Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans Alessia Omenetti, … , Detlef Schuppan, Anna Mae Diehl J Clin Invest. 2008;118(10):3331-3342. https://doi.org/10.1172/JCI35875. Research Article Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis. Find the latest version: https://jci.me/35875/pdf Related Commentary, page 3263 Research article Hedgehog signaling regulates epithelial- mesenchymal transition during biliary fibrosis in rodents and humans Alessia Omenetti,1 Alessandro Porrello,2 Youngmi Jung,1 Liu Yang,1 Yury Popov,3,4 Steve S. Choi,1 Rafal P. Witek,1 Gianfranco Alpini,5,6 Juliet Venter,6 Hendrika M. Vandongen,1 Wing-Kin Syn,1 Gianluca Svegliati Baroni,7 Antonio Benedetti,7 Detlef Schuppan,3 and Anna Mae Diehl1 1Division of Gastroenterology, Department of Medicine, Duke University Medical Center, and 2Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA. 3Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, USA. 4Laboratory of Liver Research, Department of Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany. 5Central Texas Veterans Health Care System, Temple, Texas, USA. 6Department of Medicine, Scott & White Hospital and Texas A&M Health Science Center College of Medicine, Temple, Texas, USA. 7Department of Gastroenterology, Università Politecnica delle Marche, Ancona, Italy. Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryo- genesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis. Introduction of Hh-responsive progenitor cells (30–33). Hh ligands are sol- Biliary fibrosis is an outcome of chronic biliary injury in both uble, lipid-modified morphogens that interact with patched humans and rodents (1). A number of different cell types are (Ptc), a membrane-spanning receptor on the surface of Hh- thought to contribute to matrix deposition during chronic cho- responsive cells. This ligand-receptor interaction prevents Ptc lestasis (1–5), including myofibroblastic hepatic stellate cells from inhibiting its coreceptor, smoothened (Smo). Activated (MF-HSCs) (6–8), portal fibroblasts (9–12), and fibrocytes derived Smo, in turn, initiates a series of intracellular events that cul- from bone marrow (13). Immunostaining of serial liver sections minate in activation and nuclear localization of glioblastoma from patients with primary biliary cirrhosis (PBC) recently demon- (Gli) family transcription factors. This promotes transcription strated expression of vimentin and other mesenchymal markers in of Hh-responsive genes, including several components of the proliferating bile ductules within fibrotic portal tracts, leading the Hh signaling pathway itself, such as Ptc, Gli1, and Gli2. Sig- authors to propose that epithelial-mesenchymal transition (EMT) naling is turned off when the level of Ptc exceeds that of Hh may also play a role in the pathogenesis of biliary fibrosis (14, 15). ligands, permitting “free” Ptc to interact with and inhibit Smo EMT has been implicated in repair of injury in other adult tissues, (34). Hh signaling is also abrogated by Hh-interacting pro- such as the kidney (16, 17). It is also an important mechanism tein (Hhip), a factor that binds to Hh ligands and blocks Hh for tissue morphogenesis during fetal development (18–20) and ligand–Ptc interactions (35, 36). tumor metastasis in adults (19–23). Hedgehog (Hh) family ligands Recently, we demonstrated hepatic accumulation of Hh regulate EMT during the latter circumstances (22, 24–29). ligands and activation of the Hh signaling pathway in the livers Hh signaling controls tissue construction and remodeling by of bile duct–ligated (BDL) rodents (37, 38) and patients with regulating the viability and migratory activity of various types PBC (39). In the present study we evaluated the hypothesis that the Hh pathway promotes EMT in adult bile ductular cells (i.e., cholangiocytes) by analyzing liver samples from patients and Nonstandard abbreviations used: Aqp-1, aquaporin-1; BDL, bile duct ligation/bile duct–ligated; Bmp7, bone morphogenetic protein 7; CK-7, cytokeratin-7; EMT, epi- rats with chronic biliary fibrosis and manipulating Hh pathway thelial-mesenchymal transition; Gli, glioblastoma; GO, gene ontology; Hh, hedgehog; activity in primary cholangiocytes, cholangiocyte cells lines, and Hhip, Hh-interacting protein; Hmga2, high-mobility group AT-hook 2; HSC, hepatic mice subjected to biliary injury. The findings, linking Hh path- stellate cell; Igf1r, insulin-like growth factor I receptor 1; MF-HSC, myofibroblastic hepatic stellate cell; PBC, primary biliary cirrhosis; Ptc, patched; QRT-PCR, quantita- way activation with EMT in all systems, strongly support our tive RT-PCR; R-Y, Roux-en-Y biliary-enteric anastomosis; Smo, smoothened. hypothesis and demonstrate a novel paracrine mechanism by Conflict of interest: The authors have declared that no conflict of interest exists. which induction of EMT may contribute to hepatic fibrogenesis Citation for this article: J. Clin. Invest. 118:3331–3342 (2008). doi:10.1172/JCI35875. during chronic cholestasis. The Journal of Clinical Investigation http://www.jci.org Volume 118 Number 10 October 2008 3331 research article Figure 1 Bile ductular cells express the EMT marker S100A4 in patients with PBC. S100A4 immunostaining in representative sections from control subjects (NL) undergoing resection of metastases from colorectal can- cer (A, inset) and patients with PBC (A). Immunostaining of PBC livers for S100A4 (B) and epithelial CK-7 (C) demonstrated colocalization of both markers in rare ductular cells (D). Ductular appearing cells that expressed CK-7 without or with S100A4 were counted in 20 fields at ×20 magnification in all controls and patients. Data are displayed as mean numbers of CK-7/S100A4 double-positive cells relative to num- bers of CK-7 single-positive cells per field (E) and mean fold differenc- es in numbers of CK7/S100A4 double-positive cells in PBC patients versus controls (F). **P < 0.005. Original magnification, ×100 (A), ×20 (A, inset), ×40 (B–D), ×100 (D, inset). Results (Figure 1C). Colocalization of CK-7 and S100A4 was rarely dem- EMT occurs in bile ductular cells of patients with PBC. Liver sections onstrated in ductular cells in controls and also occurred in fewer from control subjects without chronic liver disease who were than 20% of CK-7–positive ductular cells in PBC patients (Figure 1, undergoing resection of colorectal metastases and patients with D and E). However, PBC livers had about 12-fold more double- PBC were examined for expression of S100A4 (also called fibro- positive ductular cells than controls (Figure 1F), confirming an blast-specific protein–1 [FSP-1]), a marker of fibroblastic trans- earlier report (15) that EMT is induced during chronic cholestatic formation of epithelial cells (17, 20, 40, 41). Unlike control