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Intrahepatic cholangiocarcinoma (ICC) is a devastating liver cancer with extremely high intra- and inter-tumoral molecular heterogeneity, partly due to its diverse cellular origins. We investigated clinical relevance and the molecular mechanisms underlying hepatocyte (HC)-driven ICC development.Expression of ICC driver genes in human diseased livers at risk for ICC development were examined. The sleeping beauty and hydrodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SRY-box transcription factor 9 (SOX9) and yes-associated protein 1 (YAP1) in HC-driven ICC. We identified DNA methyltransferase 1 (DNMT1) as a YAP1 target, which was validated by loss- and gain-of-function studies, and its mechanism addressed by chromatin immunoprecipitation sequencing.Co-expression of AKT and Notch intracellular domain (NICD)/YAP1 in HC yielded ICC that represents 13% to 29% of clinical ICC. NICD independently regulates SOX9 and YAP1 and deletion of either, significantly delays ICC development. Yap1 or TEAD inhibition, but not Sox9 deletion, impairs HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate switch through the repression of HC-specific genes regulated by master regulators for HC differentiation, including hepatocyte nuclear factor 4 alpha, hepatocyte nuclear factor 1 alpha, and CCAAT/enhancer-binding protein alpha/beta. DNMT1 loss prevented NOTCH/YAP1-dependent HC-driven cholangiocarcinogenesis, and DNMT1 re-expression restored ICC development following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in a subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development.We identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-to-BEC/ICC conversion, which may be relevant in cholestasis-to-ICC pathogenesis in the clinic.

YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/β-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and β-Catenin cascades in iCCA.Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, β-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine β-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or β-Catenin. Immunostaining of total and nonphosphorylated/activated β-Catenin staining was performed in mouse and human iCCAs.We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, β-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by β-Catenin. Importantly, activated/nonphosphorylated β-Catenin was detected in more than 80% of human iCCAs.YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with β-Catenin. β-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and β-Catenin pathways in cholangiocarcinogenesis.

BACKGROUND AND AIMS: Following mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood. APPROACH AND RESULTS: By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process through the FXR-PTEN (phosphatase and tensin homolog)–PI3K (phosphoinositide 3-kinase)–AKT-mTOR (mammalian target of rapamycin) axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation. CONCLUSIONS: FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given the clinical trials and use of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic.

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Background and Aims: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC‐to‐hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. Approach and Results: Here, using zebrafish models of LPC‐mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC‐to‐hepatocyte differentiation through the mitogen‐activated ERK kinase (MEK)‐extracellular signal‐regulated kinase (ERK)–sex‐determining region Y‐box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC‐to‐hepatocyte differentiation as well as genetic suppression of the EGFR‐ERK‐SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short‐term treatment with EGFR inhibitors resulted in better liver recovery over the long term. Conclusions: The EGFR‐ERK‐SOX9 axis suppresses LPC‐to‐hepatocyte differentiation during LPC‐mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/β-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation., The liver possesses the ability to regenerate following sudden injury. Here, the authors use single-cell RNA-sequencing and in situ transcriptional analyses to identify a new phase of liver regeneration in mice aimed at maintaining essential functions throughout the regenerative process.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of β-catenin, one with β-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of β-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel β-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF., eLife digest The liver has an incredible capacity to repair itself or ‘regenerate’ – that is, it has the ability to replace damaged tissue with new tissue. In order to do this, the organ relies on hepatocytes (the cells that form the liver) and bile duct cells (the cells that form the biliary ducts) dividing and transforming into each other to repair and replace damaged tissue, in case the insult is dire. During long-lasting or chronic liver injury, bile duct cells undergo a process called ‘ductular reaction’, which causes the cells to multiply and produce proteins that stimulate inflammation, and can lead to liver scarring (fibrosis). Ductular reaction is a hallmark of severe liver disease, and different diseases exhibit ductular reactions with distinct features. For example, in cystic fibrosis, a unique type of ductular reaction occurs at late stages, accompanied by both inflammation and fibrosis. Despite the role that ductular reaction plays in liver disease, it is not well understood how it works at the molecular level. Hu et al. set out to investigate how a protein called β-catenin – which can cause many types of cells to proliferate – is involved in ductular reaction. They used three types of mice for their experiments: wild-type mice, which were not genetically modified; and two strains of genetically modified mice. One of these mutant mice did not produce β-catenin in biliary duct cells, while the other lacked β-catenin both in biliary duct cells and in hepatocytes. After a short liver injury – which Hu et al. caused by feeding the mice a specific diet – the wild-type mice were able to regenerate and repair the liver without exhibiting any ductular reaction. The mutant mice that lacked β-catenin in hepatocytes showed a temporary ductular reaction, and ultimately repaired their livers by turning bile duct cells into hepatocytes. On the other hand, the mutant mice lacking β-catenin in both hepatocytes and bile duct cells displayed sustained ductular reactions, inflammation and fibrosis, which looked like that seen in patients with liver disease associated to cystic fibrosis. Further probing showed that β-catenin interacts with a protein called CTFR, which is involved in cystic fibrosis. When bile duct cells lack either of these proteins, another protein called NF-B gets activated, which causes the ductular reaction, leading to inflammation and fibrosis. The findings of Hu et al. shed light on the role of β-catenin in ductular reaction. Further, the results show a previously unknown interaction between β-catenin, CTFR and NF-B, which could lead to better treatments for cystic fibrosis in the future.

Liver regeneration after most forms of injury is mediated through the proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) arising from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC-to-hepatocyte differentiation in patients with chronic liver disease could serve as a potentially new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development and has also been implicated in regulating LPC differentiation during regeneration. SRY-related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown in a zebrafish model of LPC-driven liver regeneration that inhibition of Hdac1 activity through MS-275 treatment enhances sox9b expression in LPCs and impairs LPC-to-hepatocyte differentiation. Therefore, we hypothesized that inhibition of Notch signaling would promote LPC-to-hepatocyte differentiation by repressing sox9b expression in zebrafish. We ablated the hepatocytes of Tg(fabp10a:CFP-NTR) larvae and blocked Notch activation during liver regeneration through treatment with γ-secretase inhibitor LY411575 and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC-specific Sox9b overexpression impaired LPC-to-hepatocyte differentiation. Our results establish the Notch-Sox9b signaling axis as inhibitory to LPC-to-hepatocyte differentiation in a well-established in vivo LPC-driven liver regeneration model.

Intrahepatic cholangiocarcinoma (ICC), a disease of poor prognosis, has increased in incidence. It is challenging to treat due to intra- and inter-tumoral heterogeneity, which in part is attributed to diverse cellular origin. Indeed, co-expression of AKT and NICD in hepatocytes (HCs) yielded ICC, with similarity to proliferative, Notch-activated, and stem cell-like subclasses of clinical ICC. NICD regulated SOX9 and YAP1 during ICC development. Yap1 deletion or TEAD inhibition impaired HC-to-biliary epithelial cell (BEC) reprogramming and ICC proliferation; Sox9 loss repressed tumor growth; and Yap1-Sox9 combined loss abolished ICC development in AKT-NICD model. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directed HC-to-BEC/ICC fate-switch. DNMT1 loss prevented Notch-dependent HC-to-ICC development, and DNMT1 re-expression restored ICC development following TEAD repression. Coexpression of DNMT1 with AKT was sufficient to induce hepatic tumor development including ICC. Thus, we have identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-driven ICC development.SIGNIFICANCEWe evaluated the clinical relevance of hepatocyte-driven ICC model and revealed critical but distinct roles of YAP1 and SOX9 in AKT-NICD-driven hepatocyte-derived ICC. We also identified NOTCH-YAP1/TEAD-DNMT1 axis as a critical driver for hepatocyte-to-ICC reprogramming, which might have biological and therapeutic implications in ICC subsets.

SummaryYAP1 regulates cell plasticity during liver injury, regeneration and cancer, but its role in liver development is unknown. YAP1 activity was detected in biliary cells and in cells at the hepato-biliary bifurcation in single-cell RNA-sequencing analysis of developing livers. Hepatoblast deletion ofYap1led to no impairment in Notch-driven SOX9+ ductal plate formation, but prevented the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, the mice survived for 8 months with severe cholestatic injury and without any hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggested extrahepatic biliary proliferation likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurred through hepatocyte adaptation via reduced metabolic and synthetic function including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

After liver injury, regeneration manifests as either (1) hepatocytes proliferating to restore the lost hepatocyte mass or (2) if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) dedifferentiating into liver progenitor cells (LPCs), which subsequently differentiate into hepatocytes. Following pharmacogenetic ablation of hepatocytes in Tg(fabp10a:CFP-NTR) zebrafish, resulting in severe liver injury, signal transducer and activator of transcription 3 (Stat3) and its target gene and negative regulator, socs3a, were upregulated in regenerating livers. Using either Stat3 inhibitors, JSI-124 and S3I-201, or stat3 zebrafish mutants, we investigated the role of Stat3 in LPC-driven liver regeneration. Although Stat3 suppression reduced the size of regenerating livers, BEC dedifferentiation into LPCs was unaffected. However, regenerating livers displayed a delay in LPC-to-hepatocyte differentiation and a significant reduction in the number of BECs. While no difference in cell death was detected, Stat3 inhibition significantly reduced LPC proliferation. Notably, stat3 mutants phenocopied the effects of Stat3 chemical inhibitors, although the mutant phenotype was incompletely penetrant. Intriguingly, a subset of socs3a mutants also displayed a lower number of BECs in regenerating livers. We conclude that the Stat3/Socs3a pathway is necessary for the proper timing of LPC-to-hepatocyte differentiation and establishing the proper number of BECs during LPC-driven liver regeneration.

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae-derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δomp43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae. To study the differences in proteomic expression between WT and Δomp43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tldD, efp, ntrX, pdhA, purB, and ATPA mRNA expression and decreases in Rho and yfeA mRNA expression were confirmed in Δomp43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.

The liver is a complex organ performing numerous vital physiological functions. For that reason, it possesses immense regenerative potential. The capacity for repair is largely attributable to the ability of its differentiated epithelial cells, hepatocytes and biliary epithelial cells, to proliferate after injury. However, in cases of extreme acute injury or prolonged chronic insult, the liver may fail to regenerate or do so suboptimally. This often results in life-threatening end-stage liver disease for which liver transplantation is the only effective treatment. In many forms of liver injury, bipotent liver progenitor cells are theorized to be activated as an additional tier of liver repair. However, the existence, origin, fate, activation, and contribution to regeneration of liver progenitor cells is hotly debated, especially since hepatocytes and biliary epithelial cells themselves may serve as facultative stem cells for one another during severe liver injury. Here, we discuss the evidence both supporting and refuting the existence of liver progenitor cells in a variety of experimental models. We also debate the validity of developing therapies harnessing the capabilities of these cells as potential treatments for patients with severe and chronic liver diseases.

Following massive hepatocyte ablation in zebrafish, biliary epithelial cells can extensively give rise to hepatocytes through liver progenitor cells (LPCs). The zebrafish liver injury model is an important system to elucidate the molecular mechanisms underlying LPC-driven liver regeneration. Here, we describe a chemical screening method using the zebrafish model for identifying small molecules that can modulate LPC-driven liver regeneration.

SUMMARY Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifur-cation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes., Graphical Abstract, In brief Molina et al. demonstrate that loss of Yap1 in developing livers leads to absence of intrahepatic bile ducts, preventing bile secretion from liver to small intestines. Yap1 knockout mice are small and jaundiced and exhibit adaptation via altering bile acid transport and metabolism, but they eventually die around 8 months of age.

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.

Bromodomain and extraterminal (BET) proteins recruit key components of basic transcriptional machinery to promote gene expression. Aberrant expression and mutations in BET genes have been identified in many malignancies. Small molecule inhibitors of BET proteins such as JQ1 have shown efficacy in preclinical cancer models, including affecting growth of hepatocellular carcinoma. BET proteins also regulate cell proliferation in nontumor settings. We recently showed that BET proteins regulate cholangiocyte-driven liver regeneration. Here, we studied the role of BET proteins in hepatocyte-driven liver regeneration in partial hepatectomy (PHx) and acetaminophen-induced liver injury models in mice and zebrafish. JQ1 was injected 2 or 16 hours after PHx in mice to determine effect on hepatic injury, regeneration, and signaling. Mice treated with JQ1 after PHx displayed increased liver injury and a near-complete inhibition of hepatocyte proliferation. Levels of Ccnd1 mRNA and Cyclin D1 protein were reduced in animals injected with JQ1 16 hours after PHx and were even further reduced in animals injected with JQ1 2 hours after PHx. JQ1-treated zebrafish larvae after acetaminophen-induced injury also displayed notably impaired hepatocyte proliferation. In both models, Wnt signaling was prominently suppressed by JQ1. Our results show that BET proteins regulate hepatocyte proliferation-driven liver regeneration, and Wnt signaling is particularly sensitive to BET protein inhibition.

Summary Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-??-catenin signaling. Activating ??-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between ??-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were ??-catenin-mutated liver tumors. Genetic disruption of ??-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in ??-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-??-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are ??-catenin mutated and GS positive.

Background & Aims Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. Methods We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of β-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). Results Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of β-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. Conclusions Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Background & Aims During liver regeneration, hepatocytes are derived from pre-existing hepatocytes. However, if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) become the source of new hepatocytes. We recently reported on a zebrafish liver regeneration model in which BECs extensively contribute to hepatocytes. Using this model, we performed a targeted chemical screen to identify important factors that regulate BEC-driven liver regeneration, the mechanisms of which remain largely unknown. Methods Using Tg(fabp10a:CFP-NTR) zebrafish, we examined the effects of 44 selected compounds on BEC-driven liver regeneration. Liver size was assessed by fabp10a: DsRed expression; liver marker expression was analyzed by immunostaining, in situ hybridization and quantitative PCR. Proliferation and apoptosis were also examined. Moreover, we used a mouse liver injury model, choline-deficient, ethionine-supplemented (CDE) diet. Results We identified 10 compounds that affected regenerating liver size. Among them, only bromodomain and extraterminal domain (BET) inhibitors, JQ1 and iBET151, blocked both Prox1 and Hnf4a induction in BECs. BET inhibition during hepatocyte ablation blocked BEC dedifferentiation into hepatoblast-like cells (HB-LCs). Intriguingly, after JQ1 washout, liver regeneration resumed, indicating temporal, but not permanent, perturbation of liver regeneration by BET inhibition. BET inhibition after hepatocyte ablation suppressed the proliferation of newly generated hepatocytes and delayed hepatocyte maturation. Importantly, Myca overexpression, in part, rescued the proliferation defect. Furthermore, oval cell numbers in mice fed CDE diet were greatly reduced upon JQ1 administration, supporting the zebrafish findings. Conclusions BET proteins regulate BEC-driven liver regeneration at multiple steps: BEC dedifferentiation, HB-LC proliferation, the proliferation of newly generated hepatocytes, and hepatocyte maturation.

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.

North Korea is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and South Korea, they have not been reported from North Korea. To screen for zoonotic tick-borne pathogens in North Korea, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis (H.) longicornis were collected and assayed individually for selected tick-borne pathogens. A total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella (B.) grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%) based on 16S ribosomal RNA and ITS species-specific nested polymerase chain reaction. Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae, respectively. All products were sequenced and demonstrated 100% identity and homology with previously reported sequences from other countries in GenBank. This is the first report of the detection of tick-borne pathogens in the North Korea and suggests that farm animals may act as reservoirs for zoonotic tick-borne pathogens.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

The aim of this study was to investigate the characteristics of canine uropathogenic Escherichia coli (UPEC) and the interaction between canine UPEC and human bladder epithelial cells. Ten E. coli isolates collected from dogs with cystitis were analyzed for antimicrobial resistance patterns, the presence of virulence factors, and biofilm formation. The ability of these isolates to induce cytotoxicity, invade human bladder epithelial cells, and stimulate an immune response was also determined. We observed a high rate of antimicrobial resistance among canine UPEC isolates. All virulence genes tested (including adhesins, iron acquisition, and protectin), except toxin genes, were detected among the canine UPEC isolates. We found that all isolates showed varying degrees of biofilm formation (mean, 0.26; range, 0.07 to 0.82), using a microtiter plate assay to evaluate biofilm formation by the isolates. Cytotoxicity to human bladder epithelial cells by the canine UPEC isolates increased in a time-dependent manner, with a 56.9% and 36.1% reduction in cell viability compared with the control at 6 and 9 h of incubation, respectively. We found that most canine UPEC isolates were able to invade human bladder epithelial cells. The interaction between these isolates and human bladder epithelial cells strongly induced the production of proinflammatory cytokines such as IL-6 and IL-8. We demonstrated that canine UPEC isolates can interact with human bladder epithelial cells, although the detailed mechanisms remain unknown. The results suggest that canine UPEC isolates, rather than dogspecific pathogens, have zoonotic potential.

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).

The prevalence of tick-borne encephalitis virus (TBEV) in southern Korea was determined by collecting ticks using tick drags. A total of 4,077 of 6,788 ticks collected were pooled (649 pools) according to collection site, species, and developmental stage and assayed for TBEV. The TBEV protein E and NS5 gene fragments were detected using RT-nested PCR in six pools of nymphs collected from Jeju Island (2,491 ticks). The minimum field detection rates for TBEV were 0.17% and 0.14% for Haemaphysalis longicornis and Haemayphysalis. flava nymphs, respectively. The 252 bp NS5 and 477 bp protein E gene amplicons were sequenced. Phylogenetic analysis showed that the NS5 and protein E genes of the Jeju strain were clustered with Western subtype (98.0% and 99.4% identity, respectively). The Western subtype of TBEV is endemic in Korea, including Jeju Island. The study of vector and zoonotic host susceptibility to TBEV is required to better understand its potential impact on public health.