Evidence supporting a role for EMT in repair of chronic liver injury is growing (14
). The current study demonstrates that activation of the Hh pathway promotes EMT in cholangiocytes. The Hh-regulated transcription factor Gli2 was demonstrated in nuclei of ductular cells that coexpressed epithelial and mesenchymal markers in the livers of humans with chronic biliary fibrosis. Hh pathway activity and markers of EMT also increased in the livers of rats when biliary fibrosis was induced by BDL. Primary cholangiocytes isolated from such rats exhibited both Hh pathway activation (as evidenced by downregulation of the Hh inhibitor Hhip
and upregulation of Ptc and Gli2
, Hh target genes), and gene expression changes typical of cells undergoing EMT. Moreover, when placed in Transwell cocultures with Hh-producing MF-HSCs or treated with conditioned medium from these cells, immature cholangiocytes acquired a migratory phenotype and increased expression of various mesenchymal markers, as well as several factors that promote EMT, while concomitantly repressing expression of epithelial markers and EMT inhibitors. These effects were abrogated by adding Hh-neutralizing antibodies to the MF-HSC–conditioned medium, demonstrating that paracrine Hh signaling induces EMT in cultured cholangiocytes. A similar process likely regulates EMT in vivo, as gene expression changes of EMT and liver fibrogenesis were amplified after BDL in Ptc mice that have an impaired ability to turn off Hh signaling following biliary injury (54
). Together, these findings support the concept that EMT contributes to the pathogenesis of biliary fibrosis and identify the Hh pathway as an important mediator of this response.
Our results in liver cells are consistent with the acknowledged actions of Hh signaling in other systems. For example, the Hh pathway is known to regulate EMT during fetal development, and this process is required for migration of Hh-responsive cells during tissue morphogenesis (48
). Cell migration is also a key feature of cancer invasion and metastasis, and Hh signaling has been shown to play a pivotal role in cancer cell EMT (29
). Hh pathway activation has also been shown to be necessary for invasion/migration of nonmalignant cells in adults. For example, progesterone-mediated induction of Hh ligand production by uterine stromal cells is required for embryo implantation (61
). Hh signaling also controls the migration of basal epidermal cells to more superficial aspects of adult skin (63
). Thus, the new evidence for Hh-mediated induction of EMT in some bile duct epithelial cells during chronic biliary injury suggests that migration of these cells might be involved in remodeling of adult livers with cholestatic damage.
The proximal branches of the intrahepatic biliary tree house the progenitor compartment in adult livers (42
), and mobilization of cells from this compartment occurs during the regeneration of chronically injured livers (65
). Liver epithelial progenitors are heterogeneous (66
). This is thought to reflect their derivation from more primitive, bipotent progenitors that are capable of differentiating along either the hepatocytic or biliary lineages (42
). Some of the bipotent progenitors have been shown to exhibit fibroblast-like features that are stabilized by treatment with the profibrogenic cytokine TGF-β. The immediate progeny of these bipotent progenitors also exhibit considerable plasticity, as TGF-β treatment of such immature hepatocytic cells in culture causes them to revert to the fibroblastic phenotype (i.e., undergo EMT). Other manipulations induce mesenchymal-epithelial transitions (MET), by which the fibroblastic cells differentiate into bile ductular or hepatocytic cells (68
). Assuming that immature liver cells behave similarly when confronted with TGF-β during fibrosing liver injury, one would predict that EMT occurs in certain types of liver cells when the liver microenvironment becomes enriched with TGF-β and subsides as TGF-β levels decline. Our data in the BDL/R-Y rat model of reversible cholestatic liver damage support this concept.
In non-liver cells, the mechanisms by which TGF-β induces EMT are being delineated, and emerging evidence demonstrates crosstalk with the Hh signaling pathway at several levels. TGF-β1 induces EMT by initiating ALK5-dependent events that lead to activation of Smad-3 and eventual repression of E-cadherin by factors such as Snail and Twist (22
). Recently, Hh ligand-receptor interactions that result in Smo activation were shown to enhance this process in gastric cancer cells (25
). Smo activation results in nuclear localization of Gli family transcription factors that are known to induce expression of both Snail and Twist (22
). Hence, when TGF-β is present, canonical Hh pathway activation stimulates expression of factors that promote EMT by downregulating the epithelial adhesion molecule E-cadherin (69
). Recently, we reported that TGF-β1 treatment induces expression of Hh ligands in liver cells (71
), identifying a mechanism that helps to explain why both factors accumulate during liver fibrosis. TGF-β has also been shown to induce EMT via noncanonical (i.e., Hh ligand–independent) activation of Gli transcription factors (72
Our immunohistochemical analyses of liver samples from humans and rats with fibrosing cholestatic liver damage localize EMT to ductular cells with nuclear expression of Gli2, providing the first in vivo evidence to our knowledge that EMT in non-neoplastic adult liver cells is likely to proceed via mechanisms that drive this process in other cell types. During the onset and relief of biliary obstruction in our rat model, expression of collagen gene expression (37
) and EMT markers paralleled variations in Hh ligand expression. Moreover, RNA analysis of primary cholangiocytes from BDL rats demonstrated that these cells strongly downregulate Hhip
, an Hh ligand antagonist, when undergoing EMT. Therefore, in aggregate, our findings support a role for canonical (i.e., Hh ligand/receptor-initiated) Hh signaling in biliary EMT. This concept is further supported by the evidence for increased EMT following BDL in mice with haploinsufficiency of the Smo inhibitor Ptc, as well as data indicating that treatment with Hh-neutralizing antibodies prevented induction of EMT genes in cultured cholangiocytes. The demonstration that Hh signaling modulates EMT in bile ductular cells opens novel areas for research and has important diagnostic and therapeutic implications for patients with various types of chronic cholestatic liver disease.