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

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