(Received for publication, November 1, 1994; and in revised form, January 1, 1995)
From the
Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.
Cellular growth and differentiation are modulated by a number of
peptide growth factors. Accumulated evidence supports that growth
control cascade involves growth factors and their receptors in both
tumor and non-tumor cells(1) . According to the autocrine
hypothesis, many tumor cells synthesize and release excess amounts of
certain growth factors, resulting in the abnormal proliferation of
tumor cells due to mitotic stimulation by the growth factors that they
produced(2, 3) . On the other hand, several growth
inhibitors have been purified so
far(4, 5, 6, 7, 8) . Loss
or reduction of the susceptibility of cells to growth inhibitors is
also argued about the mechanism of malignant
transformation(3) . Cellular proliferation is regulated by the
interplay of growth regulators in both positive and negative fashions.
In addition, certain tumor cell growth inhibitors also act as factors
that stimulate the growth or induce the differentiation of other types
of cells(4, 6, 7, 8, 9) .
During the screening of conditioned media of cultured cells, we
detected an activity which induced the morphological changes in the
human epitheloid carcinoma cell line HeLa, in the conditioned medium of
NIH3T3/clone T7, a cell clone of mouse fibroblast NIH/3T3 that was
adapted in protein-free medium and then acquired tumorigenicity. The
conditioned medium also inhibited the growth of several tumor cells.
Here we report the purification, structure, and biological activities
of this novel growth regulator, namely, epiregulin, which is a new
member of the epidermal growth factor (EGF) ()family.
Rat hepatocytes were isolated as
described(41) . Hepatocytes were seeded in collagen-coated
24-well plates at a density of 2.5 10
cells/well in
1 ml of DF medium supplemented with 10% FBS. After 6 h, the medium was
replaced with 0.5 ml of DF medium supplemented with 5% FBS, and cells
were incubated with epiregulin or other growth factors for 48 h.
[
H]Thymidine (Amersham) was added to 2.5
µCi/ml during the final 16-h period of incubation. Cells were
harvested by trypsinization, and the radioactivities were counted with
a Betaplate system (Pharmacia).
Human aortic smooth muscle cells
(Kurabo Co., Osaka) were plated, as were rat hepatocyte culture, at a
density of 5 10
cells/well. After 24 h, the medium
was replaced with DF medium supplemented with 2% FBS, and cells were
incubated with epiregulin or other growth factors for 48 h.
Incorporation of [
H]thymidine into the cellular
DNA was determined. In these assays, two independent experiments on
triplicate plates were carried out.
Figure 1: Morpholical changes in Hela cells induces by epiregulin. Hela cells were cultured in the absence (A) or the presence (B) of 100 ng/ml epiregulin for 15 h in serum-free medium.
Figure 2: Identification of epiregulin. A, SDS-PAGE of purified epiregulin. Purified preparation of epiregulin was analyzed on a 20% SDS-polyacrylamide gel and stained with silver. Lane 1, molecular size marker (46 kDa, ovalbumin; 30 kDa, carbonic anhydrase; 21.5 kDa, trypsin inhibitor; 14.3 kDa, lysozyme; 6.5 kDa; aprotinin; 3.4 kDa, insulin chain A; Amersham); lane 2, 150 ng of epiregulin. B, immunoblot detection of epiregulin in the conditioned medium of NIH/3T3 clone T7 cells. The samples were subjected to Western blot analysis. Lane 1, 40 ng of epiregulin; lane 2, 40 ng of epiregulin was immunoprecipitated with anti-epiregulin rabbit IgG; lane 3, 5 ml of conditioned medium was immunoprecipitated with anti-epiregulin rabbit IgG; lane 4, 5 ml of conditioned medium was immunoprecipitated with normal rabbit IgG. The arrowhead indicates the mobility of epiregulin on SDS-PAGE.
Table 1summarizes the results of purification. Approximately 5 and 10 µg of epiregulin, respectively, were recovered in P-I and P-II from 60 liters of the conditioned medium. The overall purification was 13,000-fold, and the recoveries were 1.6 and 3.0%, for P-I and P-II, respectively. The large scale purification was carried out three times. The ratio of recoveries in the two fractions varied in each preparation of the conditioned medium. Two µg of P-I and very small amounts of P-II were obtained from 40 liters; while 5 µg of P-I and 5 µg of P-II were obtained from 100 liters of the conditioned medium.
Epiregulin was immunoprecipitated from the conditioned
medium of NIH3T3/clone T7 culture with anti-epiregulin polyclonal
antibody and subjected to Western blot analysis (Fig. 2B). The immunoreacted band migrated in similar
fashion to the chemically synthesized epiregulin. The upper
intense bands were nonspecific and were presumably rabbit IgG, since
they were also observed in the lanes in which synthetic epiregulin was
applied and in those in which normal rabbit IgG was used for
immunoprecipitation instead of anti-epiregulin rabbit IgG. Ten µg
of anti-epiregulin rabbit IgG did not recognize the plate coated with
500 ng of mouse EGF in the enzyme-linked immuno-solid assay (data not
shown).
Figure 3: Amino acid sequence of epiregulin. The solid line shows the peptide fragments (B-I-III) obtained by the cyanogen bromide cleavage of purified epiregulin. The assignments of sequence for residues are indicated by arrows. An analysis of the amino acid composition of about 30 pmol of purified epiregulin supported this sequence.
Figure 4:
Sequence comparison of epiregulin and
EGF-related polypeptides. Dashes indicate gaps introduced for
better alignments. The EGF-related peptides are numbered relative to
the N termini of their mature forms on the left of each line. The
sequence of heregulin (HRG) begins only with amino acid 177 of
proHRG-(15) . Boxed residues indicate amino acids
identical to those of epiregulin. Numbers in parentheses indicate percent homology with epiregulin. The sequences are those
of vaccinia virus growth factor (VGF) (36, 37) , betacellulin (BTC)(17) ,
TGF-
(38) , EGF(39) , heparin binding EGF-like
growth factor (HB-EGF)(40) , and amphiregulin (AR)(13) . The amino acid sequence of epiregulin was
deposited in Japanese International Protein Information (accession
number JT0747).
Figure 5: Growth inhibitory activities of epiregulin for tumor cell lines. HeLa (cross-hatched bar), A549 (hatched bar), and A431 (stippled bar) cells were cultured with various concentrations of epiregulin for 5 days. Values are cell number relative to those for the control culture to which epiregulin was not added.
On the other hand, epiregulin, as well as
TGF- and recombinant hepatocyte growth factor, stimulated DNA
synthesis of rat primary hepatocytes at concentrations of 0.8-20
ng/ml, while EGF induced only an approximately 7-fold increase at
similar concentrations (Fig. 6A). The profiles of
dose-dependent stimulatory activity by epiregulin were similar to those
for TGF-
, but not to those for EGF. Maximal stimulation was
observed at 10 ng/ml of EGF and at 20 ng/ml of TGF-
, but the
stimulation was increased by epiregulin up to concentrations above 20
ng/ml. Therefore, epiregulin is one of the most potent mitogens known
for hepatocytes. The stimulatory effect of epiregulin was additive with
those of hepatocyte growth factor or TGF-
at concentrations of
4-20 ng/ml. On addition of both EGF and epiregulin to the culture
medium, the growth stimulatory effect was lower than that with
epiregulin alone. (
)
Figure 6:
Growth stimulatory activities of
epiregulin. A, stimulation of DNA synthesis in the primary
culture of rat hepatocytes. B, stimulation of proliferation of
Balb/3T3 clone A31 cells. C, stimulation of DNA synthesis of
human vascular smooth muscle cells. Cells were incubated with various
concentrations of epiregulin (), human hepatocyte growth factor
(
), mouse EGF (
), or rat TGF-
(
). Stimulation
index values are relative to those of control cultures. One stimulation
index unit = 4.1
10
cpm (A), 2.1
10
cells (B), 8.5
10
cpm (C). The methods are described under
``Experimental Procedures.''
Epiregulin displayed higher potency as a mitogen for mouse fibroblast Balb/3T3 A31 cells at concentrations above 10 ng/ml than did EGF (Fig. 6B). Epiregulin stimulated the proliferation of Balb/3T3 A31 cells 13-fold at 10 and 18-fold at 100 ng/ml, while EGF induced only an approximately 9-fold increase at these concentrations. However, the stimulation by epiregulin was less potent than EGF at concentrations less than 10 ng/ml.
Epiregulin also stimulated DNA synthesis on human aortic
smooth muscle cells (Fig. 6C). The concentrations
required for maximum stimulation by epiregulin was higher than those by
EGF and TGF-. On the other hand, epiregulin had no significant
effects on the DNA synthesis of human endothelial cells from the aortic
and the umbilical vein, nor did EGF or TGF-
(data not shown).
Binding of epiregulin to the EGF receptor was demonstrated. Although
50 ng/ml unlabeled EGF and TGF- completely competed out binding of
I-EGF to A431 cells, the purified epiregulin exhibited
only about 20% competition at the same concentration. To ascertain if
epiregulin can interact with EGF receptors on A431 cells, the effect of
synthetic epiregulin in the binding of
I-EGF to A431
cells was investigated (Fig. 7). Unlabeled synthetic epiregulin
competed with
I-EGF binding to A431 cells in a
dose-dependent fashion at concentrations above 10 ng/ml to 1 µg/ml.
Fifty % inhibition of binding of
I-EGF was observed at
122 ng/ml of epiregulin, but at 8 ng/ml of EGF. These findings suggest
that epiregulin has a lower affinity for EGF receptors on A431 cells
than does EGF.
Figure 7:
Competition with I-EGF in
binding to A431 cells.
I-EGF (2 ng, 10
cpm)
was added to the confluent A431 cultures with various amounts of
epiregulin (
), mouse EGF (
), or rat TGF-
(
).
Epiregulin was originally identified as a tumor growth
inhibitor which induced morphological changes in HeLa cells. In
addition to the well-characterized EGF and TGF-, several new
members of the EGF family including amphiregulin(13) ,
heparin-binding EGF-like growth factor(14) , heregulin/neu
differentiation factor(15, 16) , and betacellulin (17) have been purified. Epiregulin, as well as, other members
of the EGF family, mediated signals stimulating or inhibiting the
proliferation of target cells of various types. However, the biological
characteristics of epiregulin differed from those of other members of
the EGF family in the following respects: 1) epiregulin was more potent
than EGF as a mitogen for rat primary hepatocytes at concentrations
above 3 ng/ml and for Balb/3T3 A31 cells at concentrations above 10
ng/ml, but less potent as a mitogen for human aortic smooth muscle
cells. 2) All of the recently identified members of the EGF family
exhibit the ability to bind to heparin-Sepharose
columns(13, 14, 15, 16, 17) .
However, epiregulin did not bind to a heparin-Sepharose column even in
the absence of NaCl in 20 mM Tris-HCl buffer at pH 7.4 (data
not shown). Epiregulin, as well as EGF and TGF-
, appears to have
no binding sites for heparin. 3) A purified preparation of epiregulin
and synthetic epiregulin both exhibited much weaker competition than
EGF with
I-EGF in the binding to A431 cells in which the
EGF receptor was overexpressed. Epiregulin, as well as amphiregulin,
may bind to the EGF receptor with low affinity(13) .
Alternatively, the other receptors which bind EGF-related ligads,
HER2/erbB2(18, 19, 20) ,
HER3/erbB3(21, 22) , and HER4/erbB4(23) , may
serve as receptors for epiregulin.
Epiregulin was a more potent
mitogen for rat hepatocytes than was EGF. TGF- (24) and
heparin-binding EGF-like growth factor (25) each act as potent
mitogens for primary cultures of rat hepatocytes by virtue of exerting,
respectively, autocrine and paracrine effects via the same high
affinity EGF receptor. Hepatocyte growth factor, the most potent
hepatotrophic factor (26) yet known, acts in endocrine and
paracrine fashion during regeneration of the
liver(27, 28) . Epiregulin stimulated DNA synthesis in
rat hepatocytes, as did hepatocyte growth factor and TGF-
as well.
Thus, epiregulin may together with these hepatotrophic factors play an
important role in the regeneration of the liver.
Epiregulin was
derived from the tumor cell line NIH/3T3 clone T7, which is highly
tumorigenic in athymic mice. TGF- and amphiregulin are originally
derived from the conditioned media of murine sarcoma virus-transformed
cells and the phorbol 12-myristate 13-acetate-treated human mammary
adenocarcinoma MCF7 cells,
respectively(29, 30, 31, 32) . They
act as autocrine growth factors for certain tumor cell
lines(31, 32) . In transgenic animals carrying the
expression vector for TGF-
, a variety of neoplastic lesions are
detected, the type of which depends on the strain of mouse and the
promoter of the vector(33, 34, 35) . The
possibility remains that epiregulin as well as TGF-
and
amphiregulin may play roles in the initiation or progression of tumors.