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LIVER AND BILIARY TRACT

Bile acids activate EGF receptor via a TGF--dependent mechanism in human cholangiocyte cell lines

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

	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

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.

	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 170–190 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.

View larger version (90K): [in this window] [in a new window]   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.

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.

View larger version (57K): [in this window] [in a new window]   Fig. 2. EGFR ligands are constitutively expressed in KMBC cells. KMBC cells, serum-starved for 24 h before bile acid treatment, were treated with DC (200 µM) for each indicated time period. Total cellular RNA was isolated, and RT-PCR for each EGFR ligand was performed. Control KMBC cells maintained in serum-containing media (S), LX2 human stellate cells (Lx), Huh-7 cells (Hu), Hep3B cells (3B), and HepG2 cells (G2) were also maintained in serum-containing media and used for EGFR ligand RT-PCR controls. TGF-, transforming growth factor-; HB-EGF, heparin-binding EGF.

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.

View larger version (49K): [in this window] [in a new window]   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.

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.

View larger version (46K): [in this window] [in a new window]   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.

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.

View larger version (9K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 E_2. 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.

	ACKNOWLEDGMENTS

 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59427, the Palumbo Foundation, and the Mayo Foundation.

	FOOTNOTES

 

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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This Article Abstract Full Text (PDF) All Versions of this Article: 285/1/G31    most recent 00536.2002v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (37) Citing Articles via Google Scholar Google Scholar Articles by Werneburg, N. W. Articles by Gores, G. J. Search for Related Content PubMed PubMed Citation Articles by Werneburg, N. W. Articles by Gores, G. J.