m.erriah-undergraduate lab project report
TRANSCRIPT
0
Exploring the expression of neuregulin 4 in a range of adult rat
tissues
Mélanie Erriah
School of Biosciences
University of Kent at Canterbury
Final Year Project (Module BI600)
2013
Supervisor: Professor Bill Gullick
Laboratory Project
1
Abstract
The neuregulins (NRGs) are a family of epidermal growth factor-like (EGF-like) ligands that
bind to the ErbB family of receptor tyrosine kinases. They are involved in the regulation of
growth, differentiation and survival of a variety of cell types. To date, four members have
been identified: NRG1, NRG2, NRG3 and NRG4. Though most of the NRGs have been
characterised, little is known about the distribution of NRG4 in mammalian tissues. The aim
of this study was to determine the expression and localisation of NRG4 in a range of adult rat
tissues. This was ascertained by immunohistochemistry using a rabbit polyclonal antibody
that specifically targeted the protein including its five isoforms: A1, A2, B1, B2 and B3. The
specificity of this antibody had been tested by ELISA, Western and immunoblotting when it
was first made by to ensure that it showed no cross-reaction to other related immunising
peptides. NRG4 was detected in high levels in the lungs, heart and reproductive organs but
was weakly expressed in the brain, liver and thymus. At the cellular level, NRG4 was
abundant in epithelial and endocrine cells such as those making up the salivary gland ducts
and pancreatic islets of Langerhans respectively. All cells positive for NRG4 exhibited diffuse
cytoplasmic staining but no nuclear staining. The data which showed a widespread
distribution of NRG4 in various murine organs, could be used in future studies to determine
whether certain diseases such as cancer are caused or accompanied by alterations in the
expression of NRG4.
Keywords:
Neuregulin 4; ErbB4/HER4; Epidermal growth factor receptor; Tyrosine kinase;
Immunohistochemistry.
2
Table of contents
Abbreviations ............................................................................................................................ 4
Introduction .............................................................................................................................. 5
Materials and methods ........................................................................................................... 12
Production of affinity-purified anti-NRG4 127 antibodies .................................................. 12
Bradford Assay ...................................................................................................................... 13
Direct enzyme-linked immunosorbent assay (ELISA) ......................................................... 13
Wax embedding and tissue sectioning ................................................................................... 14
Immunohistochemical staining .............................................................................................. 15
Results ..................................................................................................................................... 17
Affinity column purification of anti-NRG4 127 antibodies ................................................. 17
Immunoreactivity of anti-NRG4 127 antibodies .................................................................. 18
Optimisation of anti-NRG4 127 antibodies for immunohistochemistry .............................. 20
Anti-NRG4 127 antibody blockade with antigenic peptide .................................................. 21
Detection of NRG4 in adult rat tissues ................................................................................. 22
Expression of NRG4 in epithelial tissue ............................................................................... 25
Distribution of NRG4 in the brain ........................................................................................ 27
NRG4 localisation in the pancreas ....................................................................................... 28
Discussion ................................................................................................................................ 30
Presence of NRG4 in heart tissue ......................................................................................... 30
NRG4 and the reproductive system ...................................................................................... 31
3
Expression of NRG4 in epithelial tissue ............................................................................... 32
Localisation of NRG4 in the brain ....................................................................................... 32
Presence of NRG4 in endocrine tissue ................................................................................. 33
Acknowledgements ................................................................................................................. 35
References ............................................................................................................................... 36
4
Abbreviations
AA Arachidonic Acid
AR Amphiregulin
BSA Bovine Serum Albumin
BTC Betacellulin
CNS Central Nervous System
DAB 3,3'-Diaminobenzidine
EGFR Epidermal Growth Factor Receptor
ELISA Enzyme-Linked Immunosorbent Assay
EPR Epiregulin
HB-EGF Heparin Binding-EGF
IMS Industrial Methylated Spirit
MAP Mitogen-Activated Protein
NRG Neuregulin
OPD O-Phenylenediamine Dihydrochloride
PA Phosphatidic Acid
PBS Phosphate Buffered Saline
PBST Phosphate Buffered Saline Tween
PI3-K Phosphatidylinositol-3-Kinase
PLCγ Phospholipase C gamma
RT Room Temperature
RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
STAT Signal Transducer and Activator of Transcription
TGF-α Transforming Growth Factor-α
TMD Transmembrane Domain
5
Introduction
Cell signaling is a vital aspect of normal cell-to-cell communication, required by multicellular
organisms during tissue development to respond and adapt to changes in the environment. It
involves sending and receiving signals that are important in the regulation of many cellular
processes. An example is cell migration, a very complex process that allows cells to reach
particular destinations during embryonic development, conserve the cellular architecture of
self-renewing tissues and during wound healing as well as defend to body against invading
pathogens (1, 2). There are many classes of molecules involved in the transmission of signals
between cells, one of them are the ErbB receptors, named due to their homology to the
erythroblastoma viral protein, v-ErbB (3). These are a diverse set of Type I receptor tyrosine
kinases widely distributed throughout the animal kingdom which regulate a variety of cellular
processes like proliferation, inhibition of apoptosis, and differentiation (4, 5). In vertebrates
there exist four different family members, ErbB 1/epidermal growth factor receptor (EGFR),
ErbB2/neu/HER2, ErbB3/HER3 and ErbB4/HER4 all of which exist as homologous
transmembrane proteins (1, 4). ErbB receptors regulate the intracellular effects of ligands that
are more numerous and varied than the receptors themselves (6). To date, eleven ligands have
been characterized and classified into four groups: those that interact exclusively with EGFR
(EGF, Transforming Growth Factor Alpha-TGFα and amphiregulin-AR); those that bind to
EGFR and the HER4 receptor (heparin binding-EGF, betacellulin-BTC and epiregulin-EPR);
those which interact with either HER3 and HER4 (NRG1 and NRG2) and those which bind
only to HER4 (NRG3 and NRG4) (7, 8) (Fig. 1). ErbB receptor ligands are characterised by
the presence of a splice site located between the coding region for the fourth and fifth cysteine
residues and the position of the EGF-like binding domain near the transmembrane domain
(TMD) of the ligand. They are generally derived from the proteolytic cleavage of a wide
6
range of multidomain transmembrane proteins and all contain a conserved epidermal growth
factor (EGF) domain (4).
Fig. 1. Binding specificities of the four ErbB receptors. There exists four categories of ligands that
bind to the ErbB family of receptors: EGF, TGF-α and AR bind to ErbB1; HB-EGF, BTC and EPR
bind to ErbB1 and ErbB4; NRG1 and NRG2 bind to ErbB3 and ErbB4; NRG3 and NRG4 bind to
ErbB4. Adapted from (9).
The EGF is a 53 amino acid long polypeptide in its mature proteolytically processed form and
of size 6045Da derived from the proteolytic processing of its transmembrane precursor
prepro-EGF. EGFs are characterised by a conserved sequence known as the EGF motif which
consists of six cysteine residues forming three intramolecular disulphide bonds that are
essential for ligand binding to members of the HER receptor tyrosine kinase family (10). The
EGFR is derived from a polypeptide precursor of 1210 amino acids by cleavage of the N-
terminal region resulting in a 1186-residue protein that is inserted into the plasma membrane.
7
The sequence similarity between the four ErbB receptors ranges from 53% for EGFR and
ErbB3 to 64% for EGFR and ErbB2. X-ray crystallography analyses have shown that ErbB3
and ErbB4 ligands have the same mode of binding as that employed by EGF and TGFα to the
EGFR. The EGFR can also form heterodimers with its three homologues, ErbB2, ErbB3 and
ErbB4 depending on ligand binding (1, 6, 8). ErbB2 does not have a growth-factor ligand but
is the preferred dimerisation partner for other receptors (11).
Binding of the ligand to the EGFR receptor forms a 2:2 ligand to receptor configuration
resulting in a conformational change that exposes a region of the extracellular part of the
receptor called the dimerisation arm which then interacts with analogous dimerisation arms of
other activated HER receptors (12, 13) (Fig. 2). The transmembrane and kinase domains are
thought to help in stabilising receptor dimerisation (1). Another factor also promoting
receptor dimerisation include localisation of the receptor to caveolae, which make up about
one tenth of the plasma membrane, hence boosting its effective concentration (14).
Dimerisation of the receptor triggers activation of its intrinsic tyrosine kinase domain which
then transphosphorylates tyrosine residues present in the intracellular region of the opposite
receptor. Activation of the EGFR kinase results in the relocalisation of EGFR from caveolae
to the main membrane component and clustering of EGFR dimers into clathrin-coated pits
which are then internalised. Receptor internalisation and recycling controls the strength and
duration of intracellular EGFR signaling, which can be regulated by heterodimerisation at the
plasma membrane and by association with intracellular signaling ligands. Interestingly,
EGFRs can dimerise in the absence of fully functional tyrosine kinase domains. But in the
monomeric state the kinase tends to take on an inactive conformation, which explains its
reduced kinase activity (15).
8
Fig. 2. Cartoon representation of EGF-induced dimerisation of the EGFR extracellular region.
Binding of EGF (blue circle) to the monomeric receptor induces a conformational change which
exposes the dimerisation arm. The latter can then bind to an analogous dimerisation arm of another
HER receptor, resulting in intracellular signaling. Adapted from (16).
ErbB signaling is mediated by the transphosphorylation of tyrosine residues which then
function as binding sites for a range of downstream signaling molecules such as enzymes
including phosphatidylinositol-3-kinase (PI3-K) and phospholipase C gamma (PLCγ) and
adaptor proteins like Grb2 that act as intermediates in other pathways activated by ErbBs (14)
(Fig. 3). PI3-Ks have an important role in a variety of cellular functions such as cell growth,
survival and adhesion (1). Moreover the lipid products derived from the EGF/PI3-K pathway
are also thought to regulate cell adhesion and contribute to the remodeling of the actin
cytoskeleton, a crucial step in determining cell polarity during chemotaxis (17). On the other
hand, association of Grb2 to phosphorylated tyrosines is responsible for initiating the
mitogen-activated protein (MAP) kinase pathway (9). This is a very important pathway that
regulates cell differentiation, proliferation and death (18). Finally, it was also found that
ErbB2/ErbB4 heterodimers were the only receptor combination capable of activating Stat5, a
member of the signal transducer and activator of transcription (STAT) family and an
important regulator of cell proliferation and survival (9, 19).
9
Fig. 3. ErbB receptor signaling network. Binding
of the ligand to the EGFR induces its dimerisation.
This results in cross-phosphorylation of tyrosine
residues in its cytoplasmic domain leading to
activation of signaling cascades including the PLCɣ,
PI3-K, STAT and MAP kinase pathways. Adapted
from (20).
The neuregulins (NRGs) also known as heregulins, are an important subclass of polypeptide
ligands for ErbB receptor tyrosine kinases (12). The neuregulin family of genes has four
members: NRG1, NRG2, NRG3, and NRG4 (5). The NRG1 gene is approximately 1.4 Mb in
size which represents about 1/2000th of the genome, but the protein is only encoded by less
than 0.3% of the gene. Due to multiple alternative splicing and promoters, the NRG1 gene can
generate at least 15 different NRG1 isoforms which are classified into six families I-VI (21).
However only the EGF-like domain contained in those isoforms is required for activation of
ErbB receptor-tyrosine kinases (22). The signaling pathway used by NRG1 and NRG2
involve binding of the NRG to the extracellular domain of the receptor tyrosine kinases
ErbB3 or ErbB4 (or only ErbB4 in the case of NRG3 and NRG4), leading to the formation of
ErbB homo or heterodimers, which in turn activate different intracellular signaling pathways
in a ligand-dependant manner leading to various cellular responses that include stimulation or
inhibition of proliferation, apoptosis, cell migration, differentiation, and adhesion (6).
10
Neuregulin receptors are widely expressed in the postnatal nervous system and are believed to
be essential for the initial differentiation and survival of oligodendrocyte precursors as well as
for the stabilisation of neuromuscular synapses (23, 24). NRG1, the best characterised
member of the family, has a key role in neuronal development and regulation of synaptic
plasticity which allows the brain to respond and adapt to changes in the environment (25).
NRG2 has been found to have an important function in cellular growth, differentiation and
migration in a range of cell types such as epithelial, neuronal and glial cells (1). In cell
culture, neuregulins promote survival and growth of cardiac myocytes, and protect them from
anthracycline toxicity (26). In fact, NRGs are required for vital cell-to-cell communication in
both the adult and developing heart. In the adult, the endothelium of cardiac capillaries is
thought to be responsible for paracrine NRG signaling (5).
The human NRG4 gene is situated on the short arm of chromosome 15 at position 24.2. Five
alternatively spliced isoforms of NRG4 have been described to date, each sharing the first two
thirds of the EGF domain but differing at their COOH terminus: NRG4 A1, NRG4 A2, NRG4
B1, NRG4 B2 and NRG4 B3 (7, 27) (Fig. 4). The A variants encode a transmembrane domain
(TMD) and are transported to the cell membrane where they are thought to act in a juxtacrine
manner or are released in a soluble form via a tightly regulated proteolytic processing system.
The B variants on the other hand, do not encode a TMD and are released into the cytoplasm
as soluble proteins following their synthesis (28). NRG4 was discovered to be expressed at
the mRNA level in the pancreas and at the protein level in human breast and prostate cancer
(7, 29). Recent evidence suggests that NRG4 could have a role in the differentiation of the
somatostatin-expressing δ-cells found in islets of Langerhans in the pancreas (30). While
variable cytoplasmic levels of NRG4 have been detected in prostate cancer cells, only a
11
fraction exhibited NRG4 in the membrane. Unfortunately in both cases, high levels of the
protein usually pointed towards a poorer prognosis (27).
Fig. 4. Amino acid sequences of the five NRG4 isoforms. The amino acids highlighted in yellow
indicate the residues common to all NRG4 isoforms. The red and green rectangles designate the EGF-
like receptor binding domain and transmembrane domain respectively. The amino acids underlined in
black represent the synthetic peptide sequence used to generate the rabbit pan anti-NRG4 127
polyclonal antibody used in this study. Adapted from (27).
This study aimed to compare the expression and distribution of NRG4 in a range of healthy
adult rat tissues. This was done using a specific polyclonal pan anti-NRG4 127 antibody
raised in rabbits to detect the protein from formalin-fixed paraffin embedded rat tissues. The
data generated through this set of experiments has allowed us to document the levels of
NRG4 expression in various adult rat tissues. These findings could then be used further to
investigate the role of NRG4 more extensively in diseases such as cancer by observing any
discrepancies in the levels and distribution of NRG4 in the affected tissues.
A1 MPTDHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCEEVFLPGSSIQTKSNLF
EAFVALAVLVTLIIGAFYFLCRKGHFQRASSVQYDINLVETSSTSAHHSHEQH [115aa]
A2 MPTDHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCEEVFLPGSSIQTKSNLF
EAFVALAVLVTLIIGAFYFLCRCGNTCM [90aa]
B1 MPTDHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRK [36aa]
B2 MPTDHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCS [35aa]
B3 MPTDHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCSLHENENDNNEDLYDDLLPLNE [56aa]
EGF-like receptor binding domain
TMD
TMD
EGF-like receptor binding domain
12
Materials and Methods
Production of affinity-purified anti-NRG4 127 antibodies
The rabbit anti-NRG4 127 polyclonal antibodies made to a synthetic peptide,
PTDHEEPCGPSHKS recognised the homologous N-terminal of all the NRG4 isotypes (27)
(Fig. 4). The N-terminal methionine was not included in the immunising peptide as it was
assumed that it would be absent in the mature protein. The anti-peptide sera were affinity
purified using affinity column purification according to the following protocol. 5ml of Reacti-
Gel HW-65F agarose beads (Pierce Biotechnology Inc, Rockford, USA) were washed thrice
with 50mM sodium borate buffer pH 9 centrifuging at 2000 rpm for 2min between washes.
5mg of NRG4 127 antigenic peptide was added and rotated overnight at 4°C. The beads were
washed six times with storage buffer (100mM sodium phosphate buffer pH 8 with 0.05%
sodium azide), then 100mM glycine buffer pH 2.2 and twice again with storage buffer as
done previously. 4ml of immunised rabbit serum was added to the peptide coated beads and
rotated overnight at 4°C. 20µl of the serum sample was kept as ‘before’ sample for ELISA.
The next day, the supernatant was removed and 20µl was kept as ‘after’ sample for ELISA.
The beads were washed four times with storage buffer then loaded into an empty disposable
column. The column was washed with 10ml of storage buffer after which 500µl fractions
were collected in numbered Eppendorf tubes. Eight fractions with storage buffer were initially
collected as a baseline followed by ten fractions eluted with glycine buffer. The absorbance at
280nm of each fraction was measured simultaneously to determine the fractions containing
the affinity purified antibody which were immediately neutralised with 20µl 1M disodium
phosphate buffer pH 8. The antibody fractions were then dialysed against 1L of PBS
overnight at 4°C after which they were diluted in PBS to a concentration of 50µg/ml,
aliquoted and stored at -20°C.
13
Bradford Assay
This assay was performed to determine the yield of the affinity purification and estimate the
antibody concentration. The Bradford assay was done according to the Bio-Rad microtiter
plate protocol (Bio-Rad Laboratories, Hertfordshire, UK). Six dilutions of an IgG stock
solution were prepared at 10, 20, 30, 40, 50 and 60 µg/ml in PBS to be used as protein
standards from which a model curve could be plotted, and loaded in triplicates on a microtiter
plate at 50µl per well. The sample solution containing the dialysed antibody of unknown
concentration was diluted 1:3, 1:5 and 1:10 with PBS similarly loaded onto the plate. A
negative control containing PBS only was also included. 50µl of Dye Reagent Concentrate
(Bio-Rad Laboratories) diluted 1:5 with distilled water was dispensed in each well and mixed
by pipetting up and down. The plate was incubated at room temperature (RT) for 5 min before
reading the absorbance at 620nm in a MRXTM
Dynatech plate reader (Dynatech Laboratories
Inc., Chantilly, USA). A standard curve was then drawn and used to estimate the
concentration of the affinity purified antibodies.
Direct enzyme-linked immunosorbent assay (ELISA)
This assay was used to assess the immunoreactivity of the affinity purified anti-NRG4 127
antibody as well as the immune response of the pair of rabbits immunised with the NRG4
antigenic peptide. NRG4 127 antigenic peptide and a related NRG4 immunising peptide
NRG4 123 were diluted to a final concentration of 10µg/ml in coating buffer (200mM sodium
bicarbonate buffer pH 9.0). 50µl of the antigenic peptide was pipetted in each well of a
NuncTM
96-well microtiter plate (Fischer Scientific, Leicestershire, UK) which was then
covered and incubated overnight at 4°C. The plate was washed thrice with PBS + 0.05%
Tween-20 (PBST). 200µl blocking buffer (1% BSA in PBST) was added per well to block
any non-specific protein binding sites and incubated for 1 hour at room temperature. The
14
‘before’ (original serum sample from pair of immunised rabbits) and ‘after’ (serum incubated
with peptide coated beads) serum samples, affinity purified and control antibodies were
serially diluted in PBS at: 1:50, 1:200, 1:800, 1:3200, 1:12800 and added to the microtiter
plate in quadruplet at 50µl per well. The plate was incubated for 2 hrs at room temperature.
The plate was then washed four times with PBST. 50µl of the secondary antibodies: goat anti-
rabbit IgG-peroxidase (Sigma-Aldrich, Dorset, UK) were added per well at a dilution of 1/500
in blocking buffer. The plate was further incubated for 1 hr at RT then washed thrice with
PBST. Two pairs of SIGMAFAST™ OPD (O-Phenylenediamine dihydrochloride) tablets
(Sigma-Aldrich) were dissolved in 40ml of distilled deionised water and 200µl was added to
each well to detect peroxidase activity. After sufficient colour development, 50µl of 25%
sulphuric acid was added to each well to stop the reaction. The absorbance was then read at
492nm against a reference filter of 620nm in a MRXTM
Dynatech plate reader.
Wax embedding and tissue sectioning
The rat tissues previously fixed and stored in 10% neutral-buffered formalin (4%
formaldehyde in PBS), were cut in small pieces and dehydrated in a graded Industrial
Methylated Spirit (IMS) series: 30%, 60%, 90%, 100% for 2 hrs each with constant agitation,
then in 100% IMS overnight. The dehydrated tissues were then infiltrated in a graded series of
Histoclear in IMS: 50%, 100% for 2 hrs each then placed in molten wax overnight at RT. The
tissues were transferred to fresh molten wax and incubated at 65°C for 2 hrs before being
orientated in embedding moulds with fresh wax and allowed harden overnight. The blocks of
wax were then removed from the moulds, trimmed and mounted onto plastic cassettes using
molten wax. The tissues were sectioned at 5µm thickness with a Shandon Finesse 325 manual
rotary microtome (Thermo Scientific, Cheshire, UK) onto SuperFrost Plus adhesion coated
15
glass slides (Thermo Scientific) and incubated at 40°C for 30min before being processed for
immunohistochemistry.
Immunohistochemical staining
The tissues were deparaffinised and rehydrated in Histo-Clear (National Diagnostics,
Yorshire, UK) then through descending grades of ethanol up to water. Endogenous activity
was blocked by incubating sections in 3% hydrogen peroxide in distilled water for 10 min
after which the slides were washed in phosphate buffered saline (PBS) pH 7.4. Primary
antibodies diluted in blocking buffer were added to the sections and incubated for 90 min at
room temperature or overnight at 4°C in a humidified chamber. Negative controls were also
performed simultaneously for each tissue section to assess the presence of any non-specific
staining. This included omission of the primary antibody and blockade of the primary
antibody with the NRG4 127 antigenic peptide. After primary antibody incubation, the
sections were washed in PBS. The sections were then treated with biotinylated goat anti-
mouse and rabbit IgG (Diagnostic BioSystems, California, USA) for 25 min followed by
peroxidase-conjugated streptavidin (Diagnostic BioSystems) for 25 min washing in PBS
between each step. Finally the visualisation step was carried out using DAB (3,3'-
diaminobenzidine) chromogen/substrate kit (Diagnostic BioSystems). 50µl of concentrated
DAB chromogen solution was diluted in 1ml of DAB substrate buffer which was applied to
the sections for 10 sec, giving a brown end-product at the site of the target antigen. The
sections were then rinsed in distilled water before being counterstained for 30 sec with Gill II
hematoxylin (Merck Millipore, Nottingham, UK), dehydrated and mounted under coverslips
with DPX mountant (Sigma-Aldrich). The tissue sections were evaluated by microscopy
based image analysis and photographs were taken using a Leica Leitz DMRB microscope
with PL Fluotar 200X objective. The same procedure was followed for the
16
immunohistochemical staining of tissue array slides (Super Bio Chips, Seoul, Korea). These
are slides containing 23 cores of formalin-fixed 8 week old Sprague-Dawley rat tissue from a
variety of organs.
17
Results
Affinity column purification of anti-NRG4 127 antibodies
Following washing of the affinity purification column with storage buffer, 500µl fractions
were collected sequentially in numbered Eppendorf tubes. The first eight fractions were
collected as baseline and did not contain the antibody of interest. After elution of unbound
material, the eluent was changed to 100mM glycine buffer to allow elution of column bound
antibodies. The absorbance of each fraction was measured at 280nm to locate the fractions
containing the rabbit polyclonal anti-NRG4 127 antibodies. Two main peaks were obtained
with the fractions 11 and 13 that corresponded to the point at which the affinity purified
antibodies were eluted from the column (Fig. 5). Fraction 13 was consequently selected for
further analysis due to its highest antibody content as indicated by the high absorbance value
at 280nm. The concentration of the affinity purified antibodies was then determined by a
Bradford assay and subsequent comparison to a standard curve consisting of known
concentrations of purified IgG (Fig. 6). The final yield of anti-NRG4 127 antibodies purified
from 4ml of immunised rabbit serum was estimated to be around 60μg.
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 5 10 15
Ab
sorb
an
ce 2
80
nm
Fraction number
18
Fig. 5. Spectrum of absorbance at 280nm of each fraction collected during the affinity column
purification. The column was equilibrated with storage buffer during which the baseline fractions
were collected. The eluent was then changed to 100mM glycine buffer to allow elution of bound anti-
NRG4 127 antibodies observed in fractions 11, 12 and 13.
Fig. 6. Standard curve used to estimate the concentration of the affinity purified anti-NRG4 127
antibodies. A Bradford assay was done on a range of IgG samples of known concentration and the
absorbance at 620nm was linearly plotted against the protein concentration in μg/ml.
Immunoreactivity of anti-NRG4 127 antibodies
The specificity of the anti-NRG4 127 antibody to its respective antigen had been previously
confirmed by ELISA, Western blotting and immunoblotting against four other related
immunising peptides when it was first made. These experiments demonstrated that the
antibody reacted only with its respective peptide sequence and did not exhibit any cross-
reaction with the other related sequences (27). An ELISA was done with the new batch of
affinity purified anti-NRG4 127 antibodies to assess their reactivity to the antigenic NRG4
127 peptide against which they were raised and to another related NRG4 immunising peptide,
y = 0.002x + 0.012
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0 10 20 30 40 50 60 70
Ab
sorb
an
ce 6
20
nm
Protein Concentration (μg/ml)
19
NRG4 123. The ‘before’ and ‘after’ serum samples were also included for comparison and to
determine the efficiency of the affinity column purification which was estimated to be around
60% (Fig. 7). The affinity of anti-NRG4 127 towards its antigenic peptide was slightly higher
than towards the NRG4 123 peptide which indicates that the antibody is slightly more specific
to the peptide against which it was raised. On the other hand, the anti-NRG4 123 antibody
reacted very strongly to its antigenic peptide NRG4 123, showing a very good positive
control.
Fig. 7. Graph showing the reactivity of anti-NRG4 127 and 123 antibodies against NRG4 127
and 123 immunising peptides. A direct ELISA was performed using affinity purified rabbit
polyclonal antibodies against NRG4 127 and 123 peptides. The plate was coated with the two NRG4
peptides at 10µg/ml and incubated with the serum, control antibodies and affinity purified antibodies
at the following dilutions: 1:50, 1:200, 1:800, 1:3200 and 1:12800. The reactivity was detected with
peroxidase-labelled goat anti-rabbit IgG and the absorbances were measured at 492nm against a
reference filter of 620nm.
20
Optimisation of anti-NRG4 127 antibodies for immunohistochemistry
In order to determine the antibody concentration which would give the best
immunohistochemical staining results, an experiment was performed using different
concentrations of anti-NRG4 127 antibodies at 5, 10 and 15µg/ml. Rat kidney tissue was
chosen for this purpose for their high level of NRG4 expression as seen from previous
immunohistochemical experiments (data not shown). The staining obtained at 10 and 15µg/ml
was too intense and made it impossible to distinguish the counterstained NRG4-negative
structures (Fig. 8A, B). An appropriate balance of protein expression and background staining
was achieved at 5µg/ml of the antibody and was therefore the concentration chosen for
subsequent immunohistochemical staining using anti-NRG4 127 (Fig. 8C).
Fig. 8. Immunohistochemical staining of rat kidney tissue at different concentrations of anti-
NRG4 127. The photographs show 5µm tissue sections through the renal cortex with glomeruli [1]
surrounded by distal and proximal convoluted tubules. The sections were incubated with the primary
antibodies for 90min at RT then with DAB chromogen for 10 sec. Intense NRG4 staining was
observed at 15µg/ml [A] and 10µg/ml [B] of anti-NRG4 127 antibody which completely obscured
NRG4 negative cells and structures. A concentration of 5µg/ml [C] showed a very good contrast
between NRG4 positive and negative cells and structures. Counterstaining was done using Gill II
hematoxylin. Original magnification, x100.
A B C
1 1
1
21
Anti-NRG4 127 antibody blockade with antigenic peptide
As a negative control, it was important to test whether the antibody retained its
immunoreactive properties against NRG4 when blocked with its antigenic peptide prior to
immunohistochemistry. A peptide block was therefore performed using the NRG4 peptide
against which the anti-NRG4 127 antibody was raised. Rat testis tissue was chosen for its low
to moderate NRG4 expression observed from previous immunohistochemical staining
experiments (data not shown). In the negative control, the tissues were incubated with
blocking buffer instead of the primary antibody. In the positive control, the antibody was
rotated with PBS for 1 hr instead of the antigenic peptide. The negative control did not show
any non-specific brown staining indicating a good background (Fig. 9A). The positive control
showed some NRG4 staining, particularly the interstitial Leydig cells and the germinal
epithelium lining the seminiferous tubules (Fig. 9B). The peptide block was free of any NRG4
staining, indicating that the antibodies were successfully blocked by the antigenic peptide
(Fig. 9C).
Fig. 9. Blockade of NRG4 127 antibodies with antigenic peptide shown on rat testis tissue. The
photographs show 5µm sections through the rat testis tissue with seminiferous tubules [1] surrounded
by patches of Leydig cells [2]. The antigenic peptide was added to the primary antibodies at a
concentration of 5mg/ml and rotated for 1hr at room temperature. The antibodies were then added to
the sections at a concentration of 5μg/ml for 90min at RT and the sections were then incubated with
DAB chromogen for 10 sec. Negative control without anti-NRG4 127 showed absence of brown
A B C
1
1
1 2
22
staining indicating a good background [A]. Positive control with NRG4 127 showed moderate staining
of Leydig cells and the seminiferous tubule germinal epithelium [B]. Peptide block of anti-NRG4 127
showed no staining indicating a complete blockade of the antibody by the antigenic peptide [C].
Counterstaining was done using Gill II hematoxylin. Original magnification, x200.
Detection of NRG4 in adult rat tissues
It was previously demonstrated that NRGs, particularly NRG1 are highly expressed in human
heart muscle where they are thought to have a vital role in cell signaling and cardiovascular
development, however not much is known about their level of NRG4 expression (5, 31). An
immunohistochemical study was carried out on a wide range of adult rat tissues using anti-
NRG4 127 antibodies (which detect all the NRG4 splice variants) to determine their level of
NRG4 expression. A tissue array containing 23 cores of formalin-fixed 8 week old Sprague-
Dawley rat tissue was used for this study (Table 1). The staining with anti-NRG4 127 was
highly variable in intensity but remained cytoplasmic in all tissues tested. Among the tissues
that showed weak (-/+) NRG4 staining were the liver, pons and thymus (Fig. 10A-C). The
adrenal gland, spleen and stomach showed moderate (+/++) staining (Fig. 10D-F). NRG4
expression was mainly localised to the cortex in the adrenal gland and goblet cells lining the
crypts in the stomach tissue. Strong (++/+++) staining was observed in the heart, prostate and
uterus tissue (Fig. 10G-I). NRG4 staining was mainly present in the fibromuscular stroma but
not in the convoluted epithelium lining the prostate glands. The uterus showed the presence of
NRG4 which was highly expressed in the myometrium and epithelium lining the endometrial
glands but absent in endometrium and perimetrium. The nervous and digestive system
showed the lowest level of NRG4 expression whereas the reproductive, respiratory and
urinary systems showed the highest. These observations may suggest a direct link between
23
cell metabolic activities and level of NRG4 expression due to the need for rapid cell-to-cell
communication, especially in tissues like heart muscle.
Organ system Tissue NRG4 127 expression
Cardiovascular/Musculoskeletal
Heart +++
Skeletal muscle, abdominal wall +
Skin, ear lobe ++
Digestive
Salivary gland ++
Liver +
Pancreas ++
Stomach +
Ileum +
Colon +
Immune Spleen +
Thymus -
Nervous
Cerebrum -
Cerebellum +
Pons -
Renal
Adrenal gland ++
Kidney, cortex ++
Kidney, medulla +++
Reproductive
Prostate +++
Testis ++
Epididimis +
Uterus ++
Respiratory Lung +++
24
Table 1. Table showing the expression of NRG4 in a variety of rat tissues. The negative sign [-]
indicates that the tissue did not show any brown staining and that NRG4 was absent. The positive sign
[+] indicates that brown staining was observed and NRG4 was present. The number of positive signs
represents the strength of NRG4 staining in each tissue. All tissues tested were taken from a single
tissue array slide.
Fig. 10. Examples of weak, moderate and strong NRG4 immunohistochemical staining in
various rat tissues. Antibodies were added to the tissues array at a concentration of 5μg/ml for 90min
at RT and the sections incubated with DAB chromogen for 10 sec. The liver [A], pons [B] and thymus
[C] exhibited no or very weak staining with anti-NRG4 127 which detects all NRG4 isoforms.
Moderate staining was observed in the adrenal gland [D], particularly the cortex [1] but weaker in the
medulla [2]; spleen [E] and stomach [F]. The goblet cells lining the crypts [3] in the stomach
expressed moderate levels of NRG4 compared to the stroma which was weakly positive for NRG4.
G H I
A B C
1
2
3
4
5
6
7
9
8
D E F
25
The heart [G], prostate [H] and uterus [I] showed very high levels of NRG4 expression. The prostate
showed NRG4 staining of fibromuscular stroma [4] surrounding the gland [5] but was absent in the
convoluted epithelium [6]. In the uterus, the myometrium [7] and endometrial gland epithelium [8]
were strongly positive for NRG4 compared to the perimetrium [9]. Counterstaining was done using
Gill II hematoxylin. Original magnification, x200.
Expression of NRG4 in epithelial tissue
ErbB1 was shown to have a very important role in epithelial cell development in several
organs such as the intestines, lung and skin. It was discovered that knockout of the ErbB1
gene in mice led to abnormalities in cell proliferation, migration and differentiation of
epithelial cells (3). To address whether NRG4 was also present in that tissue type, a range of
normal rat tissues were immunohistochemically stained using the anti-NRG4 127 affinity
purified antibody which reacts to all known NRG4 isoforms. Cytoplasmic staining of NRG4
observed in most tissues tested was variable in intensity but localised to epithelial tissue (Fig.
8). The anti-NRG4 antibody was strongly positive in the cortical area of the kidney, primarily
the cells making up the proximal and distal convoluted tubules (Fig. 11A). Some instances of
membrane staining along the apical surface of the cells forming the proximal convoluted
tubules were also observed. Glomeruli on the other hand did not demonstrate any NRG4
staining. The lung tissue showed strong staining of alveolar epithelial cells and pneumocytes
(Fig. 11B). NRG4 staining of the salivary gland indicated that the protein is specifically
localised to epithelial cells making up the striated ducts (Fig. 11C). No NRG4 staining was
observed in the serous acini making up the stroma. The skin tissue showed moderate to strong
staining in the epidermis, endothelial cells and adipose tissue (Fig. 11D). The dermal
fibroblasts showed weaker NRG4 staining while cartilage did not show any. No nuclear
staining was observed in any of the tissues. The level of NRG4 expression in the four tissues
26
tested was quite significant, indicating that there might be a direct correlation between the
amount of staining and the specific function of epithelial tissue.
Fig. 11. Detection of NRG4 in various rat tissues by immunohistochemical staining. Antibodies
were added at a concentration of 5μg/ml and the tissue array was incubated with DAB chromogen for
10 sec. The kidney [A] showed strong NRG4 staining in the proximal [1] and distal [2] convoluted
tubules but was absent in glomeruli [3]. The lung section [B] showing alveoli [4], pneumocytes [5]
and alveolar epithelium [6] exhibited strong cytoplasmic staining of pneumocytes and epithelium
lining the alveoli. The salivary gland [C] showed epithelial NRG4 staining of striated ducts [7] within
NRG4 negative serous acini [8]. The skin section [D] showing epidermis [9], dermis [10], hypodermis
[11], adipose tissue [12], blood vessel [13], sebaceous gland [14] and cartilage [15] exhibited strongly
positive NRG4 staining in the epidermis, adipose tissue and endothelial cells of blood vessels. The
A B
1 5
4
6
8
7
2
3
C D
9
10
11
12
13
14
15
27
cartilage was free of any NRG4 staining. Counterstaining was done with Gill II hematoxylin. Original
magnification, x200 for all tissues except skin taken at x100.
Distribution of NRG4 in the brain
The presence of NRGs in the mammalian central nervous system (CNS) has been well
characterised by reverse transcriptase-polymerase chain reaction (RT-PCR) and
immunohistochemistry (32). In order to determine whether NRG4 is present in the brain, an
immunohistochemical staining experiment was performed on formalin-fixed rat cerebrum and
cerebellum tissues. The cerebral cortex showed no brown staining with the anti-NRG4 127
antibodies, indicating the absence of NRG4 (Fig. 12A). The cerebellum on the other hand,
showed strong NRG4 staining of a specific layer of cells between the molecular and granular
layer, known as Purkinje cells while the rest of the tissue showed weak NRG4 expression
(Fig. 12B). NRG4 staining was diffuse and homogenous in the cytoplasm and no nuclear
staining was observed. The expression of NRG4 in the cerebellum appears to be cell specific,
suggesting that NRG4 might have a particular function in Purkinje cells.
Fig. 12. Immunohistochemical staining of rat cerebrum and cerebellum tissues using anti-NRG4
127. The antibodies were added at a concentration of 5μg/ml for 90min at RT and the tissue array
incubated with DAB chromogen for 10sec at RT. The cerebrum [A] showing neuronal cells [1]
A B
1
2 4
5 3
28
surrounded by neuropil [2] exhibited no NRG4 staining. The cerebellum [B] showed NRG4 staining
that was very weak in the molecular [3] and granular [4] layer but strongly positive in Purkinje cells
[5]. Original magnification, x100.
NRG4 localisation in the pancreas
It was previously demonstrated that NRG4 is expressed in high levels in the endocrine
pancreas and is restricted exclusively to somatostatin producing δ-cells (30). In order to test
whether these observations were reproducible, an immunohistochemical staining experiment
was done on a section of formalin-fixed rat pancreas. Concurrent with the literature, the
pancreas exhibited moderate NRG4 staining which was exclusively limited to islets of
Langerhans (Fig. 13). However from this experiment it was impossible to determine the
specific cell type expressing NRG4. On the other hand, acinar cells making up the pancreatic
stroma did not show any positive NRG4 staining.
Fig. 13. Immunohistochemical staining of rat pancreas tissue with anti-NRG4 127 antibodies.
The tissue section was incubated with the antibodies at a concentration of 5μg/ml for 90min at RT and
1
2
29
then with DAB chromogen for 10sec at RT. The pancreas showed strong staining of the islets of
Langerhans [1] but no NRG4 was present in the surrounding acinar cells [2]. Original magnification,
x200.
30
Discussion
The neuregulins (NRGs) are ligands of the family of epidermal growth factors (EGF) which
are involved in the growth, differentiation and survival of different types of cells. They have
been found to be present in moderate to high levels in a range of mammalian tissues such as
the CNS, epithelium and heart (1). However not much is known about the expression of the
most recently characterised neuregulin, NRG4. In order to detect the protein, a rabbit
polyclonal antibody (anti-NRG4 127) was generated against a specific 14 amino acid long
peptide sequence common to all five isoforms of NRG4. When it was first made, this
antibody had been thoroughly tested for its specificity to the peptide sequence against which it
was raised and showed no signs of cross-reaction with other related immunising peptides. A
previous immunohistochemical study carried out with anti-NRG4 127 revealed that it was
strongly positive in human prostate cancer tissue (27). In the current study done on formalin-
fixed normal adult rat tissues, the antibody detected high cytoplasmic levels of NRG4 in the
heart, epithelium and reproductive organs, but lower levels in the brain, liver and thymus. In
some tissues, NRG4 expression was localised to specific cells such as epithelial, endothelial
and endocrine cells. This suggests that NRG4 might have roles that are directly linked to the
particular functions of those cells.
Presence of NRG4 in heart tissue
Previous studies have shown that NRG1 plays a key role in cardiovascular development and
maintenance of the physiological functions of the adult heart. In fact, clinical trials have
shown that NRG1 can enhance survival, growth and proliferation of cardiomyocyte,
encourage angiogenesis in the heart, neutralise excessive β-adrenergic signaling and preserve
cardiac myofibril structure, making them a good drug candidate for treating cardiac injury and
heart failure patients (31). However so far only NRG1 has been reported to be involved in the
31
development and function of the heart and very little is known about the presence of NRG4 in
myocardial tissue. In order to test whether NRG4 was expressed in the heart, an
immunohistochemical staining experiment was carried out on formalin-fixed heart tissue
using the anti-NRG4 127 antibody that reacts to all five isoforms of NRG4. This experiment
revealed that NRG4 was present in high levels in cardiac myocytes and exhibited uniform
cytoplasmic staining. These observations might suggest a direct link between the level of
NRG4 expression and tissue metabolic rate. However a larger study would have to be
undertaken with a range of high and low metabolically active tissues in order to investigate
this hypothesis further.
NRG4 and the reproductive system
It was recently demonstrated that HB-EGF is expressed in the human and murine endometrial
luminal epithelium and regulates processes involved in embryo implantation including
vascular permeability, decidualisation and transcription of implantation marker genes (33).
NRG4 was also shown to be present in the uterine epithelium, suggesting that it might be
involved in signaling mechanisms that regulate implantation of the embryo (34). Previous
immunohistochemical studies have also demonstrated the presence of NRG4 in the prostate
(28). However very little is known about the distribution of NRG4 in other parts of the
reproductive system. The immunohistochemical study done to investigate the presence of
NRG4 in various tissues showed that it was relatively highly expressed in the rat reproductive
system. High levels were detected in the prostate stroma and uterine myometrium, while
moderate levels were observed in the testis germinal and Leydig cells. The cell specificity of
NRG4 expression observed in those tissues might suggest that NRG4 is essential for functions
that are particular to those cells such as prostatic development, induction of uterine
contractions, hormone secretion or sperm production. Future work could make use of RT-
32
PCR analysis to determine the mRNA expression of NRG4 or ErbB4 in other regions of the
reproductive system such as the seminal vesicle, fallopian tubes and ovaries.
Expression of NRG4 in epithelial tissue
Binding of EGF ligands to receptors of the ErbB family has been shown to influence the
growth of epithelial cells (1). Immunohistochemical analyses have demonstrated the presence
of NRG1, ErbB2 and ErbB3 in human lung and murine mammary gland epithelium and
ErbB4 in the epithelium of several organs including the kidney, salivary gland and testis (35).
NRG1 is thought to be involved in the autocrine regulation of epithelial cell proliferation and
differentiation (32). However as yet, the presence of NRG4 in epithelial cells has not been
fully described. The immunohistochemical study done with a range of adult rat tissues
revealed that NRG4 also tends to be mainly localised to the epithelium. NRG4 expression was
detected in moderate to high levels in the kidney proximal and distal convoluted tubules,
testis germinal epithelium, blood vessel endothelium, skin epidermis, salivary gland ducts and
alveolar epithelium. This suggests that NRG4 might also have a key role in mediating
intraepithelial signaling to regulate cell growth, differentiation and morphogenesis in those
tissues. Future work could look into the subcellular localisation of NRG4 in human
immortalised epithelial cell lines e.g. HeLa cells by immunofluorescence microscopy.
Localisation of NRG4 in the brain
RT-PCR analyses have demonstrated the presence of NRG2, NRG3 and ErbB4 in various
human and mouse tissues, with high expression levels observed in the brain which was
confirmed by immunohistochemistry (32). Activation of ErbB4 and subsequent PI-3K
signaling are thought to be crucial in brain development and function (36). However while the
presence of NRG2 and NRG3 in the mammalian CNS have been described, that of NRG4 is
33
not as well documented. Immunohistochemical staining of the cerebellum revealed that
NRG4 was present in relatively low levels in that tissue but was particularly concentrated in
Purkinje cells present at the interface between the molecular and granular layer. This suggests
that NRG4 might be involved in the transmission of nerve impulses or other neural functions
that are specific to those cells. It would be interesting to investigate the presence of the
different NRG4 splice variants in Purkinje cells using antibodies specific to each variant and
determining their subcellular localisation through immunofluorescence microscopy.
Presence of NRG4 in endocrine tissue
Previous studies have demonstrated the presence of NRG4 in high levels in the endocrine
pancreas, where it stimulates the development of somatostatin producing δ-cells, suggesting
that it is involved in the determining the fate of islet cells during pancreatic development (30).
However very little is known about the presence and distribution of NRG4 in other endocrine
tissues. The immunohistochemical study undertaken with a variety of tissues showed that
some tissues containing hormone-secreting cells exhibited good NRG4 staining. Moderate to
high levels of NRG4 expression were observed in the pancreas islets of Langerhans, adrenal
cortex in the adrenal gland, Leydig cells in the testis, cardiac myocytes and adipose tissue.
These findings might suggest that NRG4 plays an important role in endocrine tissue
differentiation and function. However in order to further explore their precise role in these
tissues, additional studies will have to be performed. This could involve investigating the
effects of the absence of NRG4 or ErbB4 on endocrine tissue development in knockout mice.
In summary, this study has provided us with a good insight on the expression and distribution
of NRG4 in a murine model. Future work on NRG4 might provide information on the
subcellular localisation of the protein and its splice variants which could help to more
34
precisely understand its role in each cell type and interpret any changes in their expression
patterns in disease states.
35
Acknowledgements
I would like to thank Mrs. Edith Blackburn for her generous help and support with the
laboratory work and Prof Bill Gullick for giving me the opportunity to work on this very
interesting research project.
36
References
1. Jorissen, R.N., Walker, F., Pouliot, N., Garrett, T.P.J., Ward, C.W., Burgess, A.W. (2003)
Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res 284:
31-53.
2. Kaverina, I., Krylyshkina, O., Small, J.V. (2002) Regulation of substrate adhesion
dynamics during cell motility. Int J Biochem Cell Biol 34: 746–761.
3. Citri, A., Yarden, Y. (2006) EGF–ERBB signaling: towards the systems level. Mol Cell
Biol 7: 505-516.
4. Stein, R.A., Staros, J.V. (2006) Insights into the evolution of the ErbB receptor family and
their ligands from sequence analysis. BMC Evol Biol 6: 1-17.
5. Falls, D.L. (2002) Neuregulins: functions, forms, and signaling strategies. Exp Cell Res
284: 14-30.
6. Yarden, Y., Sliwkowski, M.X. (2001) Untangling the ErbB signaling network. Nat Rev
Mol Cell Biol 2: 127–137.
7. Hayes, N.V.L., Gullick, W.J. (2008) The neuregulin family of genes and their multiple
splice variants in breast cancer. J Mammary Gland Biol Neoplasia 13: 205–214.
8. Olayioye, M.A., Neve, R.M., Lane, H.A., Hynes, N.E. (2000) The ErbB signaling network:
receptor heterodimerisation in development and cancer. EMBO J 19: 3159–67.
9. Hynes, N.E., Horsch, K., Olayioye, M.A., Badache, A. (2001) The ErbB receptor tyrosine
family as signal integrators. Endocr-Relat Cancer 8: 151-159.
10. Harris, R.C., Chung, E., Coffey, R.J. (2002) EGF receptor ligands. Exp Cell Res 284: 2-
13.
11. Miyazaki, Y., Nakanishi, Y., Hieda, Y. (2004) Tissue interaction mediated by neuregulin-
1 and ErbB receptors regulates epithelial morphogenesis of mouse embryonic submandibular
gland. Dev Dynam 230: 591-596.
37
12. Montero, J.C., Rodriguez-Barrueco, R., Ocan, A., Diaz-Rodriguez, E., Esparis-Ogando,
A., Pandiella, A. (2008) Neuregulins and Cancer. Clin Cancer Res 14: 3237-41.
13. Burgess, A.W., Cho, H.S., Eigenbrot, C., Ferguson, K.M., Garrett, T.P., Leahy, D.J., et al.
(2003) An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.
Mol Cell 12: 541-52.
14. Lemmon, M.A., Schlessinger, J. (2010) Cell signaling by receptor tyrosine kinases. Cell
141: 1117-34.
15. Ceresa, B.P. (2006) Regulation of EGFR endocytic trafficking by rab proteins. Histol
Histopathol 21: 987-993.
16. Lemmon, M.A. (2009) Ligand-induced ErbB receptor dimerization. Exp Cell Res 315:
638-648.
17. Weiner, O.D. (2002) Rac activation: P-Rex1—a convergence point for PIP(3) and
Gbetagamma? Curr Biol 12: R429–R431.
18. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B., Karandikar, M., Berman, K., Cobb,
M.H. (2001) Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and
Physiological Functions. Endocr Rev 22: 153-183.
19. Shelburne, C.P., McCoy, M.E., Piekorz, R., Sexl, V.V., Gillespie, S.R., Bailey, D.P. et al
(2002) Stat5: an essential regulator of mast cell biology. Mol Immunol 38: 1187–1191.
20. Gschwind, A., Fischer, O.M., Ullrich, A. (2004) The discovery of receptor tyrosine
kinases: targets for cancer therapy. Nat Rev Cancer 4: 361-370.
21. Buxbaum, J.D., Georgieva, L., Young, J.J., Plescia, C., Kajiwara, Y., Jiang, Y., et al.
(2008) Molecular dissection of NRG1-ERBB4 signaling implicates PTPRZ1 as a potential
schizophrenia susceptibility gene. Mol Psychiatr 13: 162-172.
22. Buonanno, A., Fischbach, G.D. (2001) Neuregulin and ErbB receptor signaling pathways
in the nervous system. Curr Opin Neurobiol 11: 287–296.
38
23. Gerecke, K.M., Wyss, J.M., Karavanova, I., Buonanno, A., Carroll, S.L. (2001) Erbb
transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat
central nervous system. J Comp Neurol 433: 86–100.
24. Park, S.K., Miller, R., Krane, I., Vartanian, T. (2001) The ErbB2 gene is required for the
development of terminally differentiated spinal cord oligodendrocytes. J Cell Biol 154: 1245–
58.
25. Steinthorsdottir, V., Stefansson, H., Ghosh, S., Birgisdottir, B., Bjornsdottir, S., Fasquel,
A.C., et al. (2004) Multiple novel transcription initiation sites for NRG1. Gene 342: 97-105.
26. Sawyer, D.W., Zuppinger, C., Miller, T.A., Eppenberger, H.M., Suter, T.M. (2002)
Modulation of anthracycline-induced myofibrillar disarray in rat ventricular myocytes by
neuregulin-1beta and anti-ErbB2: potential mechanism for trastuzumab-induced
cardiotoxicity. Circulation 105: 1551–1554.
27. Hayes, N.V.L., Blackburn, E., Smart, L.V., Boyle, M., Russell, G., Frost, T., et al. (2007)
Identification and characterisation of novel spliced variants of NRG4 in prostate cancer. Clin
Cancer Res 13: 3147–55.
28. Hayes, N.V.L., Blackburn, E., Boyle, M.M., Russell, G.A., Frost, T.M., Morgan, B.J.,
Gullick, W.J. (2011) Expression of neuregulin 4 splice variants in normal human tissues and
prostate cancer and their effects on cell motility. Endocr-Relat Cancer 18: 39-49.
29. Dunn, M., Sinha, P., Campbell, R., Levinson, N., Rampaul, R., Bates, T., et al. (2004) Co-
expression of neuregulin 1, 2, 3 and 4 in human breast cancer. J Pathol 203: 672–80.
30. Huotari, M.A., Miettinen, P.J., Palgi, J., Koivisto, T., Ustinov, J., Harari, D., et al. (2002)
ErbB signaling regulates lineage determination of developing pancreatic islet cells in
embryonic organ culture. Endocrinology 143: 4437-4446.
31. Xinhua, Y., James, P.M. (2011) Neuregulin1 as novel therapy for heart failure. Curr
Pharm Design 17: 1808-1817.
39
32. Veikkolainen, V., Vaparanta, K., Halkilahti, K., Iljin, K., Sundvall, M., Elenius, K. (2011)
Function of ERBB4 is determined by alternative splicing. Cell Cycle 10: 2647-2657.
33. Lim, H.J., Dey, S.K. (2009) HB-EGF: a unique mediator of embryo-uterine interactions
during implantation. Exp Cell Res 315: 619-626.
34. Brown, N., Deb, K., Paria, B.C., Das, S.K., Reese, J. (2004) Embryo-uterine interactions
via the neuregulin family of growth factors during implantation in the mouse. Biol Reprod 7:
2003-2011.
35. Gollamudi, M., Nethery, D., Liu, J., Kern, J.A. (2004) Autocrine activation of
ErbB2/ErbB3 receptor complex by NRG-1 in non-small cell lung cancer cell lines. Lung
Cancer 43: 135-143.
36. Carteron, C., Ferrer-Montiel, A., Cabedo, H. (2006) Characterization of a neural-specific
splicing form of the human neuregulin 3 gene involved in oligodendrocyte survival. J Cell Sci
119: 898-909.