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Characteristics of hydrogen peroxide inducible clone-5 and its potential role as a nuclear receptor coactivator
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Characteristics of hydrogen peroxide inducible clone-5 and its potential role as a nuclear receptor coactivator
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Content
CHARACTERISTICS OF HYDROGEN PEROXIDE INDUCIBLE CLONE-5 AND
ITS POTENTIAL ROLE AS A NUCLEAR RECEPTOR COACTIVATOR
by
Mengrao Zhang
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2013
Copyright 2013 Mengrao Zhang
ii
ACKNOWLEDGEMENTS
I appreciate Dr. Michael Stallcup and Dr. Zoltan Tokes for offering me good
opportunities to learn science during the MS training. Also, I want to thank Dr. Stallcup and
Dr. Rajas Chodankar for encouraging and guiding me to design and perform experiments
for the bench work. Equally, I would like to thank all the lab folks for their care and
generous help. Last but not least, I give my gratitude to my parents and sister for their
consistent support that enable me to pursue academic goal and their love that
strengthened my will to finish this thesis.
iii
TABLE OF CONTENTS
Acknowledgements ............................................................................................................ ii
Figures............................................................................................................................... iv
Abbreviations ...................................................................................................................... v
Abstract ............................................................................................................................ vii
Introduction ......................................................................................................................... 1
Genetic Feature of Hic-5 .................................................................................................... 2
Secondary Structure and Protein Binding Features ............................................................ 3
Hic-5 Function Associates with Focal Adhesion ................................................................. 4
Expression of Hic-5 ............................................................................................................ 7
Hic-5 Involvement in Signaling ........................................................................................... 9
Hic-5 as A Nuclear Receptor Coactivator ......................................................................... 11
References ....................................................................................................................... 20
iv
FIGURES
Figure 1. Schematic Diagram of Hic-5 and Its Interaction Partners .............................. 4
Figure 2. Hic-5 Localizes at Focal Adhesion Sites ........................................................ 6
Figure 3. Procedure to Isolate and Characterize A Nuclear Receptor
Coactivator .................................................................................................. 14
v
ABBREVIATIONS
AR androgen receptor
ARA55 androgen receptor associated protein 55
CAT chloramphenicol acetyltransferase
ChIP chromatin immunoprecipitation
CRP2 cysteine-rich protein 2
DBD DNA binding domain
EMT epithelial mesenchymal transition
ER estrogen receptor
FAC focal adhesion kinase
FISH fluorescence in situ hybridization
GR glucocorticoid receptor
GIT-1 G protein-coupled receptor kinase-interacting Arf GAP1
HATs histone acetyltransferases
Hic-5 hydrogen proxide inducible clone-5
HMTs histone methyltransferases
HREs hormone response elements
Id1 inhibitor of differentiation-1
IIF indirect immunofluorescence
LBD ligand binding domain
vi
MMTV mouse mammary tumor virus
MR mineralocorticoid receptor
NMTS nuclear matrix targeting sequence
NR nuclear receptor
PINCH particular interesting new cysteine and histidine-rich protein
PPARϒ peroxisome proliferator-activated receptor ϒ
PR progesterone receptor
PTP-PEST protein-tyrosine phosphotase-PEST
PYK2 proline-rich tyrosine kinase 2
RNA PolII RNA polymerase II
TGF-β1 transforming growth factor-β1
TSC-5 TGF-β stimulated clone-5
TR thyroid hormone receptor
vii
Abstract
Hydrogen peroxide inducible clone-5 (Hic-5), also named transforming growth
factor-β1 induced transcript 1(TGFB1|1), TGF-β stimulated clone-5 (TSC-5) or androgen
receptor associated protein 55 (ARA55), is a LIM domain protein. It has LIM domains at its
carboxyl half and LD motifs at its amino half, thus providing multiple protein-protein interfaces
for interaction with a large number of proteins. Like its homologous protein paxillin, Hic-5
exists at focal adhesion sites and is involved in mediating signals from the extracellular matrix.
Hic-5 also plays an important role in cell adhesion under physiological conditions. The
association of Hic-5 and diverse focal adhesion proteins including kinases, that can be
regulated by small G proteins, highlights its function as a scaffold protein for focal adhesion
dynamics. The expression of Hic-5 is selective with a preference of distribution in
mesenchymal cells and endothelial cells. Recent findings have shown aberrant Hic-5
expression in certain tissues under pathological conditions such as glomerulosclerosis and
carcinogenesis. Hic-5 is involved in several signaling pathways, which indicates that it may
be involved in regulating crosstalk between them. One signaling pathway that Hic-5
participates in is the nuclear receptor signaling which is important for the transcriptional
regulation of target genes. The regulation of nuclear receptor transcriptional activity renders
Hic-5 a unique member of paxillin protein subfamily and a potential coactivator. Various
strategies have been adopted to characterize the coactivator feature of Hic-5 over the past
decade. Initial discovery by Yang et al. demonstrated that Hic-5 is a potential coactivator for a
subset of nuclear receptors, which could partially localize in the nucleus and this research
work will be discussed.
1
Introduction
Hydrogen peroxide inducible clone-5 (Hic-5) was originally named TGF-β
stimulated clone-5 (TSC-5). In 1994, Shibanuma et al. rediscovered a gene Tsc-5 when
they sought clones that can be induced by transforming growth factor-β1 (TGF-β1) in
mouse osteoblast MC3T3-E1 cells. After treating those cells with either TGF-β1 or H2O2,
they observed Tsc-5 mRNA level increased. Moreover, TGF-β1 signaling seemed to be
partially mediated by H2O2. Therefore, they designated this clone as hydrogen peroxide
inducible clone-5 (Hic-5) (Shibanuma et al., 1994).
Over the past two decades, researchers have investigated Hic-5’s biological role
extensively based on inference from its interacting partner that is a known protein,
comparative study with its protein homolog, and analyses of clinical specimens. From
today’s picture, Hic-5 is regarded as a scaffold protein, which can interact with a growing
list of proteins. Hic-5 also seems to be a multifunctional protein. At the beginning, Hic-5
was suggested to be involved in cell senescence since an increasing protein level of Hic-5
was detected with cell culture passaging. In nontumorigenic immortalized human
fibroblasts, overexpression of Hic-5 caused cell growth retardation, senescence-like
morphology and increase of another senescence linked protein Cip1/WAF1/sdi1
(Shibanuma et al., 1997). These observations were consistent with the observations that
Hic-5 was accumulated in cells by TGF-β1 signaling, and TGF-β1 signaling was
associated cell growth inhibition. However, shortly after Hic-5’s discovery, it was
2
recognized as a focal adhesion protein that may participate in integrin signaling and
cytoskeleton dynamics (Matsuya et al., 1998; Fujita et al., 1998; Turner et al., 1999;
Denhez et al., 2002; Thomas et al., 1999; Panetti et al., 2004; Tumbarella et al., 2006). In
addition, some studies showed Hic-5’s function in gene transcription, thus leading to the
idea that Hic-5 is a coactivator and a nuclear-cytoplasmic shuttling protein (Fujimoto et al.,
1999; Yang et al., 2000). Recently, to illustrate its physiological role, a Hic-5 deficient
mouse model was generated (Kim-Kaniyama et al., 2011). It did not give rise to embryonic
lethality, and no apparent functional abnormality was observed, but it caused mild vascular
remodeling. Finally, current focuses on Hic-5 switch to its function in certain diseases such
as several cancers, glomerulosclerosis, and Alzheimer disease (Caltagarone et al., 2010;
Hornigold et al., 2010; Suga et al., 2012). Therefore, the significance of Hic-5 has been
increasingly noted.
Genetic Feature of Hic-5
Back to the early study of Hic-5, Nose’s group first applied fluorescence in situ
hybridization (FISH) for chromosomal mapping of Hic-5 in mouse. They settled the
location of Hic-5 on chromosome 7 (Thomas et al., 1999; Mashimo et al., 2000).
Genetically, Hic-5 contains 10 exons, but due to alternative translation start site, it can
include 11 exons with an additional exon, exon 1’, which indicates that there are isoforms
of Hic-5 (Mashimo et al., 2000). Two isoforms for Hic-5 have been documented, Hic-5
alpha (includes 10 exons) and Hic-5 beta (includes 11 exons). Among these isoforms,
3
Hic-5 alpha, which has a protein size about 55 kDa (Shibanuma et al., 1994; Matsuya et al.,
1998), is the dominant isoform in cells and has been wildly used in research work.
Secondary Structure and Protein Binding Features
The notable feature of Hic-5 protein is its four LIM domains at its carboxyl terminus,
and three or four LD motifs at its amino terminus. The variable number of LD motifs is due
to the uncertain existence of extra exon 1’. The LD domain is leucine and asperate-rich; on
the other hand, each module of the four LIM domains is cysteine/histidine-rich and
includes two zinc fingers. Because of Hic-5 C terminal zinc fingers and some experiments
such as western blot for nuclear extracts and immunofluorescence data indicating Hic-5 in
the nucleus, Nose’s group once hypothesized Hic-5 protein LIM domains also serve for
protein-nucleic acid interaction. Applying several rounds of immunoprecipitation for Hic-5
protein and genomic DNA fragments form mouse osteoblasts, they isolated seven
independent DNA clones that were purine-rich and/or had a long A/T track (Nishiya et al.,
1998). However, no follow up study about the Hic-5 direct binding to DNA has been
reported. In contrast, a large quantity of work has concentrated on Hic-5 biological function
study via investigating Hic-5 binding partner proteins. This path to uncover the role of Hic-5
is uneasy since various proteins were reported to interact with Hic-5. This is mainly due to
two reasons. First, both LD motifs and LIM domains are protein-protein interaction
interfaces. Second, the LD motifs and LIM domains are not equivalent since each of them
performs distinct role for protein binding as each domain is crucial for distinct groups of
4
proteins recognition. For example, LIM3 was reported to interact with nuclear receptors,
transcription factors, and Protein-tyrosine phosphotase-PEST (PTP-PEST). At the same
time, LIM2 was reported to interact with an ubiquitin ligase, Cbl-c (Ryan et al., 2012).
Figure 1. Schematic representation of Hic-5 highlighting phosphorylated residues
and interaction factors. Hic-5 is a 461-amino acid-length protein, comprising a variable
number of LD motifs at its N half, and four highly homologous LIM domains at its C half.
Both motifs and domains serve as protein-protein binding surfaces, which interact with
focal adhesion proteins, cytoskeleton proteins, and transcription factors. Phosphorylation
of Hic-5 occurs at multiple sites, which mediates Hic-5 interacting affinity towards other
proteins. AR: androgen receptor, CRP2: cysteine-rich protein 2, FAK: focal adhesion
kinase, GIT-1: G protein-coupled receptor kinase-interacting Arf GAP1, GR: glucocorticoid
receptor, PINCH: particular interesting new cysteine and histidine-rich protein, PTP-PEST:
protein-tyrosine phosphotase-PEST, PYK2: proline-rich tyrosine kinase 2.
Hic-5 function associates with focal adhesions
Early investigation of Hic-5 function mainly referred to the study of paxillin, because
Hic-5 protein belongs to paxillin family, which belongs to LIM domain protein family, and all
5
LIM domain proteins contain a consensus sequence
CX2CX16-23HX2(H/C)X2CX2CX16-21CX2(D/H/C). Furthermore, LIMs belongs to zinc finger
superfamily, which include two zinc fingers. Based on the number of LIM domains and the
location of LIM domains, LIM domain proteins are further grouped into several subfamilies.
The paxillin family contains paxillin, Hic-5, and leupaxin. Similar to Hic-5, paxillin is a LD
motif containing, LIM domain protein, which shares 72% similarity to Hic-5 (Sheila et al.,
1999). It had been reported and reviewed that paxillin is a focal adhesion protein (Turner et
al., 1990; Schaller, 2001). Therefore, the preliminary hypothesis on function of Hic-5 is
presuming that Hic-5 is also a focal adhesion protein.
By the pilot study of Hic-5 in comparison with paxillin, an array of proteins that
frequently appear at focal adhesion sites were confirmed to interact with Hic-5 as well by
biochemical methodologies. These include functional kinase such as focal adhesion
kinase (FAK), Proline-rich tyrosine kinase 2 (Pyk2), PTP-PEST, as well as structural
protein vinculin (Fujita et al., 1998; Matsuya et al., 1998; Nishiya et al., 1999; Thomas et al.,
1999). However, how the focal adhesion organizes the network spatiotemporally among
those associated proteins remains to be elucidated. Importantly, Hic-5 is a protein binding
platform for it has many LD motifs and LIM domains. Hic-5 interacts with these proteins
mainly by its LIM3 domain, but its LD3 domain was reported to bind to GIT1, an Arf
GTPase-activating protein (Nishiya et al., 2002).
6
Figure 2. Localization of Hic-5 at multiple focal adhesion sites for cell adhering to
extracellular matrix. Integrins and syndecan-4 are major transmembrane receptors for
extracellular matrix, and the cooperative work helps assembly of actin stress fibers as well
as focal adhesions. Hic-5 mediates integrin and syndecan-4 signaling via association with
other focal adhesion proteins. The direct interaction between Hic-5 and GIT-1 indicates
Hic-5 is involved in G protein-coupled receptor signaling. Phosphorylated Hic-5 inhibits
activation of Rac, which mediates endothelial growth factor signaling. Hic-5 could migrate
to actin stress fibers or nucleus with the existence of CRP2 or PINCH, respectively.
Another question is how Hic-5 exists with the highly homologous protein paxillin at
the focal adhesion site with many shared protein binding partners. It was a long standing
hypothesis that paxillin and Hic-5 have a competitive relationship for some shared focal
adhesion proteins, including FAK (Nishiya et al., 2001). A recent study by Turner’s group
7
found paxillin and Hic-5 actually participate in different stages of focal adhesion assembly
and maturation with dynamic interactions during these processes (Deakin et al., 2010;
Deakin et al., 2012). Besides assays on traditional 2D matrices, they set up 3D
cell-derived matrices to visualize subcellular environment protein-protein interaction. What
they observed was the different binding preference among Hic-5, paxillin and vinculin. In
contrast to 2D context, vinculin prefers to bind to Hic-5 in the 3D condition (Deakin et al.,
2012). Indeed, some regulatory factors for the interaction between vinculin and Hic-5 or
paxillin have been identified. Rac1 and RhoA play a role for vinculin-paxillin interaction and
vinculin-Hic-5 interaction, respectively. It was suggested there is a forward feedback by
which Hic-5 sustains activation of RhoA and RhoA promotes the expression of Hic-5 in
epithelial cells (Deakin et al., 2012).
Expression of Hic-5
Hic-5 expression has been intensively examined by western blotting in cells and
tissues to understand its involvement in certain pathological conditions, such as
carcinogenesis. Recent studies show Hic-5 is expressed in malignant primary cells as well
as cell lines. These cell lines include B16-F1 murine melanoma cells, Lovo colorectal
cancer cells, and prostate cancer PC3 cell line (Noguchi et al., 2012; Cui et al., 2006;
Nessler-Menardi et al., 2000). In addition, the Hic-5 expression depends on the stage of
tumor. For example, Hic-5 is detectable in PC3 cells but does not express in prostate
DU145 cell lines or LNCaP cells (Nessler-Menardi et al., 2000). Consistent with this, a
8
significantly lower level of Hic-5 protein in androgen-resistant prostate cancers was
observed in comparison with Hic-5 protein amounts in previously untreated prostate
cancers or benign prostatic hypertrophy. Therefore, Hic-5 could be a potential indicator for
the stage of prostate cancer progression. The fluctuation of Hic-5 expression level was
also linked to other pathological events. Compared with normal conditions, upregulation of
Hic-5 was observed in specimens of disease models including the hippocampus of
Alzheimer disease, focal segmental glomerulosclerosis (glomeruli of sclerotic kidneys)
(Caltagarone et al., 2010; Hornigold et al., 2010; Suga et al., 2012). Hic-5 expression
variation in these cells may cause a change of gene expression profile, or apoptosis, or
growth inhibition. In this sense, if it meets the requirement of sensitivity and specificity,
Hic-5 may serve as an indicator for these diseases. Also, since a pivotal role of Hic-5 in
given signaling pathways related to apoptosis (Hornigold et al., 2010), it may render Hic-5
a therapeutic target.
In fact, Hic-5 is even expressed selectively in healthy tissues (Yuminamochi, et al.,
2003). It is reported that Hic-5 is preferentially expressed in mesenchymal cells compared
to epithelial cells. For example, the prostate stromal cells, but not epithelial cells have
detectable Hic-5 at the protein level (Nessler-Menardi et al., 2000; Li et al., 2002). This
suggests Hic-5 is unnecessary for epithelial cells or is important to maintain mesenchymal
cell morphology and physiology. Several studies have investigated the role of Hic-5 in
epithelial mesenchymal transition (EMT) (Tumbarello et al., 2006; Pignatelli et al., 2011).
9
Epithelial mesenchymal transition is not only a step crucial for tumorigenesis, but also a
process important during development (Thiery et al., 2002; Larue et al., 2005). High
expression of Hic-5 along with other focal adhesion proteins was detected in embryonic
kidneys (Matsuura et al., 2011). In addition, Cai et al. found that differentiation of human
prostate stromal cells to smooth muscle cells is associated with Hic-5 expression (Cai et
al., 2005). Hic-5 is required in some fetal gene programs induced by phenylephrine in
neonatal cardiac myocytes (Yund et al., 2009). However, little study to examine the
regulation of Hic-5 gene expression during development has been done. Also, the
mechanism of Hic-5’s role in development and cell differentiation is mostly unknown.
Hic-5 involvement in Signaling
Hic-5 expression was shown to be upregulated in epithelial cells in response to
TGF-β stimulus (Shibanuma et al., 1994). Interestingly, recent studies reveal Hic-5 can
regulate TGF-β signaling as well, by negative feedback via interacting with and acting on
the TGF-β signaling downstream molecules. The R-Smad Smad3 was the first one shown
to bind to Hic-5 both in vitro and in vivo. Hic-5 was shown to block Smad3 transactivity by
using a luciferase reporter vector (Wang et al., 2005). Similarly, a recent study shows that
Smad1,5,8, the other R-Smads, interact with Hic-5 both in vitro and in vivo (Shola et al.,
2012). In this case, Hic-5 interaction with Smad1,5,8 prevents BMP induction of
expression of the downstream gene inhibitor of differentiation-1 (Id1) (Shola et al., 2012).
In addition, Hic-5 also regulates inhibitory Smads that mediate TGF-β signaling. It
10
physically interacts with Smad7 and is associated with reduced Smad7 protein (Wang et
al., 2008). However, the reduction of Smad7 seems not related to ubiquitin-induced protein
degradation.
Hic-5 mainly distributes in the cytoplasmic focal adhesion region and nuclear
periphery; however, little Hic-5 was detected in the nucleus under certain circumstances
such as hydrogen peroxide stimuli(Fujita et al., 1998; Thomas et al., 1999; Nishiya et al.,
1999; Shibanuma et al., 1994). It will be interesting to check where Hic-5 interacts with
Smads intracellularly in response to TGF-β stimuli, which will endow a better
understanding of the TGF-β signaling. Especially when it comes to the crosstalk between
AR signaling and TGF-β signaling, Hic-5 could be a pivotal molecule to decide
TGF-β-induced cell apoptosis or androgen-induced cell survival.
To date, we know of some crosstalk between steroid hormone signaling and other
canonical pathways. Examples are AR interaction with TGF-β signaling pathway, and GR
interaction with Wnt/β-catenin pathway; since Hic-5 is a coregulator for these pathways, it
could influence this cross talk. Though Hic-5 is a coactivator for AR and GR according to
transient luciferase reporter gene assays’ results, it is a repressor for TGF-β signaling and
Wnt signaling. As mentioned above, Hic-5 interferes with TGF-β signaling via two ways:
first it abolishes a key signaling protein function by targeting to regulatory Smads (i.e.,
Smad1, 3, 5, 8), which are the downstream signaling molecules for TGF-β receptor (Wang
et al., 2005; Shola et al., 2011); second, it reduces the inhibitory Smad, Smad7, at the
11
protein level but not mRNA level and in a proteasome-independent unknown mechanism
(Wang et al., 2008). Similarly, the reported mechanism for Hic-5 to block Wnt signaling is
by an interaction between Hic-5 and amino acids encoded by an alternatively spliced exon
of one of the LEF/TCF family members (Ghogomu et al, 2006). LEF/TCF transcription
factors are the downstream molecules for Wnt, which form a heterodimer with β-catenin
and work at target genes of Wnt signaling (Ghogomu et al, 2006).
Hic-5 as a Potential Nuclear Receptor Coactivator
A unique feature of Hic-5 among paxillin protein family is its involvement in
transcriptional regulation, which endows it as a potential nuclear receptor coactivator.
Nuclear receptors (NRs) are transcription factors, which participate in transcription
regulation in the nucleus. All NRs share a universal protein structure: the N terminal end is
variable, which has ligand-independent transactivation activity; the central part is a highly
conserved DNA binding domain (DBD) containing two zinc fingers, which recognize
conserved DNA sequence; between DBD and the C terminus is a linker region; finally, the
fairly conserved C terminus is a ligand binding domain (LBD), which has multiple functions
including ligand binding, dimerization, transactivation and protein-protein interaction.
The activation of NR requires specific hormone ligands, including steroids,
steroid-related hormones, amino acid derivatives, etc. Upon binding to the ligands
intracellularly, two identical NRs undergo a conformational change, forming a homodimer.
Then, the homodimer recognizes hormone response elements (HREs) via DBDs, resulting
12
in the regulation of transcription initiation of proximate genes (Sever & Glass, 2013).
Importantly, those HREs share a consensus sequence, which is RGGTCA. Genome-wide
sequence analysis reveals that these HREs reside in the enhancer regions and the
enhancer regions can be 100 kilobases away from the core promoters (Sever & Glass,
2013). Therefore, understanding the mechanism about which distant cis-elements and NR
act on core promoters and transcription machinery is one of the most interesting topics
that have been intensively studied.
An explanation is some proteins regarded to be nuclear receptor coactivators. Even
though these coactivators usually do not bind to DNA, they bridge the gap between NRs
and RNA Polymerase II (RNA Pol II) via protein-protein interaction or they can modify the
chromatin structure near promoter regions. As a result, nuclear receptors along with these
coactivators enhance target gene transcription from the basal level. Traditionally, those
coactivators can be grouped into three types according to their functions: Type I proteins
are those that have enzymatic activity to covalently modify histone in order to loosen the
chromatin structure in the promoter region. The first type includes histone
acetyltransferases (HATs) and histone methyltransferases (HMTs) such as CBP/p300,
CARM1; second type of proteins disrupts nucleosome to increase DNA accessibility in an
ATP-dependent manner. This type includes protein complexes such as SWI/SNF,
BRG1/BRM complexes; and third type of proteins, which is also named mediators, recruits
RNA Pol II. With an increasing number of potential coactivators recently isolated, this
13
classification seems to be limited. However, some basic traits should be considered to be
shared by all coactivators. First, nuclear receptor coactivators facilitate the transcription of
nuclear receptor targeted genes. Without coactivators, targeted genes can be transcribed
at basal level with activated nuclear receptors. Meanwhile, without activated nuclear
receptors, the targeted genes should not be transcribed by the coactivators alone. Second,
the coactivators should locate in the nucleus, and even in the nuclear receptor-RNA Pol II
complexes while it functions. Third, the enhanced transcription is transient. If the nuclear
receptor complex releases its coactivator, the effect on enhanced transcription will be
terminated.
The study of coactivators involves isolation and identification of them in a set
procedure. Usually, first step is to isolate nuclear receptor coactivators, and yeast two
hybrid assay turns out to be a useful tool. Furthermore, some other methods are used to
confirm the interaction between nuclear receptors and their coactivators directly or
indirectly, in vitro or in vivo. These methods include mammalian two hybrid assay, GST
pull down assay and co-immunoprecipitation assay. Next, to confirm that the coactivator
candidates potentiate the transactivation activity of the nuclear receptor, functional
reporter assay using reporter plasmids containing a nuclear receptor targeted promoter
was normally performed. The mRNA level of nuclear receptor targeted gene product was
another way to indicate the enhancement of transcription activity of nuclear receptors.
Importantly, to examine the location of coactivators, indirect immunofluorescence assay
14
was performed. A more powerful and precise tool is chromatin immunoprecipitation (ChIP),
which tests whether nuclear receptors as well as coactivators are recruited to the targeted
HREs.
Figure 3. Isolation and characterization of nuclear receptor coactivators. The yeast two
hybrid assay figure is from Yang et al. in 2000; the GST-pull down assay figure is from
Shola et al. in 2012; and the reporter assay figure is from Fujimoto et al. in 1999.
In 1999, androgen receptor associated protein 55 (ARA55) was cloned, and it was
characterized as an androgen receptor coactivator (Fujimoto et al., 1999). ARA55 can
enhance the transactivation activity of androgen receptor (AR) on a corresponding
AR-regulated promoter. Interestingly, it turned out that human ARA55 was highly
15
homologous to mouse Hic-5 as the protein sequence identity is 90.5% (Fujimoto et al.,
1999). While Hic-5 was originally shown to associate with cell senescence, Yang et al.
demonstrated Hic-5 not only bound to a nuclear receptor, the glucocorticoid receptor (GR),
it enhanced the transactivation activity of a set of nuclear receptors in a hormone
dependent manner (Yang et al., 2000). By applying indirect immunofluorescence assay
(IIF), they also found Hic-5 existed in the nucleus (Yang et al., 2000). While these
phenomena indicate Hic-5 plays a role in transcription regulation as a coactivator, this
feature needs to be further addressed because these experiments were done based on
detecting artificial transcription products such as chloramphenicol acetyltransferase (CAT)
activity and luciferase activity. Moreover, the expression reporter assays were conducted
in the artificial system by using mouse mammary tumor virus (MMTV) as a target for
nuclear receptor instead of natural HREs. The first effort towards how Hic-5 works with the
HREs region under normal physiological conditions was done by Heitzer et al. They
visualized the relationship between Hic-5/ARA55 and natural nuclear receptor targeted
gene HREs at the molecular level via adopting ChIP assay (Heitzer et al., 2006). In
addition, several differentiating or signal transduction related genes regulated by nuclear
receptors and Hic-5/ARA55 were observed (Heitzer et al., 2006). They also implied a
potential mechanism to explain how Hic-5 enhances AR transactivation. Finally,
up-regulation of AR transactivation by its coactivator Hic-5/ARA55 can be suppressed by
Pyk2 through a phosphorylation on this coactivator.
16
Hitherto, Hic-5 was shown to enhance the transactivation activity of several steroid
receptors (a type of nuclear receptors, including GR, AR, progesterone receptor (PR) and
mineralocorticoid receptor (MR)) (Fujimoto et al., 1999; Yang et al., 2000). Also, Hic-5 was
shown to physically bind to nuclear receptors such as GR, AR and peroxisome
proliferator-activated receptor ϒ (PPARϒ) (Fujimoto et al., 1999; Guerrero et al., 2004;
Drori et al., 2005). A non-nuclear receptor transcription factor, Sp1, was also reported
interacting with Hic-5 (Shibanama et al., 2004). Interestingly, Hic-5 has neither HAT nor
HMT activity. In addition, no data claimed that Hic-5 can modulate chromatin nucleosome
positioning or recruit RNA PolII to the core promoter. Therefore, even though Hic-5 shows
potential role to be a nuclear receptor coactivator, the detailed mechanisms remain to be
elucidated.
Next section will focus on discussing the potential role of Hic-5 as a coactivator for
GR through the work of Lan Yang et al. (Yang et al., 2000). In the article by Yang, they
were trying to find novel proteins, which can interact with steroid receptor GR in the
nucleus. Also, they preliminarily investigated the function of this protein in GR signaling.
The findings from this research will be helpful to illustrate how GR works as a
transcriptional factor. This work finally offers evidence that Hic-5 is a protein, which
distributes both in the cytoplasm and in the nucleus, interacts with GRτ2 activation domain
and affects the transactivation activity of GRτ2.
This research started with a yeast two hybrid screening in order to find the binding
17
partner of GRτ2. They chose GRτ2 domain as the bait due to two properties of GRτ2
domain. First, it is a nuclear matrix targeting sequence (NMTS). Second, it is an
independent transactivation domain, which can be induced by hormone. Thus, this τ2
domain is likely to function in the nucleus when GR is activated by hormones. By using
GRτ2 domain as the bait, they can maximize the possibility to find a binding partner
residing in the nucleus and participating in GR transcriptional activity. Researchers
successfully isolated mouse Hic-5 from a mouse cDNA library as a binding partner. Next,
they tested the functional region of Hic-5. Specifically, for the C terminus and N terminus of
Hic-5, which one was responsible for interaction with GRτ2 and what potential functions
the other region has. In addition, researchers were interested in the role Hic-5 played for
the steroid receptor transactivation activity. Moreover, researchers wanted to examine the
traits of Hic-5 on GR as a coactivator. For example, is it in an “all or none” manner or in a
dose dependent manner? What is the relationship between Hic-5 and other known
coactivators like GRIP1? Finally, Hic-5, as previously mentioned was originally
characterized as a focal adhesion protein, existing at the plasma membrane of cells. On
the other hand, nuclear receptor coactivators are supposed to function in the nucleus and
transcription events take place in the nucleus. So the researchers were addressing the
question about subcellular location of Hic-5.
The authors have used appropriate methods thoroughly. Yeast two hybrid assay
has been overwhelmingly used in screening new proteins interacting to a known protein of
18
interest. It was also used to test the interaction between two known proteins. However, this
assay can give rise to false positive frequently. A mammalian two hybrid assay following
this yeast two hybrid assay may help to confirm the interaction between Hic-5 and GRτ2 in
metazoan cell conditions. Authors could also perform GST-pull down assays to investigate
whether this interaction is direct. Importantly, Hic-5 is a protein with three or four LD
domains in the N terminal half and four LIM domains at its C terminal half. So researchers
adopted yeast two hybrid and mammalian two hybrid assays to examine which half is
responsible for interaction with GRτ2. They made C terminal and N terminal halves fused
to either GAL4AD or GAL4DBD. When Hic-5 fragments are fused to GAL4AD, the GRτ2
interacting region could be identified; at the same time, if Hic-5 fragments are fused to
GAL4DBD, it offered a good control to check the intrinsic transactivation activity of Hic-5.
At the same time, to make sure that the differences in results were by transactivation
activity instead of different Hic-5 fragments expression level, it is equally important to
perform a western blot. Third, the authors used functional reporter assays to test the effect
on steroid receptor transactivation activity. This assay was frequently used to examine
protein coactivator properties. In the presence of hormone, GR, AR, MR, PR, estrogen
receptor (ER), and thyroid hormone receptor (TR) recognize their response elements
MMTV-LUC, MMTV (ERE)-LUC, MMTV (TRE)-LUC, inducing the expression of luciferase.
When the corresponding coactivator of the nuclear receptor was added in the system, the
transcriptional activity of this nuclear receptor would be further enhanced. Noteworthy,
19
they provided a comprehensive understanding about Hic-5 effect on steroid receptor
transactivation activity by observing this effect over a wide range of nuclear receptor
concentrations. However, it fails to show whether Hic-5 effects on nuclear receptors are
hormone dependent or independent. It also fails to show this potential coactivator effect on
transactivation activity of nuclear receptors towards their natural HREs in endogenous
genes and what effects will the coactivator bring to certain genes. Nowadays, ChIP is
prevailing over the study of DNA-protein interactions. This technique will be helpful to
understand whether Hic-5 is in nuclear receptor-basal transcriptional machinery
complexes as well as to understand if Hic-5 is in the nuclear. Previous knowledge
indicates that without hormone stimulation, inactivated GR is arrested in the cytoplasm by
heat shock proteins such like hsp70 and hsp90, while activated GR after hormone
stimulation translocates into the nucleus. At the same time, Hic-5 was known as a focal
adhesion protein. This suggested Hic-5 could act on GR signaling in the cytoplasm instead
of in the nucleus. To get a comprehensive understanding of GR subcellular distribution,
paraformaldehyde-fixed REF-52 cells and Cos-1 cells were subjected to indirect
immunofluorescence analysis. The signal was apparent and single cell situations could be
imaged. Researchers also checked Hic-5 expression in nuclear fractions via observing the
single band in the western blot. This would be helpful to test the antibody specificity
towards Hic-5 as the size of Hic-5 was known.
20
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Abstract (if available)
Abstract
Hydrogen peroxide inducible clone-5 (Hic-5), also named transforming growth factor-β1 induced transcript 1(TGFB1|1), TGF-β stimulated clone-5 (TSC-5) or androgen receptor associated protein 55 (ARA55), is a LIM domain protein. It has LIM domains at its carboxyl half and LD motifs at its amino half, thus providing multiple protein-protein interfaces for interaction with a large number of proteins. Like its homologous protein paxillin, Hic-5 exists at focal adhesion sites and is involved in mediating signals from the extracellular matrix. Hic-5 also plays an important role in cell adhesion under physiological conditions. The association of Hic-5 and diverse focal adhesion proteins including kinases, that can be regulated by small G proteins, highlights its function as a scaffold protein for focal adhesion dynamics. The expression of Hic-5 is selective with a preference of distribution in mesenchymal cells and endothelial cells. Recent findings have shown aberrant Hic-5 expression in certain tissues under pathological conditions such as glomerulosclerosis and carcinogenesis. Hic-5 is involved in several signaling pathways, which indicates that it may be involved in regulating crosstalk between them. One signaling pathway that Hic-5 participates in is the nuclear receptor signaling which is important for the transcriptional regulation of target genes. The regulation of nuclear receptor transcriptional activity renders Hic-5 a unique member of paxillin protein subfamily and a potential coactivator. Various strategies have been adopted to characterize the coactivator feature of Hic-5 over the past decade. Initial discovery by Yang et al. demonstrated that Hic-5 is a potential coactivator for a subset of nuclear receptors, which could partially localize in the nucleus and this research work will be discussed.
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Zhang, Mengrao
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Characteristics of hydrogen peroxide inducible clone-5 and its potential role as a nuclear receptor coactivator
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Biology
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10/09/2013
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hydrogen peroxide inducible clone-5,nuclear receptor coactivator,OAI-PMH Harvest
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