Biliary organoids uncover delayed epithelial development and barrier function in biliary atresia.

BACKGROUND AND AIMS: Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. APPROACH AND RESULTS: We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, ß-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, ß-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. CONCLUSIONS: Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.

Loss of Anks6 leads to YAP deficiency and liver abnormalities.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Role of ß-catenin in development of bile ducts.

Beta-catenin is known to play stage- and cell-specific functions during liver development. However, its role in development of bile ducts has not yet been addressed. Here we used stage-specific in vivo gain- and loss-of-function approaches, as well as lineage tracing experiments in the mouse, to first demonstrate that ß-catenin is dispensable for differentiation of liver precursor cells (hepatoblasts) to cholangiocyte precursors. Second, when ß-catenin was depleted in the latter, maturation of cholangiocytes, bile duct morphogenesis and differentiation of periportal hepatocytes from cholangiocyte precursors was normal. In contrast, stabilization of ß-catenin in cholangiocyte precursors perturbed duct development and cholangiocyte differentiation. We conclude that ß-catenin is dispensable for biliary development but that its activity must be kept within tight limits. Our work is expected to significantly impact on in vitro differentiation of stem cells to cholangiocytes for toxicology studies and disease modeling.

Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.

The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and γ-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell-derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases.

Biliary atresia: A comprehensive review.

Biliary atresia presents as an obliterative cholangiopathy with neonatal jaundice and pale stools. The disease exhibits aetiological heterogeneity with a multiplicity of potential causative factors, both developmental and environmental. A number of clinical variants making up a minority of all cases can be defined relatively precisely which match suggested aetiology better although in most it still remains speculative. These include the syndromic form (BASM), the cystic form and those associated with CMV IgM antibodies. We review not only the clinical evidence for a developmental or an immune-mediated aetiology perhaps triggered by perinatal viral exposure but also several other recently suggested concepts such as microchimerism, gene susceptibility and environmental toxins.

Cholangiocytes derived from induced pluripotent stem cells for disease modeling.

PURPOSE OF REVIEW: Biliary diseases are a significant cause of morbidity and mortality. Challenges in establishing accurate in-vitro methods to model human bile duct diseases and evaluate therapies have contributed to a lack of effective medical treatments. The recent discovery of strategies to reprogram human somatic cells to a state of induced pluripotency has opened up new possibilities for studying both development and disease in a wide variety of human tissues. This review was undertaken to summarize the recent progress made in generating biliary tissue from induced pluripotent stem cells (iPSCs) and the application of this technology to biliary disease modeling. RECENT FINDINGS: Several groups have reported defined differentiation protocols that incorporate key signaling cues from normal biliary development to yield cholangiocyte-like cells from wild-type human iPSCs that demonstrate epithelial morphology in two and three-dimensional culture, cholangiocyte markers, biliary gene expression profiles, and functional attributes consistent with biliary epithelium. Key features of Alagille syndrome and polycystic liver disease can be modeled with iPSC-derived cholangiocytes, whereas the use of iPSCs from cystic fibrosis patients has facilitated not only modeling of cystic fibrosis biliary disease but also in-vitro correction of the disorder with pharmacological agents. SUMMARY: Mature, functional cholangiocytes can be derived from human iPSCs and utilized to model biliary diseases in vitro. These advances should facilitate further research to improve our understanding of the pathophysiology of cholangiopathies and evaluate novel treatments. In the future, this technology will likely form a key element of tissue replacement strategies.

Hepatic Notch2 deficiency leads to bile duct agenesis perinatally and secondary bile duct formation after weaning.

UNLABELLED: Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2(fl/fl)/Alfp-Cre(tg/-) ("Notch2-cKO") and Notch2(fl/fl)/Alfp-Cre(-/-) ("control") mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1ß, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation. CONCLUSION: Cholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.

Biliary atresia: will blocking inflammation tame the disease?

Biliary atresia is the most common cholangiopathy of childhood. With complete obstruction of segments or the entire length of extrahepatic bile ducts, the timely pursuit of hepatoportoenterostomy is the best strategy to restore bile drainage. However, even with prompt surgical intervention, ongoing injury of intrahepatic bile ducts and progressive cholangiopathy lead to end-stage cirrhosis. The pace of disease progression is not uniform; it may relate to clinical forms of disease and/or staging of liver pathology at diagnosis. Although the etiology of disease is not yet defined, several biological processes have been linked to pathogenic mechanisms of bile duct injury. Among them, there is increasing evidence that the immune system targets the duct epithelium and disrupts bile flow. We discuss how careful clinical phenotyping, staging of disease, and basic mechanistic research are providing insights into clinical trial designs and directions for development of new therapies to block progression of disease.

DNA hypomethylation causes bile duct defects in zebrafish and is a distinguishing feature of infantile biliary atresia.

UNLABELLED: Infantile cholestatic disorders arise in the context of progressively developing intrahepatic bile ducts. Biliary atresia (BA), a progressive fibroinflammatory disorder of extra- and intrahepatic bile ducts, is the most common identifiable cause of infantile cholestasis and the leading indication for liver transplantation in children. The etiology of BA is unclear, and although there is some evidence for viral, toxic, and complex genetic causes, the exclusive occurrence of BA during a period of biliary growth and remodeling suggests an importance of developmental factors. Interestingly, interferon-γ (IFN-γ) signaling is activated in patients and in the frequently utilized rhesus rotavirus mouse model of BA, and is thought to play a key mechanistic role. Here we demonstrate intrahepatic biliary defects and up-regulated hepatic expression of IFN-γ pathway genes caused by genetic or pharmacological inhibition of DNA methylation in zebrafish larvae. Biliary defects elicited by inhibition of DNA methylation were reversed by treatment with glucocorticoid, suggesting that the activation of inflammatory pathways was critical. DNA methylation was significantly reduced in bile duct cells from BA patients compared to patients with other infantile cholestatic disorders, thereby establishing a possible etiologic link between decreased DNA methylation, activation of IFN-γ signaling, and biliary defects in patients. CONCLUSION: Inhibition of DNA methylation leads to biliary defects and activation of IFN-γ-responsive genes, thus sharing features with BA, which we determine to be associated with DNA hypomethylation. We propose epigenetic activation of IFN-γ signaling as a common etiologic mechanism of intrahepatic bile duct defects in BA.

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPß.

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPß. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPß, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.

DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome.

Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.

Many essential parts of the body contain tubes: the liver for example, contains bile ducts and blood vessels. These tubes develop right next to each other, like entwined trees. To do their jobs, these ducts must communicate and collaborate, but they do not always grow properly. For example, babies with Alagille syndrome are born with few or no bile ducts, resulting in serious liver disease. Understanding the architecture of the tubes in their livers could explain why some children with this syndrome improve with time, but many others need a liver transplant. Visualising biological tubes in three dimensions is challenging. One major roadblock is the difficulty in seeing several tubular structures at once. Traditional microscopic imaging of anatomy is in two dimensions, using slices of tissue. This approach shows the cross-sections of tubes, but not how the ducts connect and interact. An alternative is to use micro computed tomography scans, which use X-rays to examine structures in three dimensions. The challenge with this approach is that soft tissues, which tubes in the body are made of, do not show up well on X-ray. One way to solve this is to fill the ducts with X-ray absorbing resins, making a cast of the entire tree structure. The question is, can two closely connected tree structures be distinguished if they are cast at the same time? To address this question, Hankeova, Salplachta et al. developed a technique called double resin casting micro computed tomography, or DUCT for short. The approach involved making casts of tube systems using two types of resin that show up differently under X-rays. The new technique was tested on a mouse model of Alagille syndrome. One resin was injected into the bile ducts, and another into the blood vessels. This allowed Hankeova, Salplachta et al. to reconstruction both trees digitally, revealing their length, volume, branching, and interactions. In healthy mice, the bile ducts were straight with uniform branches, but in mice with Alagille syndrome ducts were wiggly, and had extra branches in the centre of the liver. This new imaging technique could improve the understanding of tube systems in animal models of diseases, both in the liver and in other organs with tubes, such as the lungs or the kidneys. Hankeova, Salplachta et al. also lay a foundation for a deeper understanding of bile duct recovery in Alagille syndrome. In the future, DUCT could help researchers to see how mouse bile ducts change in response to experimental therapies.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells.

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

RhoA promotes differentiation of WB-F344 cells into the biliary lineage.

When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells in which RhoA expression was upregulated. To further investigate the role of RhoA in WB cell differentiation initiated by Matrigel treatment, we constructed constitutively active RhoA-expressing vectors and stably transfected them into WB-F344 cells. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopically active RhoA expression were found to form bile-duct-like structures even without Matrigel treatment. Besides, ROCK inhibitor Y27632 treatment eliminated luminal morphogenesis. F-actin cytoplasmic staining further verified that the RhoA-ROCK signal pathway was involved in differentiation of WB cells into the biliary lineage. In conclusion, our results suggested that the RhoA-ROCK-stress fibre system plays an obligatory role in Matrigel-induced WB-F344 cell luminal morphogenesis and further differentiation.

Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro.

Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.

Transcriptional profiling implicates TGFbeta/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts.

Bile duct morphogenesis involves sequential induction of biliary specific gene expression, bilayer generation, cell proliferation, remodeling and apoptosis. HBC-3 cells are a model system to study differentiation of hepatoblasts along the hepatocytic or bile ductular lineage in vitro and in vivo. We used microarray to define molecular pathways during ductular differentiation in response to Matrigel. The temporal pattern of expression of marker genes induced was similar to that observed during bile duct formation in vivo. Notch, HNF1beta, Polycystic kidney disease 2, Bicaudal C 1 and beta-catenin were up regulated during the time course. Functional clustering analysis revealed significant up regulation of clusters of genes involved in extracellular matrix remodeling, ion transport, vacuoles, lytic vacuoles, pro-apoptotic and anti-apoptotic genes, transcription factors and negative regulators of the cell proliferation, while genes involved in the cell cycle were significantly down regulated. Notch signaling pathway was activated by treatment with Matrigel. In addition, TGFbeta/BMP signaling pathway members including the type I TGFbeta receptor and Smads 3, 4 and 5 were significantly up regulated, as were several TGFbeta/BMP responsive genes including Hey 1, a regulator of Notch pathway signaling. SMADS 3, 4 and 5 were present in the nuclear fraction of HBC-3 cells during ductular differentiation in vitro, but not during hepatocyte differentiation. SMAD 5 was preferentially expressed in hepatoblasts undergoing bile duct morphogenesis in the fetal liver, while the TGFbeta/BMP signaling antagonist chordin, was expressed throughout the liver suggesting a mechanism by which TGFbeta/BMP signaling is limited to hepatoblasts that contact portal mesenchyme in vivo.

Cell lineages and oval cell progenitors in rat liver development.

We determined whether the formation of the hepatic primordium in the rat is associated with the expression of liver-specific markers. Further, we examined the origin of intra- and extrahepatic bile ducts and tried to establish whether there are cell types in the developing liver that might correspond to "stem-like" cells ("oval cells") that proliferate during carcinogenesis and toxic injury in adult livers. Using in situ hybridization and immunohistochemical methods, we show that alpha-fetoprotein (AFP) mRNA is detected in cells of the ventral foregut at 10.5 days of development and that the protein is first detected 1 day later. Thus, AFP transcription occurs before liver morphogenesis, and translation of the protein is first detected when liver cords are being formed, indicating that AFP expression in endodermal cells signals their commitment toward the liver lineage. Although albumin is considered a trait of differentiated hepatocytes, its mRNA was first detected just 1 day later than the AFP message. An analysis of the expression of lineage-specific cytokeratins (cytokeratins 7, 9, 18, and 19), surface markers, and histochemical determination of gamma-glutamyl transferase activity and glycogen revealed that (a) hepatoblasts undergo gradual maturation throughout liver development, (b) AFP- and albumin-containing hepatoblasts gave rise to intra- and extrahepatic bile ducts, and (c) hepatoblasts forming primitive intrahepatic bile ducts during liver development have markers similar to those expressed by stem-like cells that proliferate during liver carcinogenesis.

The delta opioid receptor 1 is expressed by proliferating bile ductules in rats with cholestasis: implications for the study of liver regeneration and malignant transformation of biliary epithelium.

In sharp contrast with the normal adult liver, the fetal human and rat livers and the liver of rats with cholestasis secondary to bile duct resection express the preproenkephalin mRNA, which codes for the endogenous opioid peptide Met-enkephalin. Furthermore, Met-enkephalin immunoreactivity is detected in hepatocytes and in proliferating bile ductules in the cholestatic rat liver. These data suggest that in cholestasis endogenous opioids may have a local effect in the cholestatic liver. As endogenous opioids exert their effect by binding to opioid receptors, the presence of opioid receptors in the cholestatic livers would support the hypothesis that Met-enkephalin plays a role in situ. Preliminary data presented in this manuscript reveals the expression of the delta opioid receptor in the liver of rats with cholestasis. This finding suggests that there is a scenario in which Met-enkephalin can bind to opioid receptors in the liver in cholestasis to exert a local effect. In vivo studies in this model of cholestasis with the use of opioid agonist and antagonist will shed light on the possible role of opioidergic regulation of liver regeneration. Studies on the effect of opiate antagonists on the evolution of cholestasis in this animal model may provide insight into the mechanisms of liver regeneration. In addition, as some conditions associated with cholestasis and bile ductular proliferation can be complicated by malignancy, the expression of the delta opioid receptor in malignant tumors of the biliary tree merits research.

Induction of MIC-1/growth differentiation factor-15 following bile duct injury.

Macrophage inflammatory peptide-1 (MIC-1)/growth/differentiation factor-15 (GDF-15) is a divergent member of the transforming growth factor-beta superfamily cloned by others and us. MIC-1/GDF-15 is expressed in the liver, breast, and colon. Studies have demonstrated a growth-inhibiting effect of MIC-1/GDF-15 on colon and breast cancer cell lines in vitro and on tumor growth in vivo. We previously reported that MIC-1 expression is rapidly induced after a wide variety of murine acute and chronic liver injuries including aniline dye administration. I hypothesized, therefore, that MIC-1/GDF-15 may be a mediator of biliary tract injury and could play a role in regulation of bile duct proliferation. C57BL/6 mice underwent surgical ligation of the common bile duct. Northern blot analysis revealed a time-dependent induction of MIC-1/GDF-15 mRNA in the liver. In situ hybridization of liver sections for MIC-1/GDF-15 expression after bile duct ligation demonstrated a zone 1 or periportal expression pattern, consistent with expression of MIC-1 in periductular hepatocytes. Northern blot analysis of liver mRNA from patients with sclerosing cholangitis or cirrhosis also demonstrated enhanced expression of MIC-1/GDF-15. MIC-1/GDF-15 is expressed after bile duct injury in mice and humans. Taken together with the previously demonstrated growth inhibitory effects of MIC-1/GDF-15 on normal and transformed cells, MIC-1/GDF-15 may play a role in regulation of bile duct proliferation and biliary tumor formation.

Bile duct enlargement by infusion of L-proline: potential significance in biliary atresia.

The normal signals of control of bile duct morphogenesis and growth are unknown. Consequently, aberrant development is poorly understood, as for example in those infants with biliary atresia or hypoplasia. A previous report by Isseroff et al., showed that a possible mechanism for the common bile duct enlargement by the parasite Fasciola hepatica was excess L-proline secreted by this parasite. This suggested to us that L-proline might be a specific growth factor for bile duct epithelium, and therefore possibly useful in the understanding and treatment of biliary maldevelopment. We now report an experimental study demonstrating the effect of L-proline on the normal biliary tree of the mouse. We also present a summary to date of an ongoing clinical restrospective study. These experimental findings suggest: (1) there may be a specific requirement for L-proline during the rapid growth of the biliary tract during neonatal life; (2) bile duct hypoplasia and atresia may result from a defect in proline metabolism; (3) proline administration during the early phase of biliary atresia may possibly be therapeutic.