Pallavi B. Limaye, 1 William C. Bowen, 1 Anne V. Orr, 1 Jianhua Luo, 1 George C. Tseng, 2 and George K. Michalopoulos 1

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1 Mechanisms of Hepatocyte Growth Factor Mediated and Epidermal Growth Factor Mediated Signaling in Transdifferentiation of Rat Hepatocytes to Biliary Epithelium Pallavi B. Limaye, 1 William C. Bowen, 1 Anne V. Orr, 1 Jianhua Luo, 1 George C. Tseng, 2 and George K. Michalopoulos 1 Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), plateletderived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/ catenin pathway, transforming growth factor beta (TGF- )/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF EGF. Most of the HGF EGF target genes were altered by LY Conclusion: Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase mediated signaling independent of AKT is a crucial component of the transdifferentiation process.(hepatology 2008;47: ) Abbreviations: BE, biliary epithelium; BEC, biliary epithelial cell; CBLD, chronic biliary liver disease; Dex, dexamethasone; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF-a, fibroblast growth factor-a; FGF-b, fibroblast growth factor-b; FGF-8b, fibroblast growth factor-8b; GF, growth factor; HGF, hepatocyte growth factor; ITS, insulin transferring selenium; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MSP, macrophage stimulating protein; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; SAPK, stress-activated protein kinase; SCF, stem cell factor; TGF-, transforming growth factor beta; VEGF, vascular endothelial growth factor. From the 1 Department of Pathology, School of Medicine and 2 Department of Biostatistics and Human Genetics, Graduate School of Public Health, University of Pittsburgh School of Medicine, Pittsburgh, PA Received August 7, 2007; accepted January 5, Supported in part by the American Liver Foundation in the form of Postdoctoral fellowship (P.B.L.). Supported by National Institutes of Health grants CA30241 and CA35373 (to G.K.M.), the Rangos Fund for Enhancement of Pathology Research. G.C.T. is supported by the National Institutes of Health (1 KL2 RR ). Address reprint requests to: George K. Michalopoulos, M.D., Ph.D., 200 Lothrop Street, BST S410, Pittsburgh, PA michalopoulosgk@upmc.edu; fax: Copyright 2008 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. Supplementary material for this article can be found on the HEPATOLOGY Web site ( 1702

2 HEPATOLOGY, Vol. 47, No. 5, 2008 LIMAYE ET AL Recent evidence suggests that hepatocytes and biliary epithelial cells (BECs), the 2 mature epithelial cells of the adult liver, although terminally differentiated, retain the potential to interchange their phenotypes on need. 1-5 Injury to the liver combined with arrest of proliferation of hepatocytes is known to lead to transdifferentiation of BECs into hepatocytes (via oval cells) and to restore the loss of hepatic parenchyma. 6-8 Hepatocyte to BEC transdifferentiation has also been reported recently from our laboratory in an experimental rodent model as well as in hepatocyte organoid cultures. 5,9,10 Transdifferentiation of hepatocytes to biliary epithelium (BE) has also been demonstrated in another culture system of 3-dimensional hepatocyte cultures. 3 Chronic biliary liver diseases (CBLD), characterized by extensive BE loss, are known to induce development of atypical ductules expressing both hepatocytic and biliaryspecific genes, indicating hepatocyte-to-be transdifferentiation in an attempt to restore degenerated bile ducts. 2,11 However, the mechanisms of hepatocyte to BEC transdifferentiation remain unknown. Understanding the mechanism of native hepatocyte to BEC transdifferentiation would be pivotal in designing future therapeutic strategies in CBLD patients. We have previously reported that rat hepatocytes transform into phenotypically distinct BE when cultured in the collagen-coated roller bottles in the presence of hepatocyte growth factor (HGF) or epidermal growth factor (EGF) or their combination. 9,10 Using the retrorsine/dipeptidyl peptidase IV (DPPIV) hybrid liver system, in which approximately half of the hepatocytes bear a dipeptidyl peptidase IV tag, it was clearly demonstrated that the biliary epithelium emerging in these cultures is derived from hepatocytes. 10 The current study is aimed at investigating the cellular and molecular mechanisms involved in the process of hepatocyte to BE transdifferentiation in rat hepatic organoid culture system established in our laboratory. We chose to use this system to investigate the signaling mechanisms controlling hepatocyte transdifferentiation to BE because it is much easier to control the experimental parameters in culture than in the whole animal setting. Both HGF and EGF are ligands of tyrosine kinase receptors c-met and epidermal growth factor receptor (EGFR), respectively. In the current study, using rat hepatic organoid cultures, we examined the ability of other tyrosine kinase receptor ligands in inducing the transformation of hepatocytes into BE. Seven growth factors were tested, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophagestimulating protein (MSP), and fibroblast growth factors (FGF-a, FGF-b, and FGF-8b), all known to play a role in development and differentiation Our study shows that none of the growth factors tested except HGF and EGF could induce hepatocyte to BEC transdifferentiation, in spite of presence of their cognate membrane receptors. To further study the downstream signaling initiated by HGF and EGF, activation of downstream mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)1/2, p38, and c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), as well as serine/threonine kinase AKT and pharmacological inhibition of these kinases was evaluated to further ascertain involvement of these pathways. Additionally, global changes in gene expression profiles were evaluated in cultures treated with HGF, EGF, and their combination compared with the rest of the growth factors (GFs). Materials and Methods Animals. Male Fisher 344 rats ( g) obtained from Charles River (Frederick, MA) were used for the studies described. The animal husbandry and all procedures performed on the rats employed for these studies were approved by the institutional animal care and use committee of the University of Pittsburgh and conducted according to National Institute of Health guidelines. Materials Collagenase for hepatocyte isolation was obtained from Boehringer Mannheim (Mannheim, Germany). Rat tail collagen (20%) was used for coating of roller bottles. General reagents were obtained from Sigma Chemical Co. (St. Louis, MO). HGF used for these studies was the 5 variant and was donated by Snow Brand Co. (Toshigi, Japan). EGF was purchased from BD Pharmingen (San Diego, CA). All the other growth factors, namely, VEGF, PDGF, SCF, MSP, FGF-a, FGF-b, and FGF-8b, were purchased from R&D Systems (Minneapolis, MN). All the rest of the chemicals were purchased from Sigma Chemicals (St. Louis, MO) unless otherwise stated. Antibodies Primary antibodies used for Western blotting are: Met (1:500, clone B-2), c-kit (1:500), Ron (1:500), Flt1 (1: 250), FGFR2, FGFR3 (1:250), phospho Ron (1:200), beta-catenin (1:200), chemokine LIX (1:500), GSK3 (1:200), and CK19 (1:400, Dako, Carpinteria, CA), (Santa Cruz Biotechnology, Santa Cruz, CA), EGFR (1: 1000), phospho-pdgfr- (1:1000), phospho FGFR (cross reacts with all 4 isoforms) (1:1000) (Cell Signaling Technology, Danvers, MA), FGFR1 (1:400), PDGFR- (1:200) and PDGFR- (1:100), and CD44 (1:200 (Abcam, Cambridge, MA), phospho PDGFR-, phospho c- kit, ERK1/2 (1:1000, Sigma Chemicals, St. Louis, MO),

3 1704 LIMAYE ET AL. HEPATOLOGY, May 2008 phospho ERK1/2 (1:1000), AKT (1:1000), phospho-akt (ser 473, 1:1000), p38 (1:1000), phospho-p38 (1:1000), JNK/SAPK (1:1500), phospho-jnk/sapk (1:1500) (Cell Signaling Technology, Danvers, MA) cyclin D1 (1:200, Neomarkers, Fremont, CA). All the pertinent secondary antibodies conjugated to horseradish peroxidase (used for western blotting) and primary antibody against actin (1:5000) were purchased from Chemicon (Temecula, CA). Secondary antibodies were used at 1:50,000 dilution. For immunohistochemistry, AE1/AE3 monoclonal antibody (1:20) was purchased from Chemicon (Temecula, CA), and the rest of the receptor antibodies were same as listed and were used at concentrations suggested by the manufacturer. Biotinylated secondary antibodies used for immunohistochemistry were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). They were used at 1:500 dilution. Isolation and Culture of Hepatic Cell Populations Hepatocytes. Rat hepatocytes were isolated by an adaptation of Seglen s calcium 2-step collagenase perfusion technique as previously described from our laboratory. 9 As previously described, these preparations are known to contain contaminant small numbers of other hepatic cellular elements, including stellate cells, Kupffer cells, and very few bile duct epithelial cells. The latter typically do not constitute more than 0.05% of the inoculated cell population. In our previous studies, we used chimeric livers with dipeptidyl peptidase IV hepatocytes as a source of hepatocyte isolation, and we found that BE developing in the cultures was derived from hepatocytes. 10 For the current studies, we used hepatocytes isolated from nonchimeric, normal, Fisher 344 male rats and used the formation of biliary epithelium as the endpoint of assessment for the different growth factors. Isolated hepatocytes were added to collagen-coated roller bottles (850 cm 2 surface) obtained from BD Biosciences (Franklin Lakes, NJ). Each bottle contained 200,000,000 freshly isolated hepatocytes in 200 ml hepatocyte growth medium, medium supplemented with 1 of the following: HGF (40 ng/ml), EGF (20 ng/ml), VEGF (10 ng/ml), PDGF (10 ng/ml), SCF (10 ng/ml), MSP (10 ng/ml), FGF-a, FGF-b, FGF 8b (25 ng/ml), or HGF EGF. Concentrations of the GFs used in the cultures were decided based on the half maximal effective concentration values provided by the manufacturer, from hepatocyte-related literature, or from previous studies of our own. The bottles were rotated at a rate of 2.5 rotations per minute and kept in an incubator maintained at 37 C, saturated humidity, and 5% CO 2. The tissue was harvested from the bottles on day 14 of the culture for further analysis. Inhibitors. To identify which signal transduction pathway is crucial for the HGF-induced and EGF-induced hepatocyte to biliary epithelium transdifferentiation, an interventional study was designed. Protein kinase inhibitors used in the study were purchased from Sigma Chemicals (St. Louis, MO). The inhibitors and their final concentrations in the cultures were as follows: PI3 kinase inhibitor LY (25 M); p38 inhibitor SB (7.5 M); mitogen-activated protein kinase kinase inhibitor PD98059 (50 M); AKT inhibitor (6.8 M). Inhibitors were added to the hepatocyte cultures from the beginning and were renewed every 2 days with fresh media and HGF EGF until the end of the experiment (14 days). The individual inhibitor concentrations were selected based on the manufacturer s recommendations and our preliminary studies. Western Blot Analysis Total cell lysates made in Ripa buffer (40 g) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4% to 12% NuPage Bis-Tris gels with 1 MOPS buffer (Invitrogen, Carlsbad, CA), then transferred to Immobilon-P membranes (Millipore, Bedford, MA) in NuPAGE transfer buffer containing 10% methanol. Membranes were stained with Ponceau S to verify loading and transfer efficiency. Membranes were probed with primary and secondary antibodies in Tris-buffered saline Tween 20 containing 5% nonfat milk, then processed with SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL) and exposed to a X-ray film (Lab Product Sales, Rochester, NY). Horseradish peroxidase conjugated secondary antibodies were used at a 1:80,000 dilution (Chemicon, Temecula, CA). Equal protein loading was ascertained by actin for each sample. Immunohistochemistry Paraffin-embedded hepatocyte organoid tissue sections (4 m thick) were used for immunohistochemical staining. Antigen retrieval was achieved by heating the slides in the microwave at high power in 1 citrate buffer for 10 minutes. The tissue sections were blocked in blue blocker for 20 minutes followed by incubation with pertinent primary antibody overnight at 4 C. The primary antibody was then linked to biotinylated secondary antibody followed by routine avidin-biotin complex method. Diaminobenzidine was used as the chromogen, which resulted in a brown reaction product. Affymetrix Chip Analyses. The Affymetrix oligonucleotide chip specific for rat (U230.2 A chip) containing 10,000 expressed sequences was used for global gene expression analyses. Sample preparation, data generation, and analyses were as reported previously. 18 Further analysis was conducted using dchip. Genes with less than 80% present calls or standard deviation (SD) less than 0.4

4 HEPATOLOGY, Vol. 47, No. 5, 2008 LIMAYE ET AL Fig. 1. Histology of the hepatocyte organoid tissues at day 14 of the culture treated with different GFs. Hematoxylin-eosin stained sections of cultures treated with HGF (B), EGF (C), HGF EGF (D), VEGF (E), PDGF (F), SCF (G), MSP (H), FGF-a (I), FGF-b (J), and FGF-8b (K). Biliary epithelium (BE) developed in HGF and EGF treatment (arrowhead). H, hepatocytes; E, endothelial cell. Biliary marker AE1/AE3 stained section of the hepatocyte organoid tissue at day 14 of the culture treated with HGF EGF (1m). AE1/AE3 positive BE (arrow). were filtered out. Statistical analysis microarrays (SAM) package was applied to these data with a false discovery rate (FDR) of 5%. R software is used to generate the hierarchical clustering dendrograms and the heatmaps. Results Formation of BE Was Observed Only in HGF, EGF, and HGF EGF Treatments in Hepatocyte Organoid Cultures Hematoxylin-eosin stained paraffin sections of the tissue obtained from hepatocyte organoid cultures showed a typical histological organization only in HGF (Fig. 1B), EGF (Fig. 1C), and HGF EGF (Fig. 1D) treatments consisting of a surface monolayer of cuboidal BE, a middle layer of hepatocytes embedded in connective tissue elements, and the basal layer of endothelial cells. None of the growth factors tested other than HGF and EGF could induce the formation of BE (Fig. 1E-K). The cultures treated with VEGF (Fig. 1E), PDGF (Fig. 1F), SCF (Fig. 1G), MSP (Fig. 1H), FGF-a (Fig. 1I), FGF-b (Fig. 1J), and FGF-8b (Fig. 1K), histologically resembled the control organoid culture (Fig. 1A) [grown in hepatocyte growth medium containing only insulin transferring selenium (ITS) dexamethasone (Dex)] as described previously comprising hepatocytes, few stellate cells, and endothelial cells, 9,10 but no BE. The presence of BE in the HGF, EGF, and HGF EGF treated organoid cultures was confirmed by the immunohistochemical staining of biliary cytokeratin marker AE1/AE3. AE1/AE3 positive brown staining was observed in the surface BE emerging in the presence of HGF EGF (Fig. 1L), HGF, and EGF (data not shown). These findings confirm that HGF and EGF are the only 2 unique GFs capable of inducing hepatocyte transdifferentiation into BE. Presence of the Cognate Membrane Receptors Detected by Western Blot Analysis and Immunohistochemistry Because, except HGF and EGF, all the other GFs tested failed to stimulate BE formation, the possibility that the inability of the GFs to stimulate BE development may be due to the lack of expression of their cognate membrane receptors in the organoid cultures was tested by Western blot analysis for the respective receptors. Western blot analysis indicates that after the ligand treatment, all the respective receptors [met (HGF receptor), EGFR (EGF receptor), Flt1 (VEGF receptor), PDGFR-, and PDGFR- (PDGF receptors), c-kit (SCF receptor), Ron (MSP receptor), and FGFR 1 through 4 (FGF receptors)] are expressed. These findings indicate that despite the availability of their cognate receptors, the GFs tested VEGF,

5 1706 LIMAYE ET AL. HEPATOLOGY, May 2008 Fig. 2. (A) Western blot analysis of different membrane receptors (met, EGFR, Flt1, PDGFR, c-kit, ron, and FGFRs) in hepatocyte organoid culture lysates obtained at day 14 from various GF treatments HGF, EGF, VEGF, PDGF, SCF, MSP, and FGFs as described in Materials and Methods. Pertinent ligand and the receptor pairs are marked with a red underline. Actin blot was used as a loading control. Forty micrograms protein was separated on 4% to 12% gradient gel. (B) Phosphorylated form of the receptors detected by western blotting with and without the respective ligand in the hepatocyte organoid cultures. (C) Immunohistochemical localization of the membrane receptors in the organoid cultures developed in the presence of ITS Dex (control). (A) Flt1, (B) PDGFR, (C) PDGFR, (D) c-kit, (E) Ron, (F) FGFR1, (G) FGFR2, (H) FGFR3, and (I) FGFR4. PDGF, SCF, MSP, FGF-a, FGF-b, and FGF-8b were unable to induce hepatocyte to BE transdifferentiation. To ensure that the membrane receptors were functional and were activated on exposure to their respective ligand, receptor phosphorylation (activation) of Flt1, PDGFR- and PDGFR-, c-kit, Ron, and FGFR was analyzed by Western blotting (Fig. 2B). The results indicate that PDGFR-, and PDGFR-, c-kit, Ron, and FGFR were activated (phosphorylated) on addition of their respective ligands (Fig. 2B). Flt1 was found phosphorylated in the control and after

6 HEPATOLOGY, Vol. 47, No. 5, 2008 LIMAYE ET AL Fig. 3. Western blot analysis of total and activated protein kinases (ERK1/2, p38, JNK/SAPK, and AKT) in hepatocyte organoid culture lysates obtained from various GF treatments HGF, EGF, VEGF, PDGF, SCF, MSP, and FGFs as described in Materials and Methods. Forty micrograms protein was separated on 4% to 12% gradient gel. Actin blot used as a loading control. VEGF treatment, although to a lesser extent (Fig. 2B). Presence of the cognate receptors and their localization was further confirmed by immunohistochemistry of the paraffinembedded control organoid cultures (exposed to ITS Dex) (Fig. 2C). Examined receptors Flt1 (Fig. 2Ca), PDGFR- (Fig. 2Cb) and PDGFR- (Fig. 2Cc), c-kit (Fig. 2Cd), Ron (Fig. 2Ce), FGFR1 (Fig. 2Cf), FGFR2 (Fig. 2Cg), FGFR3 (Fig. 2Ch), and FGFR4 (Fig. 2Ci) are detected in the hepatocytes in the organoid cultures, and their level of expression corresponds to the western blot data (Fig. 2A). Activation of AKT Kinase Secondary to HGF and EGF HGF and EGF are known to stimulate mitogen-activated protein kinases (MAPKs) (ERK1/2, p38, JNK) or serine/threonine kinase AKT, leading to cell proliferation, survival, and migration. To determine which signaling pathway activation is involved in the process of hepatocyte transdifferentiation, expression and activity (phosphorylation) of ERK1/2, p38, JNK, and AKT were studied by western blotting of the hepatocyte organoid culture lysates. Total protein for all the 4 protein kinases studied was induced in HGF, EGF, and HGF EGF treatments (Fig. 3A, C, E, and G). However, no change in the activity (phosphorylation) of the 3 MAPKs (ERK1/2, p38, JNK) was observed in the HGF and EGF treatments (Fig. 3B, D, F; lanes 2 through 4), whereas in the rest of the GF treatment MAPKs retained their phosphorylation status, the same as that of the control or increased (Fig. 3B, D, F; lanes 5 through 11). On the contrary, AKT phosphorylation was induced in the HGF and EGF treatments (Fig. 3D; lanes 2 through 4) compared with the control (Fig. 3D; lane 1) as well as rest of the GFs (Fig.

7 1708 LIMAYE ET AL. HEPATOLOGY, May D; lanes 5 through 11). A moderate increase in the AKT phosphorylation was also observed after FGF-a and FGF-b (Fig. 3H, lane 9 and 10) treatment, although it was lower than that of the HGF EGF and HGF treatments. These data suggest that AKT activation might be responsible for the derivation of BE from hepatocytes in HGF and EGF treatments. To further dissect the role of MAPKs and AKT in HGF-induced and EGF-induced hepatocyte to BEC transdifferentiation, hepatocyte organoid cultures were treated with inhibitors of MEK1/2 (PD98059), p38 (SB202190), AKT (AKT1/2 inhibitor), and PI3K (LY294002) with HGF EGF. Of these, phosphatidylinositol 3-kinase (PI3K) inhibitor showed complete suppression whereas AKT inhibitor showed partial reduction in the formation of BE, and the rest of the inhibitors tested could not prevent formation of BE in the cultures (data not shown). These data indicate that PI3K pathway independent of AKT signaling is responsible for the hepatocyte to BE transdifferentiation. Hierarchical Clustering Analysis of Microarray Data Reveals Tight Clustering of the Genes Regulated by HGF, EGF, and HGF EGF Treatments To study gene expression changes that might be involved in phenotypical transformation of hepatocytes to BE, microarray analysis (Affymetrix gene array Rat genome ) was performed, and comparisons between GFs that induce hepatocyte to BE transdifferentiation (HGF, EGF, and HGF EGF) and the GFs that failed to do so (ITS Dex, VEGF, PDGF, SCF, MSP, FGF-a, FGF-b, and FGF-8b) were made. Hierarchical clustering of the unsupervised data indicated that HGF EGF, HGF, and EGF treatments cluster on 1 branch while all the rest of the treatments cluster away from these treatments on a separate branch (Fig. 4A). Data were then processed with dchip. Genes with less than 80% present or SD less than 0.4 were filtered out, resulting in 2667 genes that were differentially expressed (Fig. 4B). Furthermore, the SAM package was applied to the data, revealing 512 up-regulated and 196 down-regulated genes under an FDR of 5% in the HGF EGF, HGF, and EGF treatments (Fig. 4C). For the quantitative interpretation of microarray data, log ratio of the gene expression values was calculated for treatments that show development of biliary epithelium versus groups that do not show development of biliary epithelium [group I/control, group II/control, and group II/group I (VEGF, PDGF, SCF, MSP, FGF-a, FGF-b, and FGF-8b, collectively referred to as group I; HGF, EGF, and HGF EGF, collectively referred to as group II)]. These results are provided as the supplemental data in Supplementary Table 1. The genes, which are altered significantly exclusively in HGF, EGF, and HGF EGF treatments compared with all of the rest of the treatments, are described below. Consistent with the observed phenotype, biliary-specific genes such as CK19, sodium-potassium transporting adenosine triphosphatase, and secretin receptor are significantly increased in the HGF, EGF, and HGF EGF treatments. Extracellular matrix related genes such as procollagen type 1 and 1, collagen 1, 2 laminin, cell surface glycoprotein CD44, matrix metalloproteinase 9, matrix metalloproteinase 12, and glypican 3 are also up-regulated in these treatments. Chemokines such as CXC (cysteine-any amino acid-cysteine) chemokine lipopolysaccharide-induced CXC chemokine (LIX), chemokine orphan receptor, interleukin 18, and osteopontin are increased in the treatments, showing development of biliary component. Genes belonging to the Wnt/ -catenin signaling pathway are increased in response to HGF, EGF, and HGF EGF treatments including Wnt4, -catenin, GSK3, and its downstream targets including versican, regucalcin, cyclin D1, and CYP2E1. Localization of -catenin was also carried out in the control (Fig. 4Ea) and HGF EGF treated cultures (Fig. 4Eb). -Catenin was found increased in the biliary epithelium in the HGF EGF treated culture (Fig. 4Eb). Liver regeneration related genes such as amphiregulin, pancreatitis-associated protein-1, annexin 1 are up-regulated in HGF, EGF, and HGF EGF treatments. TGF- family members including TGF 1 and TGF- 2, TGF- masking protein, TGF- superfamily receptor subunit, BMP6, and its transcription factor GATA4 are increased in HGF, EGF, and HGF EGF treatments. Up-regulation of TGF- was also confirmed by immunohistochemistry in HGF EGF treatment (Fig. 4Ed) compared with the control (Fig. 4Ec). TGF- was found increased in the hepatocytes in the HGF EGF treated culture (Fig. 4Eb). RAS p21 protein activator 3, urokinase-type plasminogen activator (u-pa), extracellular proteinase inhibitor, and macrophage inflammatory protein-1 alpha receptor (MIP1) are up-regulated in HGF, EGF, and HGF EGF treatments. Conversely, glutathione S-transferase Yb4, CYP1A1, cholesterol esterase, uridine diphosphate glucuronosyltransferase, guanylate cyclase 2C, stearyl-coa desaturase, and liver aldolase B are among the 192 genes that are down-regulated. In a separate gene array experiment, organoid cultures treated with ITS Dex (control), HGF EGF, and HGF EGF LY were analyzed, and a comparison was made between the log ratios of HGF EGF/control and HGF EGF LY294002/control. On addition

8 Fig. 4. (A) Hierarchical clustering of unsupervised gene array data obtained from Affymetrix Rat genome 2.0 analysis of hepatocyte organoid cultures treated with different GFs as labeled. (B) Heatmap of the genes statistically significant resulting after the dchip preprocessing (2667 genes). (C) Heatmap of the tightly clustered genes in the HGF EGF, HGF, and EGF treatments after SAM package was applied. Yellow, up-regulated (512 genes); red, down-regulated (192 genes). (D) Western blot analysis of representative proteins whose genes were up-regulated in the gene array after HGF and EGF treatments. Forty micrograms protein was separated on 4% to 12% gradient gel. Actin blot was used as a loading control. (E) Immunohistochemical localization of HGF EGF target genes. (A and B) Beta-catenin staining in control (A) and after HGF EGF treatment (B). Beta-catenin staining increased in the biliary epithelium, whereas there was less hepatocyte membranous staining in HGF EGF treatment. TGF-1 staining in control (C) and after HGF EGF treatment (D). TGF-1 staining was higher in the hepatocytes underlying the biliary epithelium in HGF EGF treatment.

9 1710 LIMAYE ET AL. HEPATOLOGY, May dehydrogenase/reductase (SDR family) member 7, stearoyl-coenzyme A desaturase 1, and CYP2A2. Collectively, these data indicate that HGF and EGF mediated gene profile alteration responsible for transdifferentiation is largely regulated by PI3 kinase. Fig. 5. Effect of PI3K inhibitor LY on the HGF EGF target genes. (A) Heatmap of the HGF EGF modulated genes with and without LY compared with the control. Gene expression is largely reversed in the presence of LY. Yellow, up-regulated (approximately 500 genes); red, down-regulated (approximately 125 genes). (B) Reversal of the HGF and EGF mediated up-regulation of the genes at the protein level on addition of PI3 kinase inhibitor LY detected by western blot. AKT inhibitor was ineffective in preventing HGF and EGF mediated up-regulation of target genes. of PI3 kinase inhibitor LY in the cultures, HGF EGF mediated up-regulation/down-regulation of their target genes was found to reverse back closer to the level of control (ITS Dex) as shown in the heatmap of the selected target genes (Fig. 5A) and the quantitative analysis (Supplementary Table 2). For this analysis, the same genes were selected that clustered together in HGF, EGF, and HGF EGF treatments (Fig. 4C and Supplementary Table 1). LY treatment abolished the upregulation of the key genes modulated by the HGF EGF treatment such as Wnt4, -catenin, CXC chemokine LIX, cyclin D1, BMP6, TGF, matrix metalloproteinase 9, osteopontin, CD44, and so forth, while it prevented the down-regulation of glutathione S-transferase Yb4, CYP1A1, CYP3A3, guanylate cyclase 2C, alpha-2u globulin, and so forth. Gene expression of secretin receptor and CK19 (markers of biliary epithelium) was significantly decreased in the HGF EGF LY treatment compared with HGF EGF treatment. There were some HGF EGF target genes those were not altered by the LY treatment, such as dual-specificity phosphatase 6, phospholipid scramblase 1, chemokine (C-X-C motif) ligand Confirmation of the Gene Array Data by Western Blotting The gene array data were corroborated by western blot analysis of selected proteins representing different classes showing up-regulation, which may be critical in the process of hepatocyte transdifferentiation (Fig. 4D). Consistent with the gene array findings, CK19, a biliary-specific protein, was found increased in HGF, EGF, and HGF EGF treatments compared with control as well as the rest of the GFs. These data also indicated that EGF is a stronger inducer of CK19 than HGF. Apart from these 3 treatments that resulted in biliary development, FGF-b treatment also led to CK19 induction, although no significant increase in the messenger RNA level detected by gene array was observed, and no histological biliary development was noticed in this treatment. Beta-catenin and its downstream targets cyclin D1 and CD44 are induced by HGF, EGF, and HGF EGF treatments. Interestingly, a smaller isoform of -catenin of approximately 70 kda was also noticed only in HGF, EGF, and HGF EGF treatments. This isoform is speculated to be the fetal form of -catenin. 19 CXC chemokine LIX induction by HGF, EGF, and HGF EGF treatments was also confirmed by the western blot analysis. On addition of the PI3 kinase inhibitor LY in the organoid cultures, HGF EGF mediated up-regulation of CK19, -catenin, truncated -catenin, and cyclin D1 was inhibited as confirmed by western blot (Fig. 5B). The AKT inhibitor that failed to prevent development of biliary epithelium could not inhibit up-regulation of these genes (Fig. 5B) except truncated -catenin, indicating that this form of -catenin may not be essential in the transdifferntiation process. Discussion The current study was aimed at the investigation of the mechanism of hepatocyte-to-bec transdifferentiation. Such a phenomenon has been demonstrated in experimental rodent models with biliary degeneration as well as in human CBLD patients. 1,2,5 Understanding the mechanism of hepatocyte-to-bec transdifferentiation is important because it holds a therapeutic potential for the rescue of CBLD patients by stimulating transdifferentiation of native hepatocytes into BECs to restore the lost biliary structure and function. The hepatocyte organoid culture system, which exhibits specific microarchitecture

10 HEPATOLOGY, Vol. 47, No. 5, 2008 LIMAYE ET AL composed of hepatic cell types, was used in this study. It served as an excellent tool in which different GFs could be added as needed to decipher their exact role in transdifferentiation. The significant finding of this study is that tyrosine kinase ligands HGF and EGF are unique in their function as promoters of hepatocyte-to-bec transdifferentiation. We only tested EGF from the array of the other EGFR ligands, and we only assume that the rest of the EGFR ligands would also be effective, but this was not directly addressed in the current study. Despite the expected redundancy in the downstream signaling of the GFs examined, including VEGF, PDGF, SCF, MSP, and FGF-a, FGF-b, and FGF-8b (known to elicit their response through tyrosine kinase associated membrane receptors), they all were unsuccessful in inducing hepatocyte-to-bec transdifferentiation despite the availability and activation of their cognate receptors. Some of these other GFs are expressed or known to play a role at different stages of liver development and regeneration; however, when added by themselves they did not trigger appropriate signaling that led to hepatocyteto-be transdifferentiation. To identify which protein kinases downstream of tyrosine kinase receptors is or are differentially regulated by HGF and EGF compared with the rest of the GFs resulting in hepatocyte transdifferentiation, western blot analysis was conducted for MAPKs (ERK1/2, JNK, and p38) and serine/threonine kinase AKT. Interestingly, though the total protein for all 3 MAPKs and AKT was induced because of the HGF and EGF treatment, significantly increased phosphorylation of only AKT was observed in the HGF and EGF treatments, indicating that transdifferentiation might be mediated through AKT signaling. In liver, hepatic stellate cells are known to undergo transdifferentiation to become collagen-producing myofibroblasts through activation of AKT. 20 In our studies, however, inhibition of AKT using a synthetic inhibitor only partially prevented formation of BE in the organoid cultures. Nonetheless, inhibition of PI3 kinase activity using its inhibitor (LY294002) completely prevented BE formation in the cultures, indicating that PI3K downstream signaling independent of AKT plays a central role in transdifferentiation of hepatocytes to BECs rather than the AKT signaling. These data suggest that PI3K pathway independent of AKT may be the main mediator for hepatocyte BEC transdifferentiation. PI3K signaling has been reported to be involved in hepatic stellate cell myofibroblastic transdifferentiation. 21 Inhibition of PI3K and downstream mammalian target of rapamycin (mtor)/ p70 ribosomal protein S6 kinase has been shown to induce the epithelial-to-mesenchymal transdifferentiation of intestinal epithelial cells. 22 Experimental evidence indicates that signals provided by HGF and EGF through MAPKs and AKT are essential for liver regeneration after partial hepatectomy or chemical injury. 26 However, our studies show that HGF-induced and EGF-induced transdifferentiation of hepatocytes into biliary cells is mediated through the PI3K pathway independent of AKT and MAPKs. Which of the downstream pathways of PI3K is involved in stimulating hepatocyte-to-bec transdifferentiation remains to be studied further. To identify the downstream targets exclusively activated or suppressed by HGF and EGF that may be necessary for hepatocyte to BEC transdifferentiation, a gene array analysis was conducted, revealing a set of approximately 700 genes solely altered by HGF and EGF compared with the other GFs and control (that do not develop BE). Of the up-regulated genes, we speculate that genes belonging to ECM, chemokines, Wnt/beta catenin, and TGF- /BMP pathways may play a central role in hepatocyte-to-bec transdifferentiation. Approximately 25% of the genes up-regulated are associated with the ECM, including different procollagen isoforms, matrix metalloproteinases, urokinase plasminogen activator, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. ECM through the associated proteins and enzymes is of great importance for the survival, differentiation, and normal function of cells within a tissue. Hepatocytes in culture maintained in the absence of matrix rapidly lose patterns of hepatocyte-specific gene expression and characteristic cellular microarchitecture; however, when matrix preparations (Matrigel) are added over dedifferentiated hepatocytes, differentiation is restored within 3 days. 27 Moreover, ECM remodeling is an essential part of liver regeneration after partial hepatectomy More recently it has been reported that transdifferentiation of hepatic stellate cells to myofibroblasts is facilitated by the matrix metalloproteinases activation cascade. 31 Based on these findings, we speculate that ECM also plays a critical role in signaling hepatocyte-to-be transdifferentiation via the proteins listed. Another family with several genes up-regulated in the gene array is TGF- /BMP. The BMPs are a family of secreted signaling molecules that can induce ectopic bone growth. Many BMPs are part of the TGF- superfamily and are found to play an important role in the embryonic liver growth. 17 TGF- was found to be increased in the hepatocytes underlying the biliary epithelium in the cultures treated with HGF EGF. This finding supports previously reported study showing that higher levels of TGF in the ductal hepatoblasts help them repress the effect of HNF6 and convert into biliary cells during development. 32 We suspect that by activating this pathway,

11 1712 LIMAYE ET AL. HEPATOLOGY, May 2008 the hepatocytes in culture recapitulate the fetal biliary specification programming to transdifferentiate into BE. The Wnt/beta-catenin pathway is known to carry out diverse functions, 1 of them being cellular differentiation. It is also known that this signaling in critical for the early embryonic 33 as well as postnatal development of liver. 34 Wnt4 and beta-catenin as well as their target genes such as cyclin D1, versican, regulcacin, and CYP2E1 were also up-regulated in response to both HGF and EGF treatments. Interestingly, a truncated isoform of -catenin was observed only in the HGF and EGF treatments. Such an isoform is detected in the embryonic development when hepatoblasts are differentiating into BECs and hepatocytes. 19 Increased expression of -catenin is observed primarily in the surface biliary epithelium in the organoid cultures treated with HGF EGF. This finding supports our previous study indicating that -catenin plays a significant role in early biliary lineage commitment during embryonic liver development. 35 Chemokines represent a large family of chemotactic peptides with a broad range of cellular targets. They are produced by all resident liver cells, including hepatocytes, 36 BECs, 37 sinusoidal endothelial cells, 38 Kupffer cells, 38 and stellate cells. 39 Chemokines that attract neutrophils typically contain a CXC motif near their NH 2 terminus, 40 and they exert their activity by binding to specific G protein coupled receptors (CXCR) on target cells. 41 In our study, CXC chemokines LIX, macrophage inflammatory protein-1 and 1 (MIP-1 and 1 ) are found up-regulated in the gene array after treatment with HGF and EGF. The chemokine receptors CXCR4, CXC orphan receptor, and LCR1 were also up-regulated. CXC chemokines have recently been implicated as mediators of hepatic inflammation in a model of obstructive cholestasis induced by bile duct ligation 37 or alpha-naphthyl isothiocyanate (ANIT). 42 In addition, CXC chemokines are shown to be involved in the process of transdifferentiation of fibroblasts 43 and endothelial cells. 44 Our data suggest that chemokines also may play a role in hepatocyte-to- BEC transdifferentiation. More definitive inhibition studies are needed to decipher their exact role in transdifferentiation. LY treatment abolished the up-regulation of the key genes modulated by the HGF EGF treatment as demonstrated by the gene array analysis such as Wnt4, -catenin, CXC chemokine LIX, cyclin D1, BMP6, TGF-, matrix metalloproteinase 9, osteopontin, and CD44, while it prevented the down-regulation of glutathione S-transferase Yb4, CYP1A1, CYP3A3, guanylate cyclase 2C, and alpha-2u globulin. 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