, by confocal microscopy, in neurons in the preoptic area and hypothalamus. IGF-I receptor immunoreactivity is detected in the cytoplasm and estrogen receptor a immunoreactivity is located in the cell nucleus.
Some neurons in the ventromedial nucleus and the arcuate nucleus are immunoreactive for both receptors. Many neurons in the hypothalamus and preoptic area show estrogen receptor a immunoreactivity only; however, most IGF-I receptor immunoreactive neurons are also estrogen receptor a
39
immunoreactive .
In the hippocampal formation, colocalization of estrogen receptor a and the IGF-I receptor is seen in many pyramidal neurons in the lateral portion of the CAl field. The number of double labeled cells is lower in the CA2 layer and no double labeling is observed in the CA3 or the dentate gyrus^^.
The cerebral cortex presents abundant immunohistochemical colocalization of IGF-I receptor and estrogen receptor a. There is abundant colocalization in layers III and IV from the frontoparietal cortex motor area and to a lesser extent in layer VI. Similarly, many neurons show colocalization of estrogen receptor a and IGF-I receptor in the primary olfactory cortex, whereas colocalization is less abundant in the posterior cingulate cortex and scarce in the frontoparietal cortex somatosensory area^^.
Estrogen receptor ß is expressed in almost all IGF-I receptor immunoreactive neurons in the preoptic area and the hypothalamus. Double labeled cells are abundant in the preoptic area and in the supraoptic, paraventricular and arcuate nuclei. In most neurons the labeling for estrogen receptor ß is observed in the cell nucleus; however, cytoplasm labeling is faint, but consistent, in cells with estrogen receptor ß in nuclei.
Colocalization of estrogen receptor ß and IGF-I receptor is observed in the
cytoplasm. In magnocellular neurons of the supraoptic and paraventricular
nuclei, estrogen receptor ß immunoreactivity is predominantly located in the
cell nucleus and there is little colocalization of estrogen receptor ß and IGF-I
receptor immunoreactivity in the cytoplasm^^.
10000
B 8000
6000
4000
2000
n^^ .^^^ pV ^ ^ o^'\ V^^'^.XX^^ n-\^^ ^^^
P^'" V0^'\.X>^^"
#
6000 5000 4000
^ 3000 2000
1000
I
* *
B
o^^ .-^^^ ov ^ ^ ^O^'^
#
xC\
Figure 1. Total number of BrdU-labeled cells (A) and total number of BrdU-labeled neurons (B) in the granule cell layer and subgranular zone of the dentate gyrus of ovariectomized rats.
Animals received a daily intraperitoneal injection of 10 mg/ml BrdU (5-bromo-2-
deoxyuridine; Sigma, St. Louis, MO.) in 0.9% NaCl solution during six consecutive days
(daily dose: 50 mg/kg b.w.). Animals were killed 24 h after the last injection of BrdU. Data
are expressed as means ± S.E.M. E2, Animals treated with 17-ß estradiol; IGF-I, animals
treated with insulin-like growth factor-I; ICI, animals treated with the estrogen receptor
antagonist ICI 182,780. The number of animals in each experimental group was: control (n =
5); estradiol (n = 6); IGF-I (n = 5); IGF-I -f ICI (n = 6); IGF-I + E2 (n = 8); IGF-I + E2 + ICI
(n = 6); ICI (n = 7). Asterisks = significant differences (*/? < 0.05, ** p < 0.01, *** p < 0.001)
versus control values.
Many pyramidal neurons of the CA3 layer from the hippocampus are immunoreactive for both estrogen receptor ß and IGF-I receptor, with overlapping colocalization in the cytoplasm. In the dentate gyrus there is cytoplasmic colocalization of both receptors in many interneurons. The colocalization of estrogen receptor ß and IGF-I receptor is low in the rest of the hippocampal fields^^.
Colocalization of IGF-I receptors with Area studied ER alpha ER beta
N,A N,A N,A N,A A N,A N,A N,A N,A N,A PREOPTIC AREA
Medial preoptic nucleus HYPOTHALAMIC NUCLEI
Supraoptic & paraventricular Ventromedial & Arcuate HIPPOCAMPUS
CAl CA2 CA3
Dentate gyrus CEREBRAL CORTEX
Primary olfactory cortex Posterior cingulate cortex Frontoparietal cortex
Motor area Layer I Layers II-V Layer VI
Somatosensory area Layers I-VI
CEREBELLAR CORTEX Molecular layer
Purkinje cell layer Granule cell layer White matter
N -
- N N N - - N N
- N N N - - - -
N,A N,A N N,A
A N A A
Table 1. Coexpression of IGF-I receptor and estrogen receptors in selected brain areas. A, Astrocytes; N, neurons. Based on ref. 39.
Estrogen receptor ß and IGF-I receptor are colocalized in the
frontoparietal cortex motor area from layer II to layer VI. The co-expression
is more abundant in layers II and III. There is scarce colocalization of
estrogen receptor ß and IGF-I receptor in the posterior cingulate region and
the frontoparietal cortex somatosensory area. A high proportion of neurons
show coexpression of estrogen receptor ß and IGF-I receptor in the primary
olfactory cortex. Cytoplasmic coexpression of the estrogen receptor ß and IGF-I receptor is observed in the Purkinje cells of the cerebellum^^.
Colocalization of immunoreactivity for estrogen receptor ß and IGF-I receptor is observed in glial cells with the morphological appearance of astrocytes in all brain areas. These glial cells are particularly abundant in the hilus of the dentate gyrus and in the white matter of the cerebellum^^.
Table 1 is a summary of the distribution of immunoreactive cells in the preotic area, the hypothalamus, the cerebral cortex, the hippocampus and the cerebellar cortex, in which colocalization of IGF-I and estrogen receptors has been observed.
The abundant coexpression of estrogen receptors with IGF-I receptors in neurons and glia in the brain indicates that interactions of the intracellular signaling pathways of IGF-I receptors and estrogen receptors are possible in many brain cells. In different cell lines, including neuroblastoma cells, IGF-I may activate estrogen receptors in the absence of estradiol'^^. Whether or not this is also valid for the brain in vivo is unknown. However, both in vitro and in vivo studies have shown that in the brain estradiol may activate the two main signal transduction cascades coupled to the IGF-I receptor: the PI3K and the MAPK signaling pathways.
In primary cultures of astroglia and in explants of cerebral cortex, estradiol induces the rapid activation of ERK"^^'"^^. The estradiol-induced activation of ERK may be detected in vivo after systemic administration of the hormone"^^. Estradiol also induces the phosphorylation of Akt in dissociated neurons from the rat cerebral cortex and the hippocampus, in cerebral cortical explants and in the adult rat brain in v/vo^^'^'*. Furthermore, IGF-I and estradiol act synergistically to increase Akt activity in the rat brain"^"^. Figure 2 shows the result of an experiment in which IGF-I was infused with an osmotic minipump into the lateral cerebral ventricle of ovariectomized rats to determine if a sustained increase in intracerebral IGF- I levels affects the activation of Akt/PKB by estrogen in the hippocampus.
Animals were infused for 6 days with IGF-I and then injected with estradiol and killed 24 h later. A significant increase in the phosphorylation of Akt/PKB was observed in rats injected with estradiol alone compared to animals treated with vehicle. The infusion of IGF-I in the lateral cerebral ventricle was, by itself, unable to increase the phosphorylation of Akt/PKB.
However, the hippocampus of animals that received the
intracerebroventricular infusion of IGF-I showed a striking increase in
Akt/PKB phosphorylation in response to estradiol compared to animals
infused with vehicle. This synergistic effect of IGF-I and estradiol was not
observed for ERKl and ERK2^^
Hippocampus
- pAkt - Akt
tt 800 ' 3
.a 600 ' g 400 ^
O
"W 2 0 0 -
c
XL
E 1
pAkt
.J
xuaj
E+l
Figure 2. Levels of phosphorylated Akt (pAkt) in the hippocampus of ovariectomized rats killed 24 h after systemic administration of estradiol (E), after 7 days of intracerebroventricular infusion of IGF-I (I) or 24 h after systemic administration of estradiol and 7 days after intracerebroventricular infusion of IGF-I (EI). Levels of basal Akt were used as a control for protein loading. C, Control rats injected with vehicles. Data are normalized to control values and represent the mean ± SEM of arbitrary densitometric units. Samples from 6 rats were analyzed for each experimental group. Asterisks represent significant differences (p
< 0.05) versus control values.
Glycogen synthase kinase 3 (GSK3), downstream of Akt, may also be a
point of interaction of estrogen and IGF-I signaling. Physiological
phosphorylation of microtubule-associated proteins by GSK3ß may be
involved in the regulation of microtubule dynamics, neuritic growth,
synaptogenesis and synaptic plasticity"^^. However, under pathological
conditions, GSK3ß may be responsible for the hyperphosphorylation of tau
in Alzheimer's disease"^^ and its inhibition is associated with the activation of
survival pathways in neurons"^^. Interestingly, estradiol regulates the activity
of GSK3 and decreases the phosphorylation of tau in the rat hippocampus in
vivo'^^ Furthermore, estradiol increases the association of tau with
phosphorylated GSK3ß, with the p85 subunit of the phosphatidylinositol 3-
kinase and with ß-catenin, another substrate of GSK3'^^. These observations
are coherent with results showing that systemic administration of estradiol to
adult ovariectomized rats results in a transient increase in tyrosine
phosphorylation of the IGF-I receptor, in a transient interaction of the IGF-I
receptor with the estrogen receptor a, but not ß, and in an enhanced
interaction of estrogen receptor a with the p85 subunit of PI3K in the brain
(Fig. 3)'*'.
IP: IGF-IR
WB! p-Tyr
WBi ERa
SSkDi*- --'A; - ^ ^ ^ ^ ^m- "*^ ***^ WB: IGF-IR C I h 3h 6h 12h 24h
30D
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17p-Estradiol
B
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Lys
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&Dkl><s— ^tt^
C Ih 3h 17p-Estradiol
WBi
WBi
WBi ERa
ERp
IQF-IR
Figure 3. (A) Estrogen treatment in vivo activates IGF-IR and induces the association
between IGF-I receptor (IGF-IR) and estrogen receptor a (ERa) in the hypothalamus of
ovariectomized rats. Animals were killed at the indicated periods of time after acute estrogen
treatment. IGF-I receptor was immunoprecipitated from total lysates. The immunocomplexes
were separated using sodium dodecyl su 1 fate-polyaery 1 amide gel electrophoresis (SDS-
PAGE), and probed with an antiphosphotyrosine antibody (p-Tyr, upper gel), an antibody
against estrogen receptor a (middle gel). To verify equal loading, membranes were probed
with an antibody against IGF-I receptor (lower gel). The histogram shows the densitometric
analysis of the Western blots. Values are the percentages of control levels and are represented
as means ± S.E.M. from three different animals. Data were analyzed by ANOVA followed by
the Bonferroni's test. Asterisks indicate significant differences (p < 0.05) versus the values of
control animals injected with vehicle (C). (B) Estrogen treatment induces the association
between IGF-I receptor and estrogen receptor a, but not estrogen receptor ß (ER ß) in the
hypothalamus of ovariectomized rats. Animals were treated with estrogen for 1 h, 3 h or with
vehicle. Hypothalamic lysates were immunoprecipitated with IGF-I receptor antibody and
probed with estrogen receptor a, estrogen receptor ß and IGF-I receptor antibodies. The
ability of these antibodies to recognize the antigens in total hypothalamic lysates (Lys) is also
shown. (C) Parallel immunoprecipitations were performed using a nonimmune (n.i.) serum to
verify the specificity of the bands detected by Western blotting.
Together these findings suggest that estrogen receptor a may affect IGF-I actions in the brain by a direct interaction with some of the components of IGF-I signaling, such as the IGF-I receptor, PI3K and GSK3ß (Fig. 4).
5.1 Functional Implications of the Interactions in the Signaling of IGF-I Receptor and Estrogen Receptor- a in the Brain
The interaction of estrogen receptor-a with the signaling pathways of IGF-I receptor in the brain may explain the interaction of estradiol and IGF-I in the regulation of different neural events. ERK and Akt may be involved in the interaction of IGF-I and estradiol in the regulation of neuronal differentiation, synaptic function, synaptic remodeling, neuroprotection, and sexual behavior^'"^^'^^. The synergistic interaction of IGF-I and estradiol in the phosphorylation of Akt may be critical for the regulation of glucose transport and metabolism^^'^^ and for neuroprotective actions. Akt regulates several transcription factors that may be involved in the control of neuronal survival, such as CREB,^^ NF-kappaB^"^, and several members of the forkhead family^^'^^. In addition, activation of Akt results in the phosphorylation of the Bcl-2 family member Bad and this may suppress Bad-induced cell death^^'^^.
Furthermore, Akt activation enhances Bcl-2 promoter activity^^ and both
IGF-I and estrogen induce Bcl-2 expression in neurons. Interestingly, IGF-I
receptor activation is necessary for the induction of Bcl-2 by estradiol in the
adult brain"^^. Also downstream of Akt, IGF-I and estradiol may interact on
the regulation of microtubule dynamics, neuritic growth, synaptogenesis,
synaptic plasticity, and neuronal survival, acting on GSK3ß and its
substrates, ß-catenin, and tau'^^
IGF-IR 0
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Neuronal diferentaton Cell survival Neuroendocrine effects Glucose metabolism
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Microtubule dynamics Neuriticgroiftrth Neuroprotection
NeuriiicgroiArth Synapto genesis Synaptic Plasticitir
Cell survival
