Am J Physiol Endocrinol Metab 307: E133–E140, 2014.
First published June 3, 2014; doi:10.1152/ajpendo.00626.2013.
Review
2013 SOLOMON BERSON AWARD LECTURE
Extranuclear estrogen receptor’s roles in physiology: lessons from
mouse models
Ellis R. Levin
Departments of Medicine and Biochemistry, University of California-Irvine and Long Beach Veterans Affairs Medical Center,
Long Beach, California
Submitted 18 November 2013; accepted in final form 10 April 2014
membrane estrogen receptor; signal transduction; steroid receptors
the eminent endocrinologist
Hans Selye first described rapid actions of steroid hormones in
mammals (58). Rapid actions of glucocorticoids in vascular
models were not consistent with the emerging field of gene
regulation by nuclear steroid receptors. Because of technological limitations, the mediators of these rapid actions of glucocorticoids were not defined in that period. In contrast, in the
1960s and 1970s, Pietras and Szego (48, 49), along with Szego
and Davis (59), described rapid actions of estrogen, including
calcium flux and cAMP generation that occurred in seconds
after exposure of cells to this steroid. These authors provided
evidence of estrogen binding to a presumed receptor, possibly
at the cell surface, but the nature of this probable binding
protein was unknown. These observations were consistent with
the later description and isolation of a membrane protein that
served as a receptor for plant brassinosteroids, bona fide
steroids that mediate flowering and fertility. The brassinosteroid receptor was found to be a plasma membrane tyrosine
kinase (64), establishing that the ancient and conserved function of steroids from plants to humans results from actions at
the membrane. These rapid signals seemed unlikely to emanate
from nuclear receptors, whose transcriptional function was
defined as being much slower.
MORE THAN HALF A CENTURY AGO,
Address for reprint requests and other correspondence: E. R. Levin, Medical
Service (111-I), Long Beach VA Medical Center, 5901 E. 7th St., Long Beach,
CA 90822 (e-mail: [email protected]).
http://www.ajpendo.org
Today, we know that the distribution of steroid receptors in
vertebrates favors nuclear localization. Increased genome complexity occurs as one moves up the phylogenetic order and
probably provides the impetus for evolution to create nuclear
pools of steroid receptors. This allows binding of these receptors to promoters and especially multiple steroid response
elements at enhancer DNA throughout the genome of many
cells, regulating gene expression that is necessary for normal
development and function. Nuclear steroid receptors also serve
as a model to understand the detailed mechanisms of transcription (10), resulting in most research in this area being focused
on the nuclear receptor pool.
More recently, there has been increased interest in defining
the nature and functions of extranuclear receptors. It is now
appreciated that steroid hormone action results from the integrative actions and cross-talk between receptor pools within
cells. In this review of current understanding, I detail key
developments in the area of extranuclear estrogen receptor
(ER) that serves as a model to investigate all steroid hormone(s) action.
Nature of Extranuclear ER
Using antibodies to classical “nuclear” ER␣, a protein
with comparable features was identified in the vicinity of the
plasma membrane by immunohistochemistry (34). Furthermore, the ability of estrogen to bind to a protein at the
membrane was decreased significantly by antisense oligonucleotides to classical ER␣ (29). When ER␣-null cells
E133
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Levin ER. Extranuclear estrogen receptor’s roles in physiology: lessons from
mouse models. Am J Physiol Endocrinol Metab 307: E133–E140, 2014. First
published June 3, 2014; doi:10.1152/ajpendo.00626.2013.—Steroid receptors exist
and function in multiple compartments of cells in most organs. Although the
functions and nature of some of these receptors is being defined, important aspects
of receptor localization and signaling to physiology and pathophysiology have been
identified. In particular, extranuclear sex steroid receptors have been found in many
normal cells and in epithelial tumors, where they enact signal transduction that
impacts both nongenomic and genomic functions. Here, I focus on the progress
made in understanding the roles of extranuclear estrogen receptors (ER) in physiology and pathophysiology. Extranuclear ER serve as a model to selectively
intervene with novel receptor reagents to prevent or limit disease progression.
Recent novel mouse models and membrane ER-selective agonists also provide a
better understanding of receptor pool cross-talk that results in the overall integrative
actions of sex steroids.
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MEMBRANE ESTROGEN RECEPTORS
Additional Membrane-Localized ER
There is evidence in some organs that endogenous ER␤
also localizes to the plasma membrane (47). ER␤ was first
described in 1996 and is the second receptor that mediates
some actions of estrogen in various organs (19, 28). Many
studies have indicated that truncated forms of ER␣ or
alternative steroid-binding proteins are found in a variety of
organs. ER␣ that is 46 (22) or 36 kDa (63) in size has been
reported at the membrane, especially in breast cancer cell
lines (63). ER␣ of 46 and 36 kDa are products of alternative
promoter usage that generates the splice isoforms, and the
truncated receptors have been demonstrated in various
mammalian organs (13, 63). However, confirmation of the
presence of 46-kDa receptors at the membrane of normal
organs is not well established. Rather, abundant 66-kDa,
full-length ER␣ at the cell membrane has been identified by
many investigators in multiple animal and cell models, and
the existing evidence suggests that this form of the receptor
mediates most of the rapid actions of estrogen (15). Regarding the 36-kDa ER␣, there is no conclusive evidence from
the required multiple laboratories that this receptor performs
important functions underlying normal or abnormal physiology. Furthermore, the modest abundance of this endogenous receptor at the membrane compared with ER␣ of 66
kDa in any organs/cells suggests a limited role.
Most recently, it has been reported that an orphan G-protein-coupled receptor, GPR30, serves as an ER and mediates
E2 signaling from the membrane (12) or the endoplasmic
reticulum (56). There are few publications supporting the
idea that estrogen binds to GPR30, also designated as
GPER1. Saturation binding by H3-E2 of endogenous membrane GPR30 in classical ER␣ negative breast cancer cells
showed ⬍300 disintegrations/min specifically bound (61),
often a level of noise in receptor binding studies. In numerous ER␣-null cells that express endogenous GPR30/GPER1,
neither others nor we have seen appreciable specific binding
by or signaling in response to E2 (32, 37). Furthermore,
several laboratories have created GPR30-knockout (KO)
mice (16, 33) and report few phenotypes in response to a
variety of stresses or under basal conditions and no overlap
with the profound phenotypes of ER␣- or ER␤-KO mice.
However, in some cell types, especially cancer cells, there
may be collaboration between GPR30 and membrane ER␣
as part of a larger signalsome at the membrane, thus transmitting downstream rapid signals (3). In opposition to this
idea, some investigators have not seen a functional linkage
between membrane-localized ER␣ and GPR30 in breast
cancer and other cell types (24, 60). At present, the endogenous ligand for GPR30 has not been identified, and it
remains to be determined whether GPR30 and membrane
ER␣ meaningfully collaborate in vivo.
Trafficking to and Functioning of ER at the Membrane
Attachment of palmitic acid to an internal cysteine residue
promotes ER trafficking to the membrane (Fig. 1) (1). Palmitoylation of ER␣ at cysteine 451 (mouse)/447 (human) is
required for ⬃5% of total cellular receptors to localize to this
cellular domain (1, 39). Palmitoylation at C451 occurs from the
enzymatic actions of the palmitoyl acyltransferase proteins
(PATs) DHHC7 and -21, resulting in disulfide bond linkage of
the palmitic fatty acid with ER␣ (40). Palmitoylation also
requires additional residues as part of a nine-amino acid motif
that promotes the physical association of the PATs with ER␣
(39). Heat shock protein 27 is also important for promoting
palmitoylation, probably by opening up the ER␣ monomeric
structure to allow the DHHC PATs to gain access to the
palmitoylation motif (50). Importantly, all of these aspects are
highly conserved for the membrane localization of both progesterone and androgen receptor proteins, as we defined earlier
(39, 40, 50). ER␤ also undergoes palmitoylation and subsequent membrane localization in some cell types, enacting rapid
signals in cardiomyocytes and cardiac fibroblasts, in vitro and
in vivo (41, 42).
The process of palmitoylation occurs only on ER monomers,
and therefore, E2-induced receptor dimerization limits the
available receptors that can undergo this posttranslational modification and traffic to the membrane (40). In breast cancer cells
expressing endogenous ER␣, nonreducing gel electrophoresis
showed that ⬃85% of receptors at the membrane are monomers in the absence of E2. However, dimerization of membrane
ER␣ occurs within seconds when cells are exposed to E2, and
this structural change is required for rapid signaling (51) as
well as for transcriptional functions of nuclear ER. Rapid
signaling by both ER␣ and ER␤ first results in the activation of
discrete G␣ and G␤␥ proteins (20, 54). Linkage of ER to the
activation of specific G protein subunits occurs in a cell- and
context-related fashion to control the downstream signal transduction pathways that provide the cellular response to various
stimuli. Rapid depalmitoylation also occurs (14), perhaps to
regulate the length and type of signal transduction, but this
function has not been established in many in vitro or any in
vivo models.
Palmitoylation results in the interaction of ER with the
caveolin-1 protein that serves as the transporter of the
steroid receptor to caveolae rafts within the cell membrane
(1). Both ER isoforms have been found to be highly enriched in freshly isolated caveolae rafts (20, 52) and prob-
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were transfected with a single plasmid encoding classical
ER␣, both nuclear and membrane-localized ER were produced, and the cells responded to ligand [17␤-estradiol (E2)]
with rapid signal transduction (54). These results suggested
that the membrane ER␣ is the classical nuclear receptor
protein localized to this additional cellular domain. Rapid
signaling by E2 at the membrane was consistent with the
rapid actions of numerous steroids acting at often-undefined
receptors in a variety of cells (23). Subsequent work showed
the presence of endogenous membrane and nuclear ER␣ in
human breast cancer cells that were identical by mass
spectrometry analysis of subcellular proteins isolated from
estradiol affinity columns (37). Additionally, expression of
just the ligand-binding domain (E domain) of ER␣ resulted
in membrane localization and rapid signal transduction in
response to estrogen. The important functional consequences of engagement of membrane-localized ER by estrogenic compounds included protection against developing
metabolic bone disease (18). These results suggested that
important actions of the sex steroid might originate from
signaling by a plasma membrane-localized ER␣ pool, often
collaborating with liganded nuclear steroid receptors (38).
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MEMBRANE ESTROGEN RECEPTORS
E135
ably initiate signal transduction from this location. ER have
also been found in early endosomes, but the precise nature
of these organelles and the receptor functions that may
occur here are unclear. Caveolin-1 also serves as a scaffold
protein to bind and tether many signal molecules to the
confined space in the vicinity of the membrane caveolae
rafts. Scaffolding enhances the physical interactions of ER
and G␣ and G␤␥ subunits that promote E2/ER activation of
proximal signals in seconds, such as calcium flux, cAMP
generation, and tyrosine kinase activation (e.g. Src, Ras) as
examples. Signaling to the transactivation of growth factor
receptors in cancer cells (53) results in activation of MEK/
ERK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, impacting cell fate, proliferation, migration, and
many other processes (53, 57). Rapid signaling in nontrans-
formed cells impacts physiological processes, including the
nongenomic regulation of protein function (e.g., from phosphorylation of kinases and other enzymes) and the regulation of transcription by acting in concert with nuclear ER
(genomic impact from signaling).
Metabolic Effects of Membrane ER Signaling
Recent studies suggest that estrogen regulates glucose metabolism, at least partly from membrane ER signaling, both in
normal and transformed cells (31, 65). A recent review details
the extensive effects of estrogen in various aspects of normal
organ metabolism (25). Breast cancer is a highly glycolytic
malignancy, deriving ATP, cyclic nucleotides, NADPH, and
phospholipids from the metabolism of glucose intermediates,
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Fig. 1. Localization of estrogen receptor (ER) at the plasma membrane. The ER␣ monomer is palmitolyated by DHHC7 and DHHC21 palmitoylacyltransferase
(PAT) enzymes in Golgi, causing the physical asociation of ER␣ with caveolin-1 protein in cytoplasm. Heat shock protein 27 (Hsp27) binds to a 9-amino acid
palmitoylation motif in the ligand-binding domain of ER␣, probably opening the receptor structure for subsequent PAT binding and action. Upon binding of
caveolin-1, caveolin-1 transports ER␣ to caveolae rafts (CR) in the plasma membrane. Here, caveolin-1 serves both as a structural coat protein of the caveolae
and as a scaffold for ER␣, linker proteins (PELP1), and numerous signaling molecules. In this confined area, membrane ER␣ dimerizes in response to estrogen
and then activates various G␣ and G␤␥ proteins for selective signaling. It is not established whether endogenous ER␣ span the plasma membrane. In
hormone-responsive breast cancer cells, membrane-initiated signaling by ER␣ liberates heparin-bound epidermal growth factor (HBEGF), a ligand for epidermal
growth factor receptor (EGFR) family heterodimers, ultimately activating MEK/ERK, phosphatidylinositol 3-kinase (PI3K)/Akt, and other signaling cascades
that affect cellular actions. Effector kinases phosphorylate proteins to alter their activity (non-genomic actions), and this signaling also conditions the chromatin
environment to promote nuclear ER␣ actions impacting transcription (genomic actions). Nuclear-bound ER␣ is chaperoned by Hsp90, is not palmitoylated, and
upon E2 binding, dimerizes in the nucleus to modulate gene expression.
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MEMBRANE ESTROGEN RECEPTORS
tion of the TF prevented the ability of the Golgi-located
processing proteases S1P and S2P from physically associating with SREBP-1, therefore preventing processing (36).
Thus, we described a mechanism by which estrogen signaling exclusively from membrane receptors represses some
mRNAs and produces a metabolic phenotype, one that does
not involve nuclear ER actions (Fig. 2).
Requirement of Membrane ER␣ for Normal Organ
Development and Function
The importance of steroid hormones to the development and
functions of many organs is well established. There is growing
evidence that some actions of steroid hormones in adult animals arise from membrane ER signaling; however, it is not
clear whether extranuclear ER contribute to normal organogenesis. To investigate this potential function of membranelocalized ER␣, we developed a cystine 451 alanine ER␣knock-in mouse by homologous recombination, replacing the
endogenous esr1 gene in ES cells. This mutation prevents
palmitoylation and hence, membrane trafficking by the receptor. The resulting mice show selective loss only of membranelocalized ER␣ but demonstrate abundant nuclear steroid receptors since palmitoylation does not impact nuclear ER␣ trafficking (46). The cells from homozygous nuclear ER␣-only
(NOER) mice fail to respond to E2 with the ERK and PI3K/Akt
signaling that is seen in wild-type (WT) mouse cells. As a
result, E2 stimulation of mRNAs that are regulated by both the
mentioned rapid signaling and nuclear ER␣ binding to an
estrogen response element in the gene promoter is significantly
decreased (46).
We investigated various organ phenotypes and found the
female homozygous NOER mice to be completely infertile due
to a lack of ovulation, very low progesterone levels, and
abnormal development of both the uterus and ovaries. Key
developmental genes were stimulated by estrogen only in WT
and heterozygous NOER mice (46). In the ovary, the lack of
corpora lutea in homozygous NOER mice explained the very
low serum progesterone levels (8). The mammary glands of
only the homozygous NOER mice were poorly developed in
part due to very low progesterone receptor-␤ expression that is
important to ductal side branching (6). Additional phenotypes
were also seen, and all of these abnormalities were significantly reversed in the heterozygous NOER female mice that
were comparable with WT mice. Thus, membrane and nuclear
ER␣ pools are required for the normal development and
function of many organs mainly from coordinated regulation of
key developmental genes.
What other mechanisms underlie membrane and nuclear
ER collaboration for the regulation of key genes? Additional
effects of membrane ER␣ signaling include transcription
factor upregulation and recruitment, nuclear receptor phosphorylation that enhances transcription (7), and phosphorylation of coactivators such as the SRC family that then
transit to the nucleus, where they bind to gene promoters
and enhancers (66). Recent work has shown that p38␣
activation presumably by membrane ER␣ signaling results
in this kinase acting in the nucleus to phosphorylate nuclear
ER␣ and thereby promote interaction of the receptor with
the Skp2 protein (5). This E3 ubiquitin ligase ubiquitinates
nuclear ER, promoting its degradation, and presumably
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while also maintaining glutathione in a reduced state to thwart
oxidative stress. When glucose is plentiful and available to the
tumor cells, estrogen signals through Akt to promote glycolysis
(31). However, inadequate blood flow and therefore availability of nutrients such as glucose are known to occur for tumors
in vivo and are inadequate to maintain glycolysis in breast
tumors. We modeled this by decreasing the amount of glucose
available to cultured breast cancer epithelial cells. In this
setting, estrogen promotes a metabolic shift away from glycolysis by activating AMPK only under reduced glucose conditions (31). AMPK phosphorylates a key enzyme, pyruvate
dehydrogenase, increasing its function to process the glucose
metabolite pyruvate into acetyl-CoA. Acetyl-CoA enters the
Krebs cycle as substrate for citrate formation and to produce
electrons for the oxidative phosphorylation cycle. The latter
produces increased ATP, and the former promotes reductive
oxidation of fatty acids, thereby promoting survival of the
cancer epithelial cells (31). Membrane ER␣ and ER␤ also have
important roles in promoting insulin sensitivity and insulin
synthesis and secretion from the ␤-cells of the pancreas (4, 65).
Interestingly, high glucose and heightened insulin signaling
promote aggressive development of breast cancer in mouse
models (11, 30), suggesting a possible important function by
which estrogen/ER stimulates the development of this malignancy.
To better understand the impact of rapid signaling for cell
function, we developed a mouse that expresses the ligand
binding (E domain) of ER␣, which is targeted exclusively to
the membrane of all organs but lacks nuclear or other ER␣
receptor pools (35). Cells isolated from various organs of
this membrane ER␣-only mouse (MOER) were found to
respond to E2 with rapid signaling through various kinase
pathways, cyclic nucleotide generation, and calcium flux.
Importantly, the rapid responses were comparable with
those from E2 acting in WT mouse cells, indicating that only
the membrane receptor pool is required for most signal
transduction.
To determine whether membrane ER␣ signaling alone
could induce gene transcription, we injected ovariectomized
WT, MOER homozygous, and ER␣KO mice with 4,4=,4==(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), an
ER␣ agonist, and carried out DNA microarrays from liver
RNA. Compared with mice injected with oil as a control,
most genes that were significantly regulated by PPT were
seen only in the WT mouse livers, indicating that the nuclear
ER␣ pool is required for most messenger RNA regulation.
However, 30 mRNAs were comparably suppressed by PPT
in livers of WT and MOER mice, but the suppression was
absent in ER␣-KO, and these mRNAs were mainly the key
transcripts for cholesterol, triglyceride, and fatty acid synthesis (36). Messenger RNA (mRNA) regulation correlated
with comparable suppression by PPT of all lipid content in
the livers of the WT and MOER female mice, which was not
seen in ER␣-KO mice. Further studies indicated that membrane ER␣ signaling to AMPK activation caused a phosphorylation of the key transcription factor (TF) sterol regulatory element-binding protein-1 (SREBP-1). This phosphorylation prevented the processing of SREBP-1 to a
truncated protein that translocates to the nucleus, where the
TF acts in trans to stimulate genes that are important for
lipid synthesis. Specifically, AMPK-induced phosphoryla-
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MEMBRANE ESTROGEN RECEPTORS
E137
Fig. 2. ER␣ signaling from the membrane suppresses the expression of genes involved in lipid synthesis. Estrogen-triggered signaling in liver through the ER␣
receptor at the membrane activates a kinase cascade, including protein kinase A/liver kinase B (LKB) signaling to AMP kinase (AMPK) activation. AMPK
phosphorylates sterol regulatory element-binding protein 1 (SREBF-1), thereby preventing its NH2-terminal cleavage by S1 or S2 proteases in Golgi. Failure of
SREBF-1 processing prevents nuclear translocation of the transcription factor. Therefore, SREBF-1 is sequestered in cytoplasm and cannot augment the
expression of mRNAs involved in lipid synthesis
promotes the cycling of nuclear ER on/off gene promoters/
enhancers that is necessary for gene transcription (55).
Impact of Membrane ER for Protection Against
Cardiovascular Diseases
Increasingly, it has been shown that membrane ER␣ and
ER␤ play important roles in the prevention of cardiovascular
diseases. Several forms of acute endothelial cell injury in
arteries are mitigated by E2 action and are comparably prevented by the estrogen dendrimeric compound, which binds
only to membrane ER (9). Rapid signaling by estrogen also
prevents aspects of atherosclerosis in cells from animal models
(62). Several groups have shown that ER␤ mediates the ability
of estrogen to prevent cardiac hypertrophy that results from
Gq␣ signaling by angiotensin II (Ang II) (2), genetic hypertension (17), or pulmonary hypertension (27). We have shown
that membrane ER␤ signaling prevents the activation of calcineurin by Ang II or endothelin-1 (43). Calcineurin is a
phosphatase that dephosphorylates the important family of
transcription factors, i.e., the NFAT family. As a result, these
transcription factors are retained in the cytoplasm of cardiomyocytes and cannot participate in activating the nuclear
hypertrophic gene program that is downstream of various
stimuli in vivo (26). Using a novel ER␤ agonist, we showed
prevention of Ang II-induced hypertension, cardiac hypertrophy, and cardiac fibrosis in WT but not ER␤-KO mice (42).
The antifibrotic properties of ER␤ were particularly impressive
and resulted from activation of cAMP and PKA in cardiac
fibroblasts that blocked TGF␤ production and signaling to
Smad phosphorylation. As a result, the Smad transcription
factors were retained in cytoplasm, and collagen and other
important genes that contribute to the fibrosis phenotype were
not stimulated by Ang II or TGF␤ (44). These mechanisms
indicate a novel function by which ER prevent cardiac hypertrophy and fibrosis and also lipid synthesis in the liver (36, 41,
44). These ER actions stem exclusively from membrane recep-
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.
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MEMBRANE ESTROGEN RECEPTORS
Perspectives
Investigators in the area of extranuclear steroid receptors
have described rapid signaling impacting a variety of steroid
hormone actions in many organs (15). This includes both
sex steroid and non-sex steroid receptors (21). In most
situations, collaboration between extranuclear and nuclear
receptor pools is necessary for normal organ functions.
However, we have described three in vivo models where
signaling from the membrane ER alone is sufficient to
prevent heart or liver pathology and may be applicable to
other situations. It is likely that when mammals adjust
rapidly to external stimuli, it is signaling from membrane
receptors to the posttranslational modification of proteins
such as enzymes that provide the mechanisms for rapid
adaptation. This could include adjustments of blood pressure to changing position or stress, metabolic adaptations to
ensure adequate glucose availability and optimal utilization,
ion exchange in cells that modulate muscle contraction, and
unfortunately, the ability of some cancers to adapt to cytotoxic therapies. This begs the larger question as to why ER
are so widely expressed, including cardiovascular, bone,
liver, and other organ systems where there is no obvious
connection to enhancing fertility. The widespread distribution of receptors in most cells of the body likely reflects the
additional actions of estrogen in younger mammals, including women, to ensure their overall health and, therefore,
their physical ability to bear and raise their offspring.
Considering that evolution and nature are highly geared
toward reproduction and survival of the species, this concept
is tenable. Extranuclear steroid receptors may then provide
the organizing signals to ensure proper cell and organ
function.
GRANTS
This work is supported by grants from the Veterans Administration Merit
Review Program and National Institutes Of Health (CA100366) and represents
the superb efforts of Ali Pedram and Mahnaz Razandi, my long-term colleagues in the laboratory.
AUTHOR CONTRIBUTIONS
E.R.L. prepared figures; E.R.L. drafted manuscript; E.R.L. edited and
revised manuscript; E.R.L. approved final version of manuscript.
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Several important mechanisms for ER␤ prevention of cardiac disease were identified recently in our studies. These
include prevention of Ang II-stimulated Akt activation through
ER␤ preserving Inpp5f phosphatase function (42). As a result,
GSK-3␤ remains active to phosphorylate and suppress the
activity of a key transcription factor, GATA4, resulting in
inhibition of hypertrophic genes. Additionally, we found that
membrane ER␤ signals to the repression of histone deacetlyase
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thus suppressing this prohypertrophic enzyme (45). At the
same time, membrane ER␤ signals to the increased production
and nuclear localization of HDACs 4 and 5 that are antihypertrophic, resulting in repression of hypertrophic genes such as
␤-myosin heavy chain. Thus, membrane ER␤ is the first
described endogenous molecule that contributes to maintaining
the default state in the heart of antihypertrophy by modulating
both class I and II HDACs.
Review
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