Cholangiopathies are a broad spectrum of disorders

Transcription

1 Activation of the Developmental Pathway Neurogenin-3/MicroRNA-7a Regulates Cholangiocyte Proliferation in Response to Injury Marco Marzioni, 1 Laura Agostinelli, 1 Cinzia Candelaresi, 1 Stefania Saccomanno, 1 Samuele De Minicis, 1 Luca Maroni, 1 Eleonora Mingarelli, 1 Chiara Rychlicki, 1 Luciano Trozzi, 1 Jesus M. Banales, 2 Antonio Benedetti, 1 and Gianluca Svegliati Baroni 1 The activation of the biliary stem-cell signaling pathway hairy and enhancer of split 1/ pancreatic duodenal homeobox-1 (Hes-1/PDX-1) in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and ductal cell neogenesis. PDX-1-dependent activation of Ngn-3 initiates the differentiation program by inducing microrna (mir)27 expression. Here we investigated the role Ngn-3 on cholangiocyte proliferation. Expression levels of Ngn-3 and mir-7 isoforms were tested in cholangiocytes from normal and cholestatic human livers. Ngn-3 was knocked-down in vitro in normal rat cholangiocytes by short interfering RNA (sirna). In vivo, wild-type and Ngn-3-heterozygous (1/2) mice were subjected to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding (a model of sclerosing cholangitis) or bile duct ligation (BDL). In the liver, Ngn-3 is expressed specifically in cholangiocytes of primary sclerosing cholangitis (PSC) patients and in mice subjected to DDC or BDL, but not in normal human and mouse livers. Expression of mir-7a-1 and mir-7a-2 isoforms, but not mir-7b, was increased in DDC cholangiocytes compared to normal ones. In normal rat cholangiocytes, sirna against Ngn-3 blocked the proliferation stimulated by exendin-4. In addition, Ngn-3 knockdown neutralized the overexpression of insulin growth factor-1 (IGF1; promitotic effector) observed after exposure to exendin-4, but not that of PDX-1 or VEGF-A/C. Oligonucleotides anti-mir-7 inhibited the exendin-4-induced proliferation in normal rat cholangiocytes, but did not affect Ngn-3 synthesis. Biliary hyperplasia and collagen deposition induced by DDC or BDL were significantly reduced in Ngn-3 1/2 mice compared to wild-type. Conclusion: Ngn-3- dependent activation of mir-7a is a determinant of cholangiocyte proliferation. These findings indicate that the reacquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes. (HEPATOLOGY 2014;60: ) Cholangiopathies are a broad spectrum of disorders that target cholangiocytes, the epithelial cells lining the biliary tree. 1 After injury, the biliary tree gains an unusual plasticity: cholangiocytes begin to proliferate and to synthesize and release a number of peptides, which are functional to the regulation of the cellular response to injury. 1 Such phenotypic and biological changes, defined as neuroendocrine-like transdifferentiation, 2 are thought to contribute significantly to disease progression and Abbreviations: asma, a smooth muscle actin; BDL, bile duct ligation; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; Hes-1, hairy and enhancer of split 1; mir, microrna; Ngn-3, neurogenin-3; NRC, normal rat cultured cholangiocytes; PCNA, proliferating cell nuclear antigen; PDX-1, pancreatic duodenal homeobox-1; PSC, primary sclerosing cholangitis. From the 1 Department of Gastroenterology and Hepatology, Universita Politecnica delle Marche, Ancona, Italy; 2 Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute Donostia University Hospital (HUD), IKERBASQUE, CIBERehd, UPV/EHU, San Sebastian, Spain. Received March 6, 2014; accepted June 5, Supported by an MIUR grant PRIN prot. 2009X84L84_003 and Ministero della Salute grant GR to Dr. Marzioni; by MIUR grant PRIN prot. 2009YNERCE_002, FIRB prot RBAP10MY35_001 to Dr. Svegliati Baroni. Dr. Banales was supported by the Spanish Ministry of Economy and Competitiveness (FIS PI12/00380) and the Spanish Association Against Cancer (AECC). 1324

2 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL perpetuation. The molecules released by proliferating cholangiocytes activate fibrogenetic and angiogenic processes, attract inflammatory cells, and favor carcinogenesis. 2 Such a chain of events is a typical pathologic response observed in several cholangiopathies, 1 in particular if cholangiocyte proliferation is intense, such as in primary sclerosing cholangitis (PSC). 3 Unfortunately, the molecular mechanisms that govern cholangiocyte biological response to injury are still under study. The basic helix-loop-helix transcription factor neurogenin-3 (Ngn-3) initiates the endocrine differentiation of the pancreas through the activation of microrna (mir) Ngn-3 is required not only for endocrine cell maturation, but also for maintaining their function and neogenesis after injury in adulthood. 7,8 Interestingly, Ngn-3 contributes to pancreas regeneration by way of ductal cell neogenesis and transdifferentiation. 6 Genetic lineage-tracing studies showed that Ngn-3 activity is controlled by pancreatic duodenal homeobox-1 (PDX-1), 4,6,9 which we have recently characterized as a major determinant of cholangiocyte biological response to injury. 10,11 Interestingly, both PDX-1 and Ngn-3 are expressed by the stem cells of the peribiliary glands. 12 The purpose of this study was to evaluate whether Ngn-3 regulates cholangiocyte biological response to experimental injury. Thus, we aimed to answer the following questions: 1) Is Ngn-3 expressed in proliferating cholangiocytes? 2) Does Ngn-3 affect cholangiocyte proliferation and regulate the synthesis of neuropeptides? 3) Are Ngn-3 effects mediated by mir-7 isoforms? 4) Does Ngn-3 affect the development and progression of liver injury in sclerosing cholangitis? Materials and Methods Expression of Ngn-3 in Cholangiocytes. The expression of Ngn-3 was evaluated by 1) immunohistochemistry in liver sections from patients affected by PSC (n 5 3) or congenital hepatic fibrosis (CHF, n 5 1) and in normal human liver (n 5 1), 2) real-time polymerase chain reaction (PCR) and immunoblots in cultured rat cholangiocytes and in cholangiocytes isolated both from normal mice and from mice fed 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 8 weeks, as a model of sclerosing cholangitis. 13 Purification of cholangiocytes from mice was performed by immune-bead purification, as previously reported. 10 Role of Ngn-3 in Cholangiocyte Proliferation and in Vascular Endothelial Growth Factor (VEGF) and IGF-1 Synthesis. Normal rat cholangiocytes (NRC) in culture, a self-maintaining, nontransfected, stable cell line, were exposed for 48 hours to sirnas against Ngn-3, or to a corresponding nontargeting (NT) RNA, in the absence or presence of exendin-4 (100 nmol/l), a selective agonist of the glucagon-like peptide-1 receptor (GLP-1R), as described. 10,11 We have previously shown that the activation of GLP-1R in cholangiocytes induces PDX-1 expression, thus stimulating proliferation and the neuroendocrine-like transdifferentiation of cholangiocytes. 10,11 Changes in cell proliferation were measured by employing the cell proliferation enzyme-linked immunosorbent assay (ELISA) bromodeoxyuridine (BrdU) assays according to the instructions provided by the vendor (Roche, Monza, Italy) 11 or by immunoblotting for proliferating cell nuclear antigen (PCNA). Changes in VEGF (A and C isoforms) and IGF-1 mrna expression were assessed by real-time PCR, as previously reported. 10 To test if PDX-1 activates Ngn-3 in cholangiocytes and to establish a potential interaction with the Notch effector Hes-1, 14 NRC were exposed for 48 hours to sirnas against Ngn-3 or PDX-1 or to corresponding NT RNAs, in the absence or presence of exendin-4 (100 nmol/l), as described above; changes in PDX-1 or Ngn-3 mrna levels were then assessed by realtime PCR. 10,11 Expression of mir-7 Isoforms in Cholangiocytes and Role in Ngn-3 Signaling. The expression of the mature mir-7 isoforms (mir-7a-1, mir-7a-2, and mir- 7b) was assessed by real-time PCR in cholangiocytes isolated from normal mice and from mice that received 8 weeks DDC; in addition, mir-7 isoforms were also assessed in NRC exposed to increasing concentrations of exendin-4 (1-100 nmol/l, 1 lmol/l) for 24 hours. To verify whether changes in mir-7 isoforms expression are dependent on Ngn-3 activation, NRC were exposed to sirnas against Ngn-3 or to a corresponding NT RNA, as described above; changes in Address reprint requests to: Marco Marzioni, M.D., Assistant Professor, Department of Gastroenterology, Universita Politecnica delle Marche, Nuovo Polo Didattico, III piano, Via Tronto 10, Ancona, Italy. m.marzioni@univpm.it; fax: Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Nothing to report.

3 1326 MARZIONI ET AL. HEPATOLOGY, October 2014 Fig. 1. Expression of Ngn-3 by reactive cholangiocytes. (A) Ngn-3 mrna (top) and protein expression (bottom) were significantly increased in cholangiocytes of 8-week DDC-treated mice, as compared to controls; *P < 0.05 versus normal mouse. (B) Normal human liver was negative for Ngn-3 immunohistochemistry (top); immunohistochemistry showed a positive staining for Ngn-3 in cholangiocytes (arrows) in liver sections from patients affected by PSC (middle and bottom). (C) Real-time PCR (top) and immunoblotting (bottom) showed increased expression of Ngn-3 in cultured cholangiocytes exposed to either exendin-4 or 10% FBS. Data are mean 6 SE of at least 3 experiments; *P < 0.05 versus basal. mir-7 isoforms expression were then assessed by realtime PCR. To verify whether mir-7 isoforms mediate the effects of Ngn-3 signaling, NRC were exposed for 36 hours to specific oligos anti-mir-7 (anti-mir-7a-5p) or to negative control oligos, in the absence or presence of exendin-4 (100 nmol/l). Changes in cell proliferation were then assessed as indicated above. Changes in Ngn- 3 expression were also assessed by real-time PCR, as described above and previously reported. 11 Effect of the Lack of Ngn-3 on Cholangiocyte Proliferation In Vivo. Adult (8 weeks) Ngn-3 1/2 or wild-type CD-1 mice were subjected to either 0.1% DDC feeding for 8 weeks or to bile duct ligation (BDL) for 2 weeks (n 5 8 per experimental group). Changes in cholangiocyte proliferation were determined by measuring differences in intrahepatic (small or large) bile duct mass (by quantitative immunohistochemistry for CK-19 and PCNA in liver sections and immunoblotting in whole liver lysates). 10,11,15 Changes in collagen deposition were assessed by quantitative histochemistry for Sirius-Red and immunohistochemistry for a smooth muscle actin (asma). 10,11 Animal study protocols were performed in compliance with local institution guidelines. Changes in IGF-1, VEGF-A/C, or Ngn-3 expression were also assessed in cholangiocytes isolated from the different experimental groups. Statistical Analysis. Data are expressed as mean 6 SE. Data obtained from the in vitro experiments are expressed as percent of basal value. The 95% confidence interval (CI) was calculated. Differences between groups were analyzed by analysis of variance (ANOVA). Differences between groups were considered significant when P < Results Ngn-3 Is Expressed in Proliferating Cholangiocytes. Both experimental and human conditions of sclerosing cholangitis were associated with Ngn-3 expression in

4 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL Fig. 2. Effects of Ngn-3 silencing in cultured cholangiocytes exposed to exendin-4. (A) Knockdown of Ngn-3 neutralized the increases in cell proliferation measured by BrdU incorporation (top) and PCNA protein expression (bottom). (B,C) Knockdown of Ngn-3 abolished the increases in IGF-1, but not in VEGF-A/C, mrna expression levels. (D) Knockdown of Ngn-3 abolished the reduction of Hes-1 induced by exendin-4, while it had no effect on PDX-1 expression. (E) Knockdown of PDX-1 neutralized the increase in Ngn-3 expression induced by exendin-4. Data are mean- 6 SE of at least 3 experiments; *P < 0.05 versus corresponding basal value. cholangiocytes. As shown by real-time PCR and immunoblotting, Ngn-3 mrna expression was markedly increased in cholangiocytes isolated from 8-week DDCtreated mice (Fig. 1A). Similarly, cholangiocytes were positive in immunostainings for Ngn-3 in sections from livers of patients affected by PSC (Fig. 1B) and in CHF (Supporting Fig. 1), but not in normal human liver. Those findings were reproducible in vitro: exposure of cultured cholangiocytes to either 10% fetal bovine serum (FBS) or GLP-1 receptor agonist exendin-4, which are pro-proliferative stimuli, 10,16 increased Ngn- 3 expression (Fig. 1C), assessed by both real-time PCR and immunoblotting. Ngn-3 Regulates Cholangiocyte Proliferation and IGF-1 Synthesis In Vitro. To verify whether Ngn-3 may affect cholangiocyte proliferation and synthesis of promitotic peptides, we measured the changes in cell growth and in IGF-1 or VEGF-A/C mrna levels in cultured cholangiocytes in the absence or presence of a specific sirnas against Ngn-3. When cultured cholangiocytes were exposed to exendin-4 (100 nmol/l), the increases in cell proliferation (measured by BrdU incorporation or PCNA protein expression) were not detected in cells Ngn-3 knocked-down with specific sirnas, as compared to cells exposed to NT sirna (Fig. 2A). Similarly, Ngn-3 knockdown neutralized the increases in IGF-1, but not VEGF-A/C, mrna levels stimulated by exendin-4 (Fig. 2B,C, respectively). The knockdown of Ngn-3 with sirnas prevented the decrease in Hes-1 mrna expression induced by exendin-4 but did not affect the exendin-4-stimulated PDX-1 expression (Fig. 2D); in contrast, the increases in Ngn-3 were neutralized when PDX-1 was knockeddown by sirnas (Fig. 2E). mir-7a, but not mir-7b, Mediates the Effects in Cholangiocytes. To study a possible role of mir-7 isoforms as mediators of Ngn-3 signal in cholangiocytes, we first evaluated their expression levels in those experimental conditions in which Ngn-3 was found

5 1328 MARZIONI ET AL. HEPATOLOGY, October 2014 Fig. 3. Expression of mir-7 isoforms in cholangiocytes and role in the Ngn-3 signaling. (A) Real-time PCR showed an increased expression of mir-7a-1 and mir-7a-2, but not mir-7b, in cholangiocytes isolated from 8-week DDC-treated mice, as compared to controls (right). mir-7a was also increased in cultured cholangiocytes exposed to exendin-4 or 10% FBS (middle); Ngn-3 knockdown neutralized the increases in mir-7a expression induced by exendin-4 (left). (B) Inhibition of mir-7 activity by a specific antisense neutralized the exendin-4-induced increases in cell proliferation, measured by BrdU incorporation (left) and PCNA protein expression (right). (C) Inhibition of mir-7 activity had no effect on Ngn-3 expression, measured by real-time PCR. Data are mean 6 SE of at least 3 experiments; *P < 0.05 versus the corresponding basal value; P < 0.05 versus basal. activated. As shown in Fig. 3A (left), the expression of mir-7a-1, and mir-7a-2 to a lower extent, was increased in cholangiocytes isolated from DDC-fed mice. In contrast, no changes were observed in mir- 7b expression. In vitro, exposure of cultured cholangiocytes to exendin-4 was associated with progressive increases in mir-7a expression (Fig. 3A, middle), thus paralleling the changes in Ngn-3 in the same experimental setup (Fig. 1). To verify that changes in mir-7a expression are Ngn-3-dependent, we assessed the changes in mir-7a expression in cells knocked-down for Ngn-3 with specific sirnas. The increases in mir-7a levels elicited by exendin-4 were no longer evident in cells exposed to the sirna for Ngn-3, as compared to cells exposed to NT sirna (Fig. 3A, right). To demonstrate that mir-7a mediates the effect of Ngn-3 in cholangiocytes, we exposed cultured cholangiocytes to specific anti-mir-7 oligos. mir-7 antisense oligos neutralized the cell proliferation stimulated by exendin-4 (Fig. 3B), but not the increase in Ngn-3 mrna expression (Fig. 3C). Ngn-3 Regulates Cholangiocyte Proliferation and Collagen Deposition in Mouse Models of Sclerosing Cholangitis. To verify whether Ngn-3 plays a role in the regulation of cholangiocyte proliferation in vivo, we reproduced two different mouse models of experimental sclerosing cholangitis in Ngn-3 1/2 mice. The increases in bile duct mass and cholangiocyte proliferation induced by 8 weeks DDC feeding were significantly reduced in Ngn-3 1/2 mice. Interestingly, the decrease in cell proliferation was particularly evident for small cholangiocytes, the subpopulation that expanded more after DDC (Fig. 4; Supporting Fig. 2A). Reduction of cholangiocyte proliferation in Ngn- 3 1/2 mice was associated with decreases in collagen deposition and in the number of asma-positive cells (Fig. 5).

6 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL Fig. 4. Role of Ngn-3 in cholangiocyte proliferation in the 8-week DDC model of sclerosing cholangitis. (A) Increases in bile duct mass (immunohistochemistry for CK-19) were significantly reduced in DDC-treated Ngn-3 1/2 mice compared to wild-type. Left: Representative images of CK- 19 immunostaining (203). Right: Quantitative analysis of CK-19 immunostaining (top) and percentage of small and large bile ducts in each experimental group (bottom). (B) Increases in cholangiocytes proliferation (immunohistochemistry for PCNA) were significantly reduced in DDCtreated, Ngn-3 1/2 mice as compared to wild-type animals. Left: Representative images of PCNA staining in cholangiocytes (arrows) (403). Right: Quantitative analysis of PCNA staining in large and small bile ducts. Data are mean 6 SE. *P < 0.05 versus corresponding value of wildtype control diet; #P < 0.05 versus corresponding value of wild-type DDC diet. Similar findings were obtained in 2-week BDL Ngn- 3 1/2 mice, as compared to wild-type mice (Figs. 6, 7; and Supporting Fig. 2B). However, and in contrast to DDC, the decrease in cell proliferation observed in Ngn-3 1/2 mice was particularly evident for large cholangiocytes, the subpopulation that expanded more after BDL. The increases in IGF-1 and Ngn-3 expression induced in cholangiocytes by DDC feeding or BDL

7 1330 MARZIONI ET AL. HEPATOLOGY, October 2014 Fig. 5. Role of Ngn-3 in collagen deposition and fibroblast activation in the 8-week DDC model of sclerosing cholangitis. (A) Collagen deposition (Sirius Red staining) and (B) fibroblast activation (immunohistochemistry for asma) were significantly reduced in DDC-treated, Ngn-3 1/2 mice compared to wild-type animals. Left: Representative images of Sirius Red (upper part; positive in red) and asma (lower part; positive in brown) staining in the different experimental groups (203). Right: Quantitative analysis of Sirius Red and asma staining. Data are mean 6 SE. *P < 0.05 versus wild-type control diet; #P < 0.05 versus wild-type DDC diet. were significantly reduced in Ngn-3 1/2 mice, whereas no differences were observed for VEGF-A/C (Fig. 8). Discussion The current study shows that: 1) Ngn-3 is expressed in proliferating cholangiocytes in experimental models of sclerosing cholangitis and in livers of PSC patients; 2) in vitro, Ngn-3 is a determinant of cholangiocyte proliferation and synthesis of promitotic peptides, such as IGF-1; 3) Ngn-3 is activated by PDX-1 and its effects are mediated by mir-7 isoforms; 4) the lack of Ngn-3 reduces cholangiocyte proliferation and collagen deposition in animal models of sclerosing cholangitis. Cell reprogramming and transdifferentiation are opening new paths in the field of cell-based therapy

8 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL Fig. 6. Role of Ngn-3 in cholangiocyte proliferation in 2-week BDL model of sclerosing cholangitis. (A) Increases in bile duct mass (immunohistochemistry for CK-19) were significantly reduced in BDL, Ngn-3 1/2 mice as compared to wild-type animals. Left: Representative images of CK-19 immunostaining (203). Right: Quantitative analysis of CK-19 immunostaining (top) and percentage of small and large bile ducts in each experimental group (bottom). (B) Increases in cholangiocytes proliferation (immunohistochemistry for PCNA) were significantly reduced in BDL, Ngn-3 1/2 mice as compared to wild-type animals. Left: Representative images of PCNA immunostaining (positive in brown) (403). Right: Quantitative analysis of PCNA immunostaining in large and small bile ducts. Data are mean 6 SE. *P < 0.05 versus corresponding value of wild-type control; #P < 0.05 versus corresponding value of wild-type 2 wk BDL. and organ regeneration. 17 This is of particular interest for organs affected by chronic disorders, for which transplantation is the sole effective therapy. 18 A potential application of those new approaches requires a detailed understanding of the mechanisms leading to organ development and their contributions in response to injury. In recent years it has been shown that hepatic stem cells and biliary tree stem cells participate in organ development and are a possible source for cell replacement in liver diseases. 12,19 Cells of the stem compartment display a hepatopancreatic commitment and may transdifferentiate towards hepatic, biliary, or pancreatic

9 1332 MARZIONI ET AL. HEPATOLOGY, October 2014 Fig. 7. Role of Ngn-3 in collagen deposition and fibroblast activation in the 2-week BDL model of sclerosing cholangitis. (A) Collagen deposition (Sirius Red staining) and (B) fibroblast activation (immunohistochemistry for asma) were significantly reduced in BDL, Ngn-3 1/2 mice as compared to wild-type animals. Left: Representative images of Sirius Red (upper part; positive in red) and asma (lower part; positive in brown) staining in the different experimental groups (203). Right: Quantitative analysis of Sirius Red and asma staining. Data are mean 6 SE. *P < 0.05 versus wild-type control; #P < 0.05 versus wild-type 2-week BDL. lineages. 20 Maturation towards the hepatic or pancreatic commitment is thought to depend on the modulation of different transcription factors, such as SOX9, SOX19, PDX-1, and Ngn-3. 12,19 Ngn-3 is a basic helix-loop-helix transcription factor that is critical for the development of the endocrine pancreas. 4-6 It functions primarily as an enhancer of the expression of lineage-committed transcription factors required for the differentiation of the endocrine progenitor cells to mature endocrine subtypes. 6 Disruption of Ngn-3 expression in the course of pancreas development abrogates islet formation. 6 Similarly, Ngn-3-positive precursors modulate the development of pancreatic ducts. 21 Interestingly, Ngn-3 signaling is recapitulated in adulthood, when it drives pancreatic regeneration and cell proliferation in response to injury. 7,22 Of note, Ngn-3 is up-regulated in proliferating pancreatic ducts, 22 which can contribute to b-cell

10 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL Fig. 8. Expression of IGF-1, VEGF-A/C, and Ngn-3 in cholangiocytes isolated from in vivo models of sclerosing cholangitis. (A) In the 8-week DDC model, the induction of IGF-1 mrna expression (left), Ngn-3 mrna (middle) and protein expression (right) stimulated by the DDC diet in wild-type mice were significantly reduced in DDC-treated, Ngn-3 1/2 mice. There were no differences in the expression of VEGF-A/C between wild-type and Ngn-3 1/2 mice treated with DDC (right). (B) Similar findings could be reproduced in the 2-week BDL model. *P < 0.05 versus the corresponding value of the wild-type control; #P < 0.05 versus the corresponding value of DDC (or BDL) wild-type. formation. 6 In the stomach, Ngn-3 is essential both for the differentiation of enteroendocrine cells and for the maintenance of the gastric epithelial cell identity. 23 In this study we demonstrated that Ngn-3 has a role in the biological response of cholangiocytes to injury. Similar to pancreatic cells, 22 Ngn-3 is hyperexpressed in proliferating cholangiocytes, both in vitro and in vivo (Fig. 1). Ngn-3 immunostaining showed positive expression in reactive cholangiocytes in livers of PSC patients, thus highlighting the activation of the Ngn-3 signaling in human disease. Cholangiocyte proliferation is driven both by the activation of intracellular signals and by the release of promitotic peptides by proliferating cells, which act in an autocrine fashion. 2 When Ngn-3 synthesis was blocked with specific sirnas, the increases in cell proliferation and IGF-1 synthesis were no longer evident (Fig. 2). Thus, Ngn-3 elicits cholangiocyte proliferation both directly and by inducing promitotic peptide synthesis. Genetic lineage-tracing studies indicate that Ngn-3 is downstream of PDX-1, a master regulator of pancreatic development and response to injury. 4,6,9,24 We have recently found that PDX-1 is also a determinant of cholangiocyte proliferation and transdifferentiation in cholestatic conditions, 10,11 being thus similar to that observed for Ngn-3. To verify if Ngn-3 expression is downstream of PDX-1 activation, we assessed the changes in PDX-1 mrna levels in cultured cholangiocytes transiently silenced for Ngn-3 expression. As shown in Fig. 2D, the increases in PDX-1 synthesis were not affected by Ngn-3 knockdown. In contrast, knockdown of PDX-1 neutralized the increases in Ngn-3 expression (Fig. 3E). Those data confirm that Ngn-3 activation in cholangiocytes is dependent on PDX-1, as well as what happens in pancreas. 4,9 Interestingly, the PDX-1 and Ngn-3 concerted and simultaneous action induces the transdifferentiation of epithelial cells towards a neuroendocrine phenotype. 25 mirnas act in concert with transcription factors to modulate gene expression and affect developmental transitions. 5 In the pancreas, mirnas regulate stem cell lineage differentiation and cell function. 26 To this

11 1334 MARZIONI ET AL. HEPATOLOGY, October 2014 regard, a major role is played by mir-7, which is activated by Ngn-3. 5 Interestingly, mir-7 is also expressed in pancreatic epithelial cells. 28 In this study we showed that mir-7a is hyperexpressed in proliferating cholangiocytes and it mediates the Ngn-3 effects. As shown in Fig. 3, inhibition of mir-7 activity does not affect Ngn-3 synthesis but neutralizes its promitotic effect. In parallel, mir-7 synthesis was substantially absent in Ngn-3 knockdown cholangiocytes. In mammals, mir- 7 is encoded by three genomic loci, by which different isoforms originate, namely, mir-7a-1, mir-7a-2, and mir-7b. Studies have shown that the expression of those isoforms correlates with their activity. 5 Our data indicate that only mir-7a isoforms (mostly mir-7a-1), but not mir-7b are expressed in proliferating cholangiocytes (Fig. 3). These data are unique of the biliary epithelium, since in the pancreas all three mir-7 isoforms mediate the effects of Ngn-3. 5 To verify whether Ngn-3 may have a significant pathophysiological role in chronic liver injury, we used two different experimental models of sclerosing cholangitis in Ngn-3 1/2 mice. Figures 4, 6, and 8 show that cholangiocyte proliferation and IGF-1 synthesis in those models were markedly reduced in Ngn-3 1/2 mice as compared to wild-type mice. In particular, the lack of Ngn-3 affected the biliary subpopulation mostly involved in the biological response, which were large cholangiocytes in the BDL and small cholangiocytes in the DDC model. 2,13 Cholangiocyte maturation may start from hepatic stem cells located in the limiting plate/canals of Hering. 19 Since the DDC model is also characterized by a prominent expansion of the stem cell compartment, 29 our findings thus cannot rule out, but rather support, a possible role of Ngn-3 1/2 for progenitor cells of the liver and their specification towards the biliary fate. 12,19 A parallel decrease in collagen deposition was also observed in Ngn-3 1/2 mice (Figs. 5, 7). The expression of Ngn-3 in quiescent or activated hepatic stellate cells is absent or negligible (Supporting Fig. 3), especially if compared to cholangiocytes. Therefore, the differences in collagen deposition among the experimental groups can be ascribed to changes in cholangiocyte proliferation, 2 rather than to a possible effect of Ngn-3 on profibrogenic cells the liver. These data represent the first in vivo finding of the role of Ngn-3 in modulating an adult organ biological response to experimental injury. In particular, the data emerging from the DDC model are of interest, since this model recapitulates several of the features of chronic cholestatic conditions, such as cholangiocyte injury and proliferation, periductal fibrosis, and activation of the stem cell compartment. 3,13 These data support the concept that the transdifferentiation of the biliary epithelium in response to injury results from the balance between the Notch and the Ngn-3 signaling. Notch and Ngn-3 signaling are dynamically linked: Notch-1-mediated activation of Hes-1 determines Ngn-3 protein destabilization and limits endocrine commitment of cell fate. 21,30,31 On the other hand, Ngn-3 can extrinsically activate the Notch-1/ Hes-1 pathway and promote pancreatic duct formation, in a sort of negative feedback. 31 In the liver, the Notch/Hes-1 signaling is essential for the proper development of bile ducts, being necessary for cell specification towards the biliary fate. 32 In Hes-1- deficient mice, bile duct development is deranged and a biliary to pancreatic conversion is observed. 32 In the current study, we found that Hes-1 down-regulation is not detectable in Ngn-3 knockdown cells. Those data support our previous finding that, in the adult liver, proliferation and transdifferentiation of injured cholangiocytes occur upon Hes-1 down-regulation and PDX-1 activation. 10 Since Ngn-3 is the effector of PDX-1, altogether these data suggest the hypothesis that the fine-tuning of the counteracting Notch/Hes-1 and PDX-1/Ngn-3 pathways is key in the regulation of the cholangiocyte biological response to injury. Such a concept may help interpret the progression of liver injury in chronic cholestatic diseases. The neo-cholangiogenesis observed in those disorders is thought to represent an attempt to regenerate the damaged bile ducts. 2 On the other hand, proliferating new ducts are not functional and sustain the fibrogenesis. 2,33 We propose that Ngn-3 is the final modulator of the equilibrium between Notch, which favors the formation of functional bile ducts, and PDX-1 signaling, which favors cell proliferation and transdifferentiation. A possible inhibition of Ngn-3 activity may thus represent a target for molecular therapies that aim to control cholangiocyte proliferation and sustain the regeneration of fully functional bile ducts. In this regard, the identification of mir-7 isoforms as effectors of Ngn-3 signaling may be of interest. Several studies have clarified the potential of mirnas as biomarkers and therapeutic targets in human disease. 34,35 In summary, our study showed that activation of Ngn-3/miR-7 signaling is a major event for the regulation of cholangiocyte proliferation in response to experimental injury. These data open novel concepts to interpret the pathophysiology of chronic cholestatic conditions.

12 HEPATOLOGY, Vol. 60, No. 4, 2014 MARZIONI ET AL References 1. Lazaridis KN, Strazzabosco M, LaRusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 2004;127: Alvaro D, Mancino MG, Glaser S, Gaudio E, Marzioni M, Francis H, et al. Proliferating cholangiocytes: a neuroendocrine compartment in the diseased liver. Gastroenterology 2007;132: Eaton JE, Talwalkar JA, Lazaridis KN, Gores GJ, Lindor KD. Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management. Gastroenterology 2013;145: Johansson KA, Dursun U, Jordan N, Gu G, Beermann F, Gradwohl G, et al. Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types. Dev Cell 2007;12: Kredo-Russo S, Ness A, Mandelbaum AD, Walker MD, Hornstein E. Regulation of pancreatic microrna-7 expression. Exp Diabetes Res 2012;2012: Rukstalis JM, Habener JF. Neurogenin3: a master regulator of pancreatic islet differentiation and regeneration. Islets 2009;1: Van de Casteele M, Leuckx G, Baeyens L, Cai Y, Yuchi Y, Coppens V, et al. Neurogenin 31 cells contribute to beta-cell neogenesis and proliferation in injured adult mouse pancreas. Cell Death Dis 2013;4:e Wang S, Jensen JN, Seymour PA, Hsu W, Dor Y, Sander M, et al. Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function. Proc Natl Acad Sci U S A 2009;106: Oliver-Krasinski JM, Kasner MT, Yang J, Crutchlow MF, Rustgi AK, Kaestner KH, et al. The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice. J Clin Invest 2009;119: Marzioni M, Saccomanno S, Agostinelli L, Rychlicki C, De Minicis S, Pierantonelli I, et al. PDX-1/Hes-1 interactions determine cholangiocyte proliferative response to injury in rodents: possible implications for sclerosing cholangitis. J Hepatol 2013;58: Marzioni M, Saccomanno S, Candelaresi C, Rychlicki C, Agostinelli L, Shanmukhappa K, et al. Pancreatic duodenal homeobox-1 de novo expression drives cholangiocyte neuroendocrine-like transdifferentiation. J Hepatol 2010;53: Cardinale V, Wang Y, Carpino G, Cui CB, Gatto M, Rossi M, et al. Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets. HEPATOLOGY 2011;54: Fickert P, Stoger U, Fuchsbichler A, Moustafa T, Marschall HU, Weiglein AH, et al. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. Am J Pathol 2007;171: Strazzabosco M, Fabris L. Development of the bile ducts: essentials for the clinical hepatologist. J Hepatol 2012;56: Mancinelli R, Franchitto A, Glaser S, Meng F, Onori P, Demorrow S, et al. GABA induces the differentiation of small into large cholangiocytes by activation of Ca(21) /CaMK I-dependent adenylyl cyclase 8. HEPATOLOGY 2013;58: Marzioni M, Alpini G, Saccomanno S, Candelaresi C, Venter J, Rychlicki C, et al. Glucagon-like peptide-1 and its receptor agonist exendin-4 modulate cholangiocyte adaptive response to cholestasis. Gastroenterology 2007;133: Guo J, Wang H, Hu X. Reprogramming and transdifferentiation shift the landscape of regenerative medicine. DNA Cell Biol 2013;32: Orlando G, Soker S, Stratta RJ. Organ bioengineering and regeneration as the new Holy Grail for organ transplantation. Ann Surg 2013;258: Cardinale V, Wang Y, Carpino G, Mendel G, Alpini G, Gaudio E, et al. The biliary tree a reservoir of multipotent stem cells. Nat Rev Gastroenterol Hepatol 2012;9: Wang Y, Lanzoni G, Carpino G, Cui CB, Dominguez-Bendala J, Wauthier E, et al. Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis. Stem Cells 2013;31: Magenheim J, Klein AM, Stanger BZ, Ashery-Padan R, Sosa-Pineda B, Gu G, et al. Ngn3(1) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium. Dev Biol 2011;359: Xiao X, Chen Z, Shiota C, Prasadan K, Guo P, El-Gohary Y, et al. No evidence for beta cell neogenesis in murine adult pancreas. J Clin Invest 2013;123: Lee CS, Perreault N, Brestelli JE, Kaestner KH. Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev 2002;16: Deramaudt TB, Sachdeva MM, Wescott MP, Chen Y, Stoffers DA, Rustgi AK. The PDX1 homeodomain transcription factor negatively regulates the pancreatic ductal cell-specific keratin 19 promoter. J Biol Chem 2006;281: Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 2008;455: Nieto M, Hevia P, Garcia E, Klein D, Alvarez-Cubela S, Bravo-Egana V, et al. Antisense mir-7 impairs insulin expression in developing pancreas and in cultured pancreatic buds. Cell Transplant 2012;21: Dumortier O, Van Obberghen E. MicroRNAs in pancreas development. Diabetes Obes Metab 2012;14(Suppl 3): Correa-Medina M, Bravo-Egana V, Rosero S, Ricordi C, Edlund H, Diez J, et al. MicroRNA mir-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas. Gene Expr Patterns 2009;9: Jakubowski A, Ambrose C, Parr M, Lincecum JM, Wang MZ, Zheng TS, et al. TWEAK induces liver progenitor cell proliferation. J Clin Invest 2005;115: Baeyens L, Bonne S, Bos T, Rooman I, Peleman C, Lahoutte T, et al. Notch signaling as gatekeeper of rat acinar-to-beta-cell conversion in vitro. Gastroenterology 2009;136: e Qu X, Afelik S, Jensen JN, Bukys MA, Kobberup S, Schmerr M, et al. Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment. Dev Biol 2013;376: Lozier J, McCright B, Gridley T. Notch signaling regulates bile duct morphogenesis in mice. PLoS One 2008;3:e Xia X, Demorrow S, Francis H, Glaser S, Alpini G, Marzioni M, et al. Cholangiocyte injury and ductopenic syndromes. Semin Liver Dis 2007;27: Vo DD, Staedel C, Zehnacker L, Benhida R, Darfeuille F, Duca M. Targeting the production of oncogenic micrornas with multimodal synthetic small molecules. ACS Chem Biol 2014 [Epub ahead of print]. 35. Dassow H, Aigner A. MicroRNAs (mirnas) in colorectal cancer: from aberrant expression towards therapy. Curr Pharm Des 2013;19: Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher s website.