Bile acids activate EGF receptor via a TGF-
-dependent mechanism in human cholangiocyte cell lines
Nathan W. Werneburg,
Jung-Hwan Yoon,
Hajime Higuchi, and
Gregory J. Gores
Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic,
and Foundation, Rochester, Minnesota 55905
Submitted 20 December 2002
; accepted in final form 21 February 2003
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ABSTRACT
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Bile acids transactivate the EGF receptor (EGFR) in cholangiocytes.
However, the mechanisms by which bile acids transactivate the EGFR remain
unknown. Our aims were to examine the effects of bile acids on EGFR activation
in human cholangiocyte cell lines KMBC and H-69. Bile acids stimulated cell
growth and induced EGFR phosphorylation in a ligand-dependent manner. Although
cells constitutively expressed several EGFR ligands, only transforming growth
factor-
(TGF-
) antisera effectively blocked bile acid-induced
EGFR phosphorylation. Consistent with the concept that matrix
metalloproteinase (MMP) activity is requisite for TGF-
membrane release
and ligand function, bile acid transactivation of EGFR and cell growth was
blocked by an MMP inhibitor. In conclusion, bile acids activate EGFR via a
TGF-
-dependent mechanism, and this EGFR activation promotes cellular
growth.
c-Src kinase; matrix metalloproteinase; ligand dependent; transactivation
CHOLANGIOCYTES ARE EPITHELIAL cells that line the intra- and
extrahepatic bile duct apparatus of the liver and biliary tract. These cell
are the targets of a number of diseases often referred to as the
cholangiopathies, which include primary biliary cirrhosis, primary sclerosing
cholangitis, biliary atresia, allograft rejection, graft vs. host disease, and
other syndromes leading to bile duct loss, sclerosis, and cholestasis
(4). Also, cholangiocarcinoma,
an often fatal neoplasm, originates from cholangiocytes
(21). Growth of normal and
neoplastic cholangiocytes is, therefore, important in the regeneration of bile
ducts and the progression of cholangiocarcinoma, respectively. Thus the
mechanisms regulating cholangiocyte growth are of both fundamental and
biomedical interest.
Bile acids have recently been shown to stimulate cholangiocyte and
cholangiocarcinoma proliferation
(2,
3). Proliferation of
cholangiocytes by bile acids occurs by a phosphatidylinositol
3-kinase-dependent pathway (1).
Phosphatidylinositol 3-kinase is stimulated by a number of mitogenic receptor
tyrosine kinases including the EGF receptor (EGFR)
(14). Bile acids have been
shown to functionally transactivate the EGFR
(19,
22), and therefore,
transactivation of the EGFR is likely an important mechanism by which bile
acids stimulate cholangiocyte proliferation. The mechanisms responsible for
bile acid-mediated EGFR transactivation remain unclear and potentially include
both ligand-dependent and -independent mechanisms
(19,
23). Ligand-independent
signaling of EGFR is well described and occurs by processes that promote
receptor oligomerization in the plasma membrane, usually overexpression
paradigms (5). However, EGFR is
usually activated by several ligands including EGF, transforming growth
factor-
(TGF-
), and heparin-binding EGF-like growth factor
(HB-EGF). These ligands are synthesized as transmembrane proteins
(6). Regulated
metalloproteinases cleave these ligands, releasing them from the cellular
membrane into the extracellular space permitting their function as autocrine
and/or paracrine ligands for EGFR
(9,
18). Interestingly, c-Src, an
intracellular tyrosine kinase, has been shown to activate the
metalloproteinases or "sheddases," which are responsible for the
release of these ligands (13,
17), and bile acid
transactivation of EGFR is also c-Src dependent
(22). These observations
suggest that bile acid stimulation of c-Src with subsequent metalloproteinase
cleavage of a membrane-bound EGFR ligand may be responsible for their
transactivation of EGFR.
The overall objective of the current study was to examine the signaling
mechanisms by which bile acids transactivate EGFR. Our specific aims were to
answer the following key questions using a human cholangiocarcinoma cell line:
1) Do bile acids activate EGFR in a ligand-dependent manner?
2) Do the cells express ligands for EGFR, and, if so, which one is
responsible for bile acid transactivation of EGFR? and 3) Is bile
acid transactivation of EGFR dependent on c-Src and matrix metalloproteinase
(MMP) activity? The results suggest bile acid stimulation of EGFR is
TGF-
-mediated and is dependent on Src kinase and MMP activities.
 |
MATERIALS AND METHODS
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Cell Line and Culture. KMBC cells, a human cholangiocarcinoma cell
line (10), were grown in DMEM
supplemented with 10% fetal bovine serum, penicillin (100,000 U/l),
streptomycin (100 mg/l), and gentamycin (100 mg/l). H69 cells are
SV40-transformed human bile duct epithelial cells and were cultured as
previously described (7). Cells
were serum-starved for 24 h before bile acid treatment to avoid the
confounding variable of serum-induced signaling. LX2, human stellate cells
(20), HUH-7, a human
hepatocellular carcinoma cell line stably transfected with the
sodium-dependent taurocholate cotransporting polypeptide (Ntcp)
(12), Hep3B, a human
hepatocellular carcinoma cell line
(11), and HepG2, a human
hepatoma cell line (8) were
used for EGFR ligand RT-PCR. Cells were cultured in the presence of 10% serum
in media employing the culture conditions described in the corresponding
references.
Immunoblot analysis. Cells were lysed for 20 min on ice with lysis
buffer [50 mM Tris·HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin,
leupetin, pepstatin, 1 mM Na3VO4, and 1 mM NaF] and
centrifuged at 14,000 g for 10 min at 4°C. Samples were resolved
by 7.5% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with
appropriate primary antibodies at a dilution of 1:1,000. Peroxidase-conjugated
secondary antibodies (Biosource International, Camarillo, CA) were incubated
at a dilution of 1:10,000. Bound antibodies were visualized using enhanced
chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) and exposed
to Kodak X-OMAT film. Primary antibodies: mouse anti-phosphotyrosine, clone
4G10, was obtained from Upstate Biotechnology (Lake Placid, NY); rabbit
anti-EGFR and goat anti-actin were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA).
Phosphorylation of EGFR. Cells were lysed in ice-cold lysis buffer
as described above and diluted with the lysis buffer to a protein
concentration of 1 mg/ml. One milligram of the cytosolic protein was incubated
with 10 µl anti-EGFR antibody (Santa Cruz) overnight at 4°C. Immune
complexes were precipitated with 100 µl of protein A/G PLUS-Agarose (Santa
Cruz) for 2 h at 4°C and then washed three times with lysis buffer.
Polypeptides were eluted from the beads by boiling for 5 min in 2x
Laemmli sample buffer. The immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose membranes, and immunoblotted with an
anti-phosphotyrosine antibody. Immune complexes on nitrocellulose were
visualized and analyzed as described above. The membrane was stripped with
stripping buffer (100 µM 2-mercaptoethanol; 2% SDS; 50 mM Tris·HCl,
pH 6.7) and reblotted with the anti-EGFR antibody as described above.
RT-PCR for EGF, TGF-
, and HB-EGF. Total RNA was
extracted from the cells using the TRIzol reagent (Invitrogen, Carlsbad, CA).
The cDNA template was prepared using oligo(dT) random primers and Moloney
Murine Leukemia Virus RT as previously described in detail. After the reverse
transcription reaction, the cDNA template was amplified by PCR with
Taq polymerase (Invitrogen) in a reaction mixture containing 20 pmol
of each primer set: EGF, 5'-GCTTCAGGACCACAACCATT-3'and
5'-TCAATCACAGACTGCTTGGC-3'; TGF-
,
5'-GAGTGCAGACCCGCCCGTGGC-3'and
5'-CCAGGAGGTCCGCATGCTCAC-3'; HB-EGF,
5'-CCACACCAAACAAGGAGGAG-3' and
5'-ATGAGAAGCCCCACGATGAC-3'. 18S internal standard primer set was
purchased from Ambion (Austin, Texas). After 2% agarose gel electrophoresis,
ethidium bromide staining was performed to detect the PCR products.
Cell proliferation. Cell proliferation was measured using the
CellTiter 96 Aqueous One Solution cell proliferation assay (Promega) on the
basis of the cellular conversion of the colorimetric reagent
3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
salt (MTS) into soluble formazan by dehydrogenase enzymes found only in
metabolically active proliferating cells. After bile acid treatment, 20 µl
dye solution was added into each well in 96-well plate and incubated for 2 h.
Subsequently, absorbance was recorded at 490-nm wavelength using an ELISA
plate reader (Molecular Devices, Sunnyvale, CA).
Materials and reagents. Deoxycholic acid (DC) and
taurochenodeoxycholic acid (TCDC) were obtained from Sigma (St. Louis, MO),
chenodeoxycholic acid (CDC) was from Calbiochem (La Jolla, CA), and the MMP
inhibitor GM6001 was from Chemicon (Temecula, CA). ZD 1839 was obtained form
AstraZeneca (Cheshire, UK).
Statistical analysis. All data represent at least three
independent experiments using cells from a minimum of three separate
isolations and are expressed as the means ± SD. Differences between
groups were compared using two-tailed Student's t-tests.
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RESULTS
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Do bile acids activate EGFR in a ligand-dependent mechanism?
Pretreatment of KMBC cells with the EGFR-neutralizing antibody, which
competitively blocks ligand binding, inhibited DC-induced tyrosine
phosphorylation of a protein with a molecular weight of 170190 kDa
(Fig. 1A).
Confirmation that this phosphorylated protein was the EGFR was obtained by
immunoprecipitation of the EGFR followed by immunoblot analyses using antisera
specific for the phosphorylated form of this receptor
(Fig. 1B). Indeed, the
DC-induced EGFR phosphorylation was inhibited by the EGFR-neutralizing
antibody. These observations demonstrate that the bile acid DC induces EGFR
activation by a ligand-dependent mechanism.

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Fig. 1. Deoxycholic acid-induced EGF receprot (EGFR) phosphorylation is prevented
by EGFR ligand domain antibody. Before deoxycholic acid (DC; 200 µM)
treatment, KMBC cells were preincubated for 2 h in the presence or absence of
an anti-EGFR ligand domain antibody (EGFR Ab, 10 µg/ml). A: cells
were lysed after 1 h of DC treatment, and immunoblot (IB) analysis was
performed using anti-phosphotyrosine (PhoTyr) antibody. Immunoblot analysis
using anti- -actin antisera was performed as a control for protein
loading. B: immunoprecipitation (IP) of the EGFR from cell lysates
was performed followed by immunoblot analyses using either anti-Pho-Tyr or
anti-EGFR Ab.
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Do KMBC cells express EGFR ligands? To determine whether KMBC
cells express EGFR ligands, we next performed RT-PCR for EGF, TGF-
, and
HB-EGF. Transcripts of all three ligands were readily amplified by RT-PCR in
KMBC cells. RT-PCR was run on all samples with ribosomal RNA 18S primers as a
standard control. As assessed by this semiquantitative approach, mRNA
expression of these ligands did not appear to be altered by DC treatment
(Fig. 2). These data indicate
that EGFR ligands are constitutively expressed in these cells.
Which EGFR ligand is responsible for bile acid-induced EGFR
phosphorylation? To identify the ligand responsible for DC-induced EGFR
phosphorylation, we pretreated KMBC cells with neutralizing antibodies against
EGF, HB-EGF, or TGF-
before incubation with DC. Among the antisera
tested, TGF-
antisera was the most effective in blocking DC-induced
EGFR phosphorylation (Fig.
3A), implicating TGF-
as the ligand mediating bile
acid-induced EGFR phosphorylation.

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Fig. 3. Bile acids stimulate EGFR phosphorylation in KMBC and H-69 cells in a
TGF- -dependent manner. Cells were treated with the bile acids DC (200
µM), chenodeoxycholic acid (CDC; 200 µM), or taurochenodeoxycholic acid
(TCDC; 200 µM) for 1 h. The cells were lysed, and immunoblot analysis was
performed for phospho-EGFR and total EGFR. The ratios were calculated by
densitometric scanning of the intensity of phosphotyrosine band relative to
the EGFR band. Data were expressed as means ± SD from 3 individual
experiments. Representative immunoblots are shown. A: KMBC cells.
Cells were also pretreated for 2 h with specific neutralizing antibodies for
EGF, HB-EGF, or TGF- (10 µg/ml) followed by bile acid treatment for
1h.*P < 0.01 for DC, CDC, TCDC, DC + EGF antisera, and DC + HB-EGF
antisera vs. control; B: H69 cells were treated for 1 h with the bile
acids described above, lysed, and immunoblot analysis was performed.
*P < 0.05 for DC and CDC vs. control.
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Is bile acid-mediated EGFR transactivation bile acid or cell-type
specific? The question of whether the effect of DC on EGFR
phosphorylation is bile acid or cell line was next addressed. First, we
examined the bile acid specificity (Fig.
3A). Indeed, we were able to demonstrate that additional
bile acids such as CDC and TCDC transactivate the EGFR in KMBC cells. Next, we
ascertained the cell specificity of our observations by determining the
effects of these bile acids on EGFR activation in the H-69 cell line. This is
a human, immortalized (SV-40), nonmalignant cell line that expresses many of
the differentiated functions of primary cholangiocytes. Indeed, DC and CDC
also both transactivated EGFR in this differentiated cell line
(Fig. 3B). Thus our
results with deoxycholate in the KMBC cell line can be generalized to
additional bile acids and a more well-differentiated cholangiocyte cell
line.
Is MMP activity necessary for bile acid-induced EGFR
phosphorylation? To determine whether MMP-mediated release of TGF-
is required for bile acid-induced EGFR phosphorylation, MMP activity was
inhibited with GM6001. Treatment of KMBC cells with this inhibitor
significantly diminished bile acid-induced EGFR phosphorylation
(Fig. 4). These observations
suggest that MMP activity is necessary for TGF-
release, which is a
prerequisite for bile acid-induced ligand-dependent EGFR activation.

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Fig. 4. Matrix metalloproteinase (MMP) activity is necessary for DC-induced EGFR
phosphorylation. KMBC cells were preincubated in the presence or absence of an
MMP inhibitor (GM6001, 25 µM) for 16 h before addition of DC (200 µM).
After 1 h exposure to DC, cells were lysed and immunoblot analysis was
performed using anti-phosphotyrosine or anti-EGFR antisera. The ratios were
calculated by densitometric scanning of the intensity of phosphotyrosine band
relative to the EGFR band. Data were expressed as means ± SD from 3
individual experiments. A representative immunoblot was shown. *P
< 0.01, vs. control or GM6001 + DC-treated cells.
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Does bile acid-induced EGFR activation promote cellular growth? We
reasoned that if bile acids activate EGFR, this might promote cellular growth.
During 4 days of culture in the presence of bile acid, cell growth was
monitored by the MTS assay (Fig.
5). DC significantly promoted cellular growth of KMBC cells, and
this was preventable by the MMP inhibitor, which can block DC-induced EGFR
activation. To further address the necessity of EGFR activation for bile
acid-enhanced cholangiocyte cell growth, we performed additional experiments
with the highly selective EGFR kinase inhibitor ZD1839 (10 µM). This
selective compound also reduced deoxycholate-induced growth demonstrating the
growth enhancement is EGFR dependent (Fig.
5). Together, these findings suggest that the growth-promoting
effect of DC is mediated by EGFR activation.

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Fig. 5. DC-induced cellular growth is EGFR dependent. Before DC (200 µM)
treatment, KMBC cells cultured in 96-well plates (2 x 103
cells/well) were incubated in the presence or absence of the MMP inhibitor
(GM6001, 25 µM) or a selective EGFR kinase inhibitor (ZD1839, 10 µM). At
day 4, a
3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethyloxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
salt assay (Promega) was performed according to the manufacturer's
instructions. All experiments were performed in 10% FBS-containing media. Data
are expressed as means ± SD from 6 individual experiments. *P
< 0.01 vs. control.
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DISCUSSION
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The principal findings of this study relate to the mechanism by which bile
acids transactivate the EGFR. The results indicate that bile acid
transactivation of the EGFR occurs by an MMP/TGF-
-dependent pathway.
The results provide new information regarding the growth-modifying potential
of bile acids. Each of these findings will be discussed further.
Although EGFR may be activated by several ligands and also
ligand-independent processes
(5,
6), bile acids appear to
transactivate the EGFR by a TGF-
ligand-dependent mechanism. Indeed,
KMBC cells express TGF-
and, more importantly, anti-TGF-
antisera inhibits bile acid-associated EGFR activation. TGF-
is
synthesized as a transmembrane-spanning polypeptide and must be cleaved by MMP
or "secretases" to function as an EGFR ligand
(13,
15). Consistent with these
concepts, an MMP inhibitor also diminished bile acid transactivation of EGFR.
This observation further strengthens the concept that bile acid
transactivation of EGFR is ligand mediated.
Previous studies from our laboratory
(22,
23) demonstrated that Src
kinase activity was necessary for bile acid-driven EGFR autophosphorylation.
However, the mechanism by which Src kinase mediated EGFR activation was not
elucidated in these studies. Prostaglandin E2 transactivates the
EGFR by Src activation of MMP, leading to TGF-
secretion
(16). Our previous studies
(22) compared with the current
documentation suggest bile acid transactivation of EGFR is similar to
prostaglandin E2. Indeed, an Src inhibitor, an MMP inhibitor, and
anti-TNF-
antigen all blocked bile acid EGFR transactivation. The
simplest interpretation of these data is a linear model by which bile acids
stimulate Src activity enhancing MMP activity generating soluble TGF-
.
Further studies will be necessary to identify the mechanisms by which bile
acids stimulate Src kinase.
Bile acids stimulate cholangiocyte growth in vivo and in vitro
(2,
3). TGF-
activation of
EGFR is a potent mitogenic stimulant in many cells
(6,
16). Bile acid-associated
TGF-
-mediated EGFR stimulation, therefore, likely contributes to the
mitogenic effects of bile acids on cholangiocytes
(2,
3). Indeed, disruption of this
pathway with the MMP inhibitor attenuated bile acid-stimulated growth of KMBC
cells. This concept has two biological implications. First, augmentation of
this signaling pathway (e.g., bile acid feeding) may potentially be useful in
diseases associated with bile duct injury. The ability to enhance
cholangiocyte proliferation in these diseases could promote bile duct healing
and function. Conversely, bile acid mitogenesis may facilitate the progression
of cholangiocarcinoma. Inhibition of this bile acid-stimulated pathway may
prove useful as an adjuvant in the therapy of cholangiocarcinoma.
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ACKNOWLEDGMENTS
|
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This work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-59427, the Palumbo Foundation, and the Mayo
Foundation.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. J. Gores, Professor
of Medicine, Mayo Medical School, Clinic, and Foundation, 200 First St. SW,
Rochester, MN 55905 (E-mail:
gores.gregory{at}mayo.edu).
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.
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Copyright © 2003 by the American Physiological Society.