From Genesis Research and Development Corporation Limited, P. O. Box 50, Auckland 1015, New Zealand and § Genometrix Incorporated, The Woodlands, Texas 77381
Received for publication, August 2, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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High throughput sequencing of a mouse
keratinocyte library was used to identify an expressed sequence tag
with homology to the epidermal growth factor (EGF) family of growth
factors. We have named the protein encoded by this expressed sequence
tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF
superfamily. Two hydrophobic regions, corresponding to a putative
signal sequence and transmembrane domain, flank a core of amino acids
encompassing six cysteine residues and two putative
N-linked glycosylation sites. Epigen shows 24-37%
identity to members of the EGF superfamily including EGF,
transforming growth factor The epidermis of mammalian skin is a complex structure, the
assembly and maintenance of which requires the regulation of a large
number of genes (1). In particular, mRNAs encoding several members
of the EGF1 superfamily of
growth factors have been localized to the proliferative compartment of
the epidermis, suggesting that they play an important role in the
maintenance of skin structure (2-4). The EGF superfamily is an
expanding group of growth factors containing several members including
EGF, TGF Just as the EGF superfamily comprises structurally similar members, so
do the receptors through which the peptides signal. In mammals, the EGF
receptor tyrosine kinase family includes c-erbB-1 (EGFR,
HER-1), c-erbB-2 (HER-2, Neu), c-erbB-3
(HER-3), and c-erbB-4 (HER-4) (19-22). c-erbB
receptor signal transduction begins with stabilization of a receptor
homo- or heterodimer through ligand binding (23). Signaling is such
that a single receptor, e.g. c-erbB-1, can bind
to several EGF superfamily ligands (EGF, TGF The Ras-MAP kinase signaling pathway is widely used by receptor
tyrosine kinases to promote diverse cellular responses including cell
growth, differentiation, and apoptosis (27, 28). In vitro analysis of EGF superfamily members has revealed that all are able to
stimulate or inhibit the proliferation of epithelia-derived normal and
transformed cell lines, and several members are able to stimulate
growth of fibroblasts, smooth muscle, and neural cell lines in culture
(11, 29-35). EGF and TGF We have identified a novel member of the EGF ligand superfamily from
mouse keratinocytes, which we have named Epigen, for epithelial mitogen. mRNA encoding Epigen
has a restricted tissue distribution, present in heart, liver, and
testis. We have purified recombinant Epigen and compared its biological
activities with that of EGF and TGF Cell Culture--
HaCaT and A431 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine
serum, 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Life Technologies, Inc.), 0.77 mM L-asparagine (Sigma), 0.2 mM
arginine (Sigma), 160 mM penicillin G (Sigma), 70 mM dihydrostreptomycin sulfate (Roche Molecular Biochemicals). HaCaT SRE cells were also supplemented with 0.5 mg/ml
geneticin (Life Technologies, Inc.).
cDNA Identification--
A population of basal keratinocytes
from the epidermis of neonatal mouse skin was used to generate a
directionally cloned cDNA library in pBK-CMV using the Zap Express
kit (Stratagene). Clones excised from the library were sequenced from
the T3 primer, and homology searches were performed using the FastA and
FastX algorithms.
Northern Blotting--
An adult mouse tissue Northern blot was
purchased from CLONTECH and probed with a
[32P]dATP end-labeled antisense oligonucleotide
corresponding to the sequence
5'-GGTCGATATAGGAACCAGCTACCTTTGCCTGCTCTGGATCTGCTG-3' according to the
manufacturer's instructions. The blot was reprobed using the control
Bacterial Construct for Protein Expression--
For bacterial
expression, the vector pET16b (Novagen) was modified to shorten the
bacterial leader sequence. The vector was digested with NcoI
and XhoI to remove the existing leader sequence. This was
replaced by the sequence
5'-ccatgggccatcaccatcaccaccatgcgaattcgctcgag-3', which encodes the
amino acid sequence MGHHHHHHANSLE. DNA encoding residues 53-103
of Epigen was polymerase chain reaction-amplified using the primers
5'-gggaattctctgaagttctctcatc-3' and 5'-gcggatccttaagcatacgaagttag-3'. The resulting DNA fragments were digested with EcoRI and
BamHI and ligated into the modified pET16b vector.
SRE Construct--
A pGL3-promoter vector (Promega)
containing the neomycin resistance gene was used to develop an SRE
luciferase reporter construct where the SRE was inserted 5' of the SV40
promoter. The following primers were used to generate the enhancer
region of the human c-fos promoter: SRE1,
5'-gatccgcagcccgcgagcagttcccgtcaatccctcccccctta-3'; SRE2,
5'-cacaggatgtccatattaggacatctgcgtcagcaggtttccacggcctttccctgtagcccta-3'; SRE3,
5'-cctaatatggacatcctgtgtaaggggggagggattgacgggaactgctcgcgggctgcg-3'; and
SRE4, 5'-gatctagggctacagggaaaggccgtggaaacctgctgacgcagatgt-3'. Primers
were annealed to prepare two double-stranded oligonucleotides: SRE1/3
and 2/4. SRE1/3 and SRE2/4 were then ligated together into the
BamHI and BglII sites of pGL3 to form the SRE. A
concatamer of eight SRE enhancers was prepared by subsequent rounds of
digestion and SRE ligation into pGL3.
Recombinant Protein Expression and Purification--
Small scale
expression of Epigen in pET16b was induced in BL21/DE3/Lys cells
according to the manufacturer's instructions. Protein induction was
monitored by analyzing whole bacterial lysates by SDS-PAGE and Western
blotting using an anti-His antibody (Roche Molecular Biochemicals)
according to the manufacturer's instructions. For purification,
expression was scaled up, and bacterial cell pellets were resuspended
in lysis buffer (20 mM Tris-HCl, pH 8.0, 10 mM
Map Kinase and c-erbB-1 Phosphorylation Assays--
For MAP
kinase phosphorylation assays, HaCaT cells were seeded in 6-well dishes
such that the cells reached 80% confluence after an overnight
incubation. Cells were then transferred into serum-free media
and incubated for a further 24 h. Recombinant Epigen, TGF SRE Activation--
HaCaT cells were stably transfected with the
concatamerized SRE construct described above, using standard
techniques. For the assay, 5 × 103 cells were
aliquoted into wells of a 96-well plate and incubated for 24 h.
Media was changed to 0.1% fetal bovine serum, and cells were
incubated for an additional 24 h. Cells were incubated in the
presence of recombinant Epigen, EGF (PeproTech), TGF HaCaT Proliferation and Inhibition Assay--
-HaCaT
proliferation assays were performed in 96-well flat-bottomed plates in
0.1 ml of Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 0.05% fetal calf serum. Recombinant Epigen,
EGF, TGF A Novel EGF Family Member Isolated from a Mouse Keratinocyte
cDNA Library--
High throughput sequencing of a mouse immature
keratinocyte library identified an expressed sequence tag of 1715 base
pairs containing a single open reading frame of 459 nucleotides (Fig. 1A). A putative translation
initiation codon was found within the sequence GAATATGG,
which matched the consensus sequence established for eukaryotic
translational initiation (47). Only 5 base pairs of sequence was found
upstream of the ATG codon in this cDNA, whereas the 3' untranslated
region was 1.25 kilobases in length and contained a poly(A)
stretch preceded by a putative polyadenylation signal (AATAAA).
Conceptual translation of the 459-base pair open reading frame produced
a predicted protein of 152 amino acids. Hydrophobicity analysis
identified a stretch of 18 amino acids, starting from the putative
initiating methionine, indicative of a signal peptide. The putative
cleavage site of the signal peptide was predicted to be located between
Ala18 and Ala19 (SignalP; Ref. 48). An
additional hydrophobic region was present between Ile111
and Cys130, suggesting the presence of a transmembrane
domain. A search against the Prosite data base (release 15.0; Ref. 49)
revealed that residues 83-94 (CRCFTGYTGQRC) matched the consensus
pattern of EGF 1 and 2 domains. In addition, the predicted protein also contained two N-linked glycosylation sites between amino
acids 36 and 39 (NWTF) and between amino acids 40 and 43 (NNTE).
Alignment of the predicted protein, which we have named Epigen,
with entries in the Swiss-Prot Database indicated that Epigen was
similar to several members of the EGF family. Over the entire Epigen
protein sequence, this homology was relatively low, displaying 26 and
29% identity to TGF mRNA Distribution of Epigen--
The pattern of Epigen
expression in adult mouse tissues was assessed by Northern blotting
using commercial poly(A)+ blots from
CLONTECH. A [32P]dATP end-labeled
synthetic oligonucleotide hybridizing to the 3' untranslated region of
mouse Epigen was used as a probe. Expression of mouse Epigen was
evident in testis and liver tissues, with some expression in heart
(Fig. 2). A longer exposure indicated that Epigen was also expressed in
lung and kidney (data not shown). As a loading control, the blot was
reprobed with a Epigen Stimulates Phosphorylation of MAP Kinase--
We assessed
whether Epigen, like other EGF family members, could activate the
Ras/MAP kinase/c-fos signal transduction pathway (26). For
this purpose, primers were designed to amplify cDNA that encoded
Epigen from residues Leu53 to Ala103.
Polymerase chain reaction products were subcloned into the bacterial expression vector pET16b, in frame with an N-terminal poly-histidine tag (Fig. 3A). The presence of
a poly-histidine tag at a similar position in TGF Epigen Activates Genes under the Control of the SRE--
We then
assessed whether Epigen activated genes under the control of the SRE.
Reporter constructs containing concatamerized SRE sequences upstream of
a luciferase gene were stably transfected into HaCaT cells as described
under "Experimental Procedures." Reporter activity was evaluated by
measuring luciferase levels. As shown in Fig.
5A, a dose-dependant increase
in luciferase level was detected when Epigen was added to the stably
transfected HaCaT cell line. A 3-fold increase in luciferase levels was
observed at the highest Epigen concentration used, with luciferase
levels returning to base line at 1.8 nM. TGF Epigen Is Mitogenic for Epithelial Cells--
To further examine
whether Epigen displays EGF and TGF c-erbB-1 Is Activated upon Epigen Stimulation--
To determine
whether c-erbB-1 played a role in the mitogenic responses
obtained, a blocking antibody to this receptor was added concurrently
with a constant amount of Epigen or TGF Here we report the molecular cloning, expression, and biological
activity of Epigen, a novel member of the EGF ligand superfamily. Epigen was identified from a cDNA library constructed from a
population of immature keratinocytes due to its homology with
epiregulin, the latest member of the EGF superfamily (11). At the amino acid level, Epigen displays all the characteristics of an EGF family
member. A putative signal sequence and transmembrane domain flank a
core of amino acids encompassing six cysteine residues and two putative
N-linked glycosylation sites. It is this core of amino acids
that is most similar to the active peptides of EGF superfamily members.
The number and positioning of the cysteine residues in Epigen is
absolutely conserved relative to the other family members. Examination
of the disulfide bonding arrangement in EGF and TGF All EGF superfamily members identified to date that bind to
c-erbB-1 have been isolated as small, secreted peptides from
culture supernatants. Subsequent analysis of the mRNA from which
they are encoded indicated that all are derived from membrane-bound precursors that are proteolytically cleaved from the plasma membrane (9, 11-16). The protein sequence of Epigen has a putative
transmembrane domain situated in an identical position to that of the
other EGF superfamily members. This suggests that Epigen is also
synthesized as a transmembrane-tethered precursor, from which an active
peptide is cleaved. However, because the TGF In silico analysis of the keratinocyte library from which
Epigen was identified indicated that expressed sequence tags encoding Epigen were present at a frequency of less than 1:10,000 sequences. Northern blot analysis of adult mouse tissues confirmed that the mRNA for Epigen was present at low levels in testis, heart, and liver tissues. The expression profiles of several EGF superfamily members overlap with the profile obtained for Epigen. For example, HB-EGF is also found predominantly in lung, brain, and heart tissues, although also in skeletal muscle, kidney, and spleen (15). Epiregulin is present in heart, lung, and smooth muscle tissues of the adult mouse. TGF Recombinant Epigen was purified from a bacterial expression system and
used in several assays designed to examine whether it behaved as a
classical EGF family member in vitro. The transformed epithelial cell line HaCaT was chosen as a model system because it is
very sensitive to the effects of EGF and TGF To examine how the signal transduction patterns observed with Epigen,
TGF Differences in the duration and intensity of MAP kinase phosphorylation
and proliferative responses are well documented for EGF superfamily
members (8, 30, 32, 33). In NIH/3T3 cells, epiregulin is more potent
than EGF at higher concentrations, whereas at low concentrations, EGF
is more potent (33). In smooth muscle cell growth, HB-EGF is
significantly more potent than EGF at all concentrations (8). These
differences can often be correlated with the affinity of each ligand
for the c-erbB receptors on specific cells, such that when
compared with EGF, HB-EGF has greater affinity for c-erbB-1
on smooth muscle cells, and epiregulin has a lower affinity for
c-erbB-1 on NIH/3T3 cells (8, 23, 32, 33). These features
are thought to allow for an enormous range and control of signal output
(23). Therefore it may be that the affinity of Epigen for
c-erbB-1 on HaCaT cells at least partially accounts for the
signal transduction patterns and growth responses obtained.
Alternatively, because Epigen was identified by molecular, rather than
biochemical, means, it is possible that native Epigen has a slightly
different amino acid composition at the N or C terminus from that of
recombinant Epigen. C-terminal residues in particular are known to have
a significant influence on receptor binding, because a C-terminally
extended recombinant AR protein was found to be 10 times more active
than truncated forms (58). The isolation of native Epigen from culture
supernatants will address these issues.
We have identified an expressed sequence tag that encodes Epigen, a
novel member of the EGF superfamily of peptide growth factors. To our
knowledge, this is the first example of an EGF-like ligand to be
identified by expressed sequence tag data base screening. Epigen
displays all the structural characteristics of an EGF family member and
behaves similarly to TGF, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was
present in testis, heart, and liver. Recombinant Epigen was synthesized
in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor
in several assays. In epithelial cells, Epigen stimulated the
phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also
stimulated the proliferation of HaCaT cells, and this
proliferation was blocked by an antibody to the extracellular domain of
the receptor tyrosine kinase c-erbB-1. Thus, Epigen is
the newest member of the EGF superfamily and, with its ability to
promote the growth of epithelial cells, may constitute a novel
molecular target for wound-healing therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Epiregulin, HB-EGF, AR, betacellulin, and the neuregulins
(5-11). These members were first identified as secreted peptides;
however, subsequent cloning of their cDNA has revealed that all are
derived from membrane-bound precursors that are proteolytically cleaved
from the plasma membrane (9, 12-16). Although members of the EGF
superfamily have relatively low homology with each other at the amino
acid level, the presence of six conserved cysteine residues in the
active peptide suggests that all have a similar tertiary structure.
Indeed, the solution structure of EGF and TGF
demonstrated that
identical disulfide linkages between these conserved cysteine residues
enable the formation of a three-looped structure (17). Central to the
function of the EGF superfamily is a conserved domain known as the EGF
motif, which is present in all EGF superfamily members identified to
date. This motif encompasses three of the six conserved cysteine
residues and contains additional residues important for tertiary
structure stabilization and receptor binding (18).
, epiregulin, AR,
HB-EGF, betacellulin; see Ref. 24 for review), and a single EGF
superfamily ligand (e.g. epiregulin) can bind to several
receptors (c-erbB-1 and B-4; Ref. 25). Tyrosine
phosphorylation of C-terminal residues of c-erbB receptors
activates the Ras-MAP kinase pathway, leading to regulation of
c-fos expression by the binding of transcription factors to
sites such as the serum inducing element and SRE within the
c-fos promoter (see Ref. 26 for review).
can promote angiogenesis in
vivo and can stimulate migration of endothelial cells and
keratinocytes in vitro, both features thought to be of
importance in epithelial wound healing (36-41). However, aberrant
activation of the EGF ligand/receptor pathway in diseases such as
psoriasis and cancer suggests that disregulation of EGF superfamily
members and their receptors may play a role in the progression of these
and other disorders (32, 42-46).
. In epithelial cells, Epigen
stimulates the phosphorylation of c-erbB-1 and MAP kinase
proteins. Epigen also activates genes under the control of the SRE. In
addition, Epigen is a mitogen for HaCaT cells, and this activity
can be significantly reduced by a blocking antibody to the receptor
c-erbB-1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin fragment provided with the blot.
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). To the lysed cells, 1% Nonidet P-40 was added, and the mixture was incubated on ice for 10 min. Lysates were further disrupted by
sonication on ice at 95 watts for 4 × 15 s and then
centrifuged for 15 min at 14,000 rpm to pellet the inclusion bodies.
The pellet containing inclusion bodies was resuspended in lysis buffer
containing 0.5% w/v CHAPS and sonicated on ice for 5-10 s. This
mixture was stored on ice for 1 h and centrifuged at 14,000 rpm
for 15 min at 4 °C, and the supernatant was discarded. The pellet
was once more resuspended in lysis buffer containing 0.5% w/v CHAPS,
sonicated, and centrifuged, and the supernatant was removed as before.
The pellet was resuspended in solubilizing buffer (6 M
guanidine HCl, 0.5 M NaCl, 20 mM Tris-HCl, pH
8.0), sonicated at 95 watts for 4 × 15 s, and then
centrifuged for 20 min at 14,000 rpm and 4 °C to remove debris. The
supernatant was stored at 4 °C until use. Recombinant Epigen was
purified using a nickel-chelating Sepharose column (Amersham Pharmacia
Biotech) following the manufacturer's recommended protocol. For
refolding, the protein solution was added to 5 times its volume of
refolding buffer (1 mM EDTA, 1.25 mM reduced
glutathione, 0.25 mM oxidized glutathione, 20 mM Tris-HCl, pH 8.0) over a period of 1 h at 4 °C.
The refolding buffer was stirred rapidly during this time, and stirring
was continued at 4 °C overnight. The refolded proteins were then
concentrated by ultrafiltration using standard protocols.
(Genzyme), or control protein (TR1002P, a secreted protein produced as
for Epigen) was added to the cells at a concentration of 18 nM and incubated for up to 20 min. Media was removed
after the indicated times, and cells were immediately lysed in 100 µl of radioimmune precipitation buffer (50 mM Tris-HCl, pH
7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, leupeptin, pepstatin). MAP kinase phosphorylation was then
assessed by SDS-PAGE and Western blot analysis using
anti-ACTIVETM MAP kinase polyclonal antibody (Promega)
following the manufacturer's instructions. Blots were stripped and
reprobed using anti-ERK1/2 antibody (Santa Cruz Biotechnology)
to ensure equal protein loading. For c-erbB-1
phosphorylation assays, A431 cells were substituted for HaCaT cells and
treated in the same manner. c-erbB-1 phosphorylation was
assessed as above, using an antibody to EGF receptor (activated form,
Transduction Laboratories) following the manufacturer's instructions.
Blots were stripped and reprobed using anti-EGF receptor (Transduction
Laboratories) to ensure equal protein loading.
, or a control
protein at concentrations titrating from 180 nM for 6 h, washed twice in phosphate-buffered saline, and lysed with 40 µl of
lysis buffer (Promega). 10 µl was transferred to a 96-well plate, and
10 µl of luciferase substrate (Promega) was added by direct injection
into each well by a Victor2 fluorometer (Wallac);
the plate was shaken, and the luminescence for each well was read at
3 × 1-s intervals.
, or a control protein was titrated into the plates from a
concentration of 12 nM, and 1 × 103 HaCaT
cells were added to each well. The plates were incubated for 5 days in
an atmosphere containing 10% CO2 at 37 °C. Cell growth
was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction. HaCaT inhibition assays were performed essentially as above except that anti-c-erbB-1 (USB) or
isotype control antibodies were titrated into wells (from 1 µg/ml)
containing a constant amount of Epigen (1.75 nM) or TGF
(0.1 nM).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cDNA and predicted protein sequence of
Epigen and alignment with other EGF family members.
A, the coding region of Epigen is highlighted in
uppercase, and the 5' and 3' untranslated regions are shown
in lowercase. The predicted protein sequence is denoted by
the one-letter amino acid code. The EGF domain and putative
polyadenylation signal are underlined. B,
multiple alignment of Epigen with the active peptides of mouse
epiregulin (GenBankTM accession number Q61521),
TGF (GenBankTM accession number P48030), EGF
(GenBankTM accession number P01132), HB-EGF
(GenBankTM accession number Q06186), betacellulin
(GenBankTM accession number Q05928), and AR
(GenBankTM accession number P31955). The predicted start
methionine of Epigen is numbered 1. For the known EGF family members,
the first amino acid of the active peptide is numbered 1. The
N-terminal extensions of AR, HB-EGF, and betacellulin were not included
in the alignment. Identical residues are highlighted in light
blue, conserved residues are highlighted in purple, and
related residues are highlighted in gray. EPR,
epiregulin; BTC, betacellulin.
and Epiregulin, respectively. This compares
favorably with the protein sequence identity between other EGF family
members, which ranges from 23 to 33%. Because EGF family members exist
in a functional form as small peptides, we aligned the functional
peptides of the EGF family with Epigen (Fig. 1B). This
revealed that a 51-amino acid internal segment of Epigen was more than
40% identical to the active peptides of EGF, TGF
, and Epiregulin.
The active peptides of the EGF family are sufficient for activity and
contain several conserved residues critical for the maintenance of this
activity. Epigen has also retained these residues, which include six
cysteines (Cys59, Cys67, Cys72,
Cys83, Cys85, Cys94), three
glycines (Gly70, Gly88, Gly91), and
an arginine and leucine (Arg93 and Leu99). The
six cysteine residues in TGF
form three disulfide bonds that are
required for optimum binding affinity to c-erbB-1 (50, 51).
Mutation of Arg93 and Leu99 was found to
abrogate binding of EGF and TGF
to c-erbB-1 (18), and
Gly91 and Arg93 are highly conserved within EGF
family members but not in proteins that contain EGF units without
growth factor activity (52). Together, the conserved features of Epigen
with members of the EGF family at the amino acid level suggested that
Epigen encoded a novel member of the EGF family. Furthermore, an
increase in sequence conservation of a 51-amino acid internal peptide
of Epigen with the functional peptides of the EGF family suggested that this peptide would encompass residues sufficient for its biological activity.
-actin fragment.
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Fig. 2.
mRNA expression of Epigen in
adult mouse tissues. A poly(A)+
CLONTECH Northern blot was probed with a
[32P]dATP end-labeled oligonucleotide probe specific to
the 3' untranslated region of Epigen. A major transcript at
approximately 1.7 kilobases was evident in several tissues.
He, heart; Br, brain; Sp, spleen;
Lu, lung; Li, liver; Ki, kidney;
Te, testis.
was found to have
no effect on its biological activity (13). The expression vector was
transformed into Escherichia coli to prepare recombinant
Epigen (Fig. 3B). We assayed for MAP kinase phosphorylation
upon stimulation of HaCaT cells by Epigen over a 20-min period. In this
assay, phosphorylated MAP kinase was detected by Western blot analysis
using an antibody that recognizes the phosphorylated forms of MAP
kinase only (Fig. 4, A-C,
panel P). As shown in Fig. 4A, an increase in MAP
kinase phosphorylation was detected following a 5-min incubation of
HaCaT cells with Epigen. The level of MAP kinase phosphorylation
steadily increased to a maximal level at 20 min of stimulation. TGF
also induced MAP kinase phosphorylation after 5 min of incubation;
however, maximal levels were reached at 10 to 15 min post-stimulation
and had begun to decrease by 20 min (Fig. 4B). A control
protein was only able to stimulate weak MAP kinase phosphorylation at
20 min when compared with Epigen and TGF
(Fig. 4C).
Reprobing each blot with an antibody recognizing total MAP kinase
verified that an equal quantity of protein from the control and test
samples had been analyzed (Fig. 4, A-C, panel
T).
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Fig. 3.
Construct design and purification of
recombinant Epigen. A, a schematic representation of
the complete coding region of Epigen (not drawn to scale). DNA encoding
amino acids 53-103 of Epigen were subcloned into a bacterial
expression vector for protein purification. The one-letter amino acid
code has been used. B, recombinant Epigen production in
whole bacterial lysates before (lane 1) and after
(lane 2) induction by
isopropyl-1-thio- -D-galactopyranoside and detection by
Western blotting using an anti-His antibody. Lane 3 shows
purified recombinant Epigen running at ~7 kDa by Coomassie staining
on an SDS-PAGE gel. G, glycosylation sites; AP,
active peptide; TM, transmembrane domain; WB,
Western blot; C, Coomassie stain.
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Fig. 4.
Induction of MAP kinase phosphorylation in
HaCaT cells by Epigen and TGF . HaCaT
cells were cultured overnight in the absence of serum and then
stimulated with 18 nM Epigen (A), TGF
(B), or control protein (C) at 37 °C for the
times indicated. Cells were processed as described under
"Experimental Procedures." Lysates were analyzed by SDS-PAGE and
Western blotting using antibodies to the phosphorylated form of MAP
kinase protein (P) or to total MAP kinase
(T).
stimulated
a 5-fold increase in luciferase levels at 18 nM, with
luciferase levels decreasing to background at 2.0 × 10
2 nM (Fig. 5B). EGF
was most potent in this assay, promoting a 4.5-fold increase in
luciferase levels at 1.8 nM (Fig. 5C).
Luciferase levels induced by this ligand had not returned to base line
even at concentrations of 2 × 10
4
nM. These results suggest that recombinant Epigen activates
a signaling pathway through which EGF and TGF
exert their biological effects, consistent with activation of c-erbB-1 or other
members of the c-erbB family.
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Fig. 5.
Induction of genes under the control of the
SRE. HaCaT cells containing concatamerized SRE sequences coupled
to a luciferase reporter gene were stimulated with various
concentrations of Epigen (A), TGF (B), or EGF
(C) for 6 h as described under "Experimental
Procedures." The mean and S.D. were calculated from three separate
wells and are represented as fold-induction of the reporter gene
relative to control. Results are representative of at least three
separate experiments.
-like activities, HaCaT cells
were assayed for their proliferative responses to Epigen. As shown in
Fig. 6A, Epigen promoted the growth of HaCaT cells in a dose-dependant manner. Maximal growth was
obtained at a concentration of 1.75 nM. To compare Epigen proliferation with that promoted by other EGF family members, EGF and
TGF
were also used to stimulate HaCaT cells. EGF was the most potent
growth factor and stimulated maximal growth at 0.01 nM
(Fig. 6C), whereas maximal growth by TGF
was obtained at
a 10-fold higher concentration of 0.1 nM (Fig.
6B).
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Fig. 6.
Induction of HaCaT growth by Epigen
(A), TGF
(B), or EGF (C). HaCaT
cells were stimulated with various concentrations of each ligand for 5 days in low serum conditions as described under "Experimental
Procedures." The mean and S.D. were calculated from three separate
wells. Background 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) levels for this assay are shown in
A. Results are representative of at least three separate
experiments.
in a growth assay. We found
that addition of anti-c-erbB-1 reduced the mitogenic effects
of Epigen by up to 60% at antibody concentrations of 250 ng/ml (Fig.
7A). The mitogenic effects of
TGF
were reduced by up to 50% at antibody concentrations of 500 ng/ml (Fig. 7B). These results suggested that
c-erbB-1 was probably involved in the transmission of
recombinant Epigen mitogenic activity. To further examine the
involvement of c-erbB-1 in Epigen-driven growth, we
assayed for c-erbB-1 phosphorylation upon stimulation of
A431 cells by Epigen and TGF
. In this assay, phosphorylated
c-erbB-1 was detected by Western blot analysis using an
antibody that recognizes the phosphorylated forms of
c-erbB-1 only. As shown in Fig. 7C, an increase
in c-erbB-1 phosphorylation was detected following a 15-min
incubation of A431 cells with Epigen or TGF
. A control protein was
not able to induce c-erbB-1 phosphorylation during this
time. Reprobing each blot with an antibody recognizing total c-erbB-1 protein verified that an equal quantity of protein
from the control and test samples had been analyzed.
View larger version (27K):
[in a new window]
Fig. 7.
Inhibition of Epigen- or
TGF -induced HaCaT growth by an antibody to
c-erbB-1 and induction of c-erbB-1
phosphorylation by Epigen and TGF
on A431
cells. HaCaT cells were grown in 1.75 nM Epigen
(A) or 0.1 nM TGF
(B) in the
presence of various concentrations of anti-c-erbB-1 antibody
(closed squares) or control antibody (open
squares). The mean and S.D. were calculated from three
separate wells, and results are represented as percent growth relative
to the isotype control antibody. Results are representative of at least
three separate experiments. C, A431 cells were stimulated
with 18 nM Epigen, TGF
, or control protein at 37 °C
for 15 min. Cells were processed as described under "Experimental
Procedures." Lysates were analyzed by SDS-PAGE and Western blotting
using antibodies to the phosphorylated form of c-erbB-1
protein or to total c-erbB-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
indicated that
the six cysteine residues form three pairs of disulfide bonds, with
substitution of any of these residues generally resulting in loss of
biological activity (18, 53). The six-cysteine structural unit is not
only conserved within the EGF superfamily but is also present in
several proteins that do not display EGF-like activities
(e.g. see Ref. 54). However, EGF family members can be
distinguished from these proteins due to the strict conservation of one
glycine and one arginine residue within this unit. The arginine residue
is absolutely required for strong binding to c-erbB-1, as
shown by extensive point mutation analysis of TGF
and EGF (18).
Epigen conforms to the characteristics described above and thus is
likely to exhibit EGF- and TGF
-like structural and receptor binding properties.
precursor is capable of binding to, and inducing phosphorylation of, c-erbB-1 while
situated on the plasma membrane (55, 56), it is possible that the
transmembrane-tethered form of Epigen is similarly active.
is present in brain, but is also present in
pituitary glands and macrophage cells (11, 33, 52, 57). Therefore, although other EGF superfamily members are expressed in tissues also
expressing Epigen, it appears that the combined tissue distribution profile and expression level of each family member is unique.
. Epigen and TGF
both
induced phosphorylation of MAP kinase in HaCaT cells over a 20-min
stimulation period. The magnitude of MAP kinase phosphorylation induced
by both ligands was similar; however, TGF
-induced phosphorylation
had reached maximal levels at 10 min and was sustained at this level,
whereas Epigen-induced phosphorylation reached maximal levels at 20 min. Phosphorylation of MAP kinases often results in the transcription
of many genes that are under the control of a common regulatory
element, the SRE. A reporter gene linked to upstream SRE enhancer
elements was induced in a dose-dependent manner by Epigen,
TGF
, and EGF. Epigen induced a 3-fold increase in reporter activity
at the highest concentration tested, whereas TGF
and EGF induced a
5- and 4.5-fold increase, respectively, in reporter activity at
optimal concentrations. Thus, the profile of MAP kinase phosphorylation
and SRE activation induced by Epigen, TGF
, and EGF was unique to
each ligand, with EGF the most potent, and Epigen the least potent.
, and EGF were translated into more specific cellular responses,
we compared the induction of HaCaT growth by Epigen with the two known
EGF superfamily members. All three proteins invoked a
dose-dependent growth response. Epigen and TGF
promoted similar levels of growth at their optimum concentration, whereas EGF-driven growth was poorer. However, the optimal EGF concentration needed to promote growth of HaCaT cells was 10-fold less than that of
TGF
, and the optimal concentration of Epigen was 10-fold greater than that of TGF
. An antibody to the extracellular domain of
c-erbB-1 efficiently blocked the growth stimulation of HaCaT cells by Epigen and by TGF
, which suggested that c-erbB-1
was involved in the Epigen signaling pathway. Further evidence for the
involvement of c-erbB-1 was obtained when Epigen was found to induce phosphorylation of c-erbB-1 in A431 cells; this
response was somewhat weaker than that induced by TGF
. Therefore,
although c-erbB-1 is clearly activated during Epigen
signaling, this activation may be a secondary event following the
binding of Epigen to another receptor. A detailed examination of Epigen
binding to c-erbB-1 and the other erbB receptors
is necessary to determine which is the primary receptor for Epigen.
and EGF in signal transduction and growth
assays. However, there are ligand-specific differences in magnitude of
growth response and concentration of ligand giving maximal response in
all the assays performed. Other cross-comparative assays show that EGF
superfamily members are able to induce their own mRNA production
and that of other family members in ligand-specific patterns (59-61).
This suggests that Epigen and the other members of the EGF family have
distinct, nonredundant functions. Members of the EGF family have a
number of overlapping functions in vitro, and this has
prompted increased analysis of the contribution each ligand makes to
growth, development, and disease. For example, triple knockout mice
bearing mutations in TGF
, Epiregulin, and AR have recently been
created, uncovering a role for AR in mammary ductal morphogenesis (62).
Current investigations in this laboratory are focused on uncovering a
specific in vivo role for Epigen in normal and aberrant
growth and development.
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ACKNOWLEDGEMENTS |
---|
We thank Jane Martin and the DNA Technologies group (Genesis R & D) for technical assistance and Dr. Alain Delcayre (AP Cells Inc., San Diego, CA) for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ291391.
To whom correspondence should be addressed. Tel.: 64-9-373-5600;
Fax: 64-9-373-2189; E-mail: l.strachan@genesis.co.nz.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M006935200
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ABBREVIATIONS |
---|
The abbreviations used are:
EGF, epidermal
growth factor;
TGF, transforming growth factor
;
HB-EGF, heparin-binding EGF-like growth factor;
AR, amphiregulin;
MAP, mitogen-activated protein;
SRE, serum response element;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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