Targeting of CYP2E1 by miRNAs in alcohol-induced intestine injury.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.

Rbpj deletion in hepatic progenitor cells attenuates endothelial responses in a mouse model of cholestatic liver disease.

Background and Aims: Since the role of hepatic progenitor cells (HPCs) constituting ductular reactions in pathogenesis remains ambiguous, we aimed to establish the in vivo cause-and-effect relationship between HPCs and angiogenesis, a process associated with chronic liver disease progression. We previously demonstrated that peritumoral ductules are associated with angiogenesis in liver tumors and forkhead box L1 (Foxl1)- expressing murine HPCs secrete angiogenic factors in vitro. Therefore, we hypothesized that HPCs are capable of remodeling the vascular microenvironment and this function of HPCs is dependent on recombination signal binding protein for immunoglobulin kappa J region (RBPJ), a key effector of the Notch signaling pathway. Approach and Results: We generated HPC-specific Rbpj conditional knockout mice using Foxl1-Cre and treated them with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet to induce cholestatic liver disease. Knockout mice displayed significant reduction of HPC proliferation and ductular reactions as well as attenuated vascular and fibrotic areas compared to control mice. Assessment of vascular endothelial growth factor A-positive areas in vivo and the effects of Rbpj shRNAs in vitro indicated that Rbpj knockout in HPCs reduces the total number of angiogenic factor-expressing cells rather than affecting angiogenic factor expression within HPCs. Single-nucleus RNA sequencing analysis indicated that conditional Rbpj knockout in HPCs induces transcriptional changes in endothelial cells and alters expression of genes involved in various functions of the endothelium. Conclusion: Our findings indicate that HPCs regulate endothelial responses to cholestatic liver disease and Rbpj deletion in HPCs attenuates these responses, identifying novel targets for modulating angiogenesis during disease progression.

Integrated analysis using ToppMiR uncovers altered miRNA- mRNA regulatory networks in pediatric hepatocellular carcinoma-A pilot study.

BACKGROUND: Pediatric hepatocellular carcinoma (HCC) is a group of liver cancers whose mechanisms behind their pathogenesis and progression are poorly understood. AIM: We aimed to identify alterations in the expression of miRNAs and their putative target mRNAs in not only tumor tissues of patients with pediatric HCC but also in corresponding non-tumorous background livers by using liver tissues without underlying liver disease as a control. METHODS AND RESULTS: We performed a small-scale miRNA and mRNA profiling of pediatric HCC (consisting of fibrolamellar carcinoma [FLC] and non-FLC HCC) and paired liver tissues to identify miRNAs whose expression levels differed significantly from control livers without underlying liver disease. ToppMiR was used to prioritize both miRNAs and their putative target mRNAs in a gene-annotation network, and the mRNA profile was used to refine the prioritization. Our analysis generated prioritized lists of miRNAs and mRNAs from the following three sets of analyses: (a) pediatric HCC versus control; (b) FLC versus control; and (c) corresponding non-tumorous background liver tissues from the same patients with pediatric HCC versus control. No liver disease liver tissues were used as the control group for all analyses. Many miRNAs whose expressions were deregulated in pediatric HCC were consistent with their roles in adult HCC and/or other non-hepatic cancers. Our gene ontology analysis of target mRNAs revealed enrichment of biological processes related to the sustenance and propagation of cancer and significant downregulation of metabolic processes. CONCLUSION: Our pilot study indicates that alterations in miRNA-mRNA networks were detected in not only tumor tissues but also corresponding non-tumorous liver tissues from patients with pediatric HCC, suggesting multi-faceted roles of miRNAs in disease progression. Our results may lead to novel hypotheses for future large-scale studies.

Mechanism of paracrine communications between hepatic progenitor cells and endothelial cells.

Hepatic progenitor cells (HPCs) are facultative tissue-specific stem cells lining reactive ductules, which are ubiquitously observed in chronic liver diseases and cancer. Although previous research mainly focused on their contribution to liver regeneration, it turned out that in vivo differentiation of HPCs into hepatocytes only occurs after extreme injury. While recent correlative evidence implies the association of HPCs with disease progression, their exact role in pathogenesis remains largely unknown. Our previous research demonstrated that HPCs expressing angiogenic paracrine factors accumulate in the peritumoral area and are positively correlated with the extent of intratumoral cell proliferation and angiogenesis in the livers of patients with liver cancer. Given the crucial roles of angiogenesis in liver disease progression and carcinogenesis, we aimed to test the hypothesis that HPCs secrete paracrine factors to communicate with endothelial cells, to determine molecular mechanisms mediating HPCs-endothelial interactions, and to understand how the paracrine function of HPCs is regulated. HPCs promoted viability and tubulogenesis of human umbilical vein endothelial cells (HUVECs) and upregulated genes known to be involved in angiogenesis, endothelial cell function, and disease progression in a paracrine manner. The paracrine function of HPCs as well as expression of colony stimulating factor 1 (CSF1) were inhibited upon differentiation of HPCs toward hepatocytes. Inhibition of CSF1 receptor partly suppressed the paracrine effects of HPCs on HUVECs. Taken together, our study indicates that inhibition of the paracrine function of HPCs through modulation of their differentiation status and inhibition of CSF1 signaling is a promising strategy for inhibition of angiogenesis during pathological progression.

ß-catenin cancer-enhancing genomic regions axis is involved in the development of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a disease that occurs in children and young adults. The development of FLC is associated with creation of a fusion oncoprotein DNAJB1-PKAc kinase, which activates multiple cancer-associated pathways. The aim of this study was to examine the role of human genomic regions, called cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs), in the development of FLC. Previous studies revealed that CEGRs/ALCDs are located in multiple oncogenes and cancer-associated genes, regularly silenced in normal tissues. Using the regulatory element locus intersection (RELI) algorithm, we searched a large compendium of chromatin immunoprecipitation-sequencing (ChIP) data sets and found that CEGRs/ALCDs contain regulatory elements in several human cancers outside of pediatric hepatic neoplasms. The RELI algorithm further identified components of the ß-catenin-TCF7L2/TCF4 pathway, which interacts with CEGRs/ALCDs in several human cancers. Particularly, the RELI algorithm found interactions of transcription factors and chromatin remodelers with many genes that are activated in patients with FLC. We found that these FLC-specific genes contain CEGRs/ALCDs, and that the driver of FLC, fusion oncoprotein DNAJB1-PKAc, phosphorylates ß-catenin at Ser675, resulting in an increase of ß-catenin-TCF7L2/TCF4 complexes. These complexes increase a large family of CEGR/ALCD-dependent collagens and oncogenes. The DNAJB1-PKAc-ß-catenin-CEGR/ALCD pathway is preserved in lung metastasis. The inhibition of ß-catenin in FLC organoids inhibited the expression of CEGRs/ALCDs-dependent collagens and oncogenes, preventing the formation of the organoid's structure. Conclusion: This study provides a rationale for the development of ß-catenin-based therapy for patients with FLC.

Cellular origins of regenerating liver and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the predominant primary cancer arising from the liver and is one of the major causes of cancer-related mortality worldwide. The cellular origin of HCC has been a topic of great interest due to conflicting findings regarding whether it originates in hepatocytes, biliary cells, or facultative stem cells. These cell types all undergo changes during liver injury, and there is controversy about their contribution to regenerative responses in the liver. Most HCCs emerge in the setting of chronic liver injury from viral hepatitis, fatty liver disease, alcohol, and environmental exposures. The injuries are marked by liver parenchymal changes such as hepatocyte regenerative nodules, biliary duct cellular changes, expansion of myofibroblasts that cause fibrosis and cirrhosis, and inflammatory cell infiltration, all of which may contribute to carcinogenesis. Addressing the cellular origin of HCC is the key to identifying the earliest events that trigger it. Herein, we review data on the cells of origin in regenerating liver and HCC and the implications of these findings for prevention and treatment. We also review the origins of childhood liver cancer and other rare cancers of the liver.

A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigate whether the neonatal liver provides a protumorigenic niche to HB development. APPROACH AND RESULTS: HB development was compared between orthotopic transplantation models established in postnatal day 5 (P5) and 60 (P60) mice (P5Tx and P60Tx models). Single-cell RNA-sequencing (sc-RNAseq) was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. CONCLUSIONS: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver that express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout (KO) in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in tumors from patients with metastatic HB, there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with prognosis of patients with HB among pediatric cancers. We demonstrated that the neonatal liver provides a prometastatic niche to HB development through the Cxcl1/Cxcr2 axis.

HDAC1-Dependent Repression of Markers of Hepatocytes and P21 Is Involved in Development of Pediatric Liver Cancer.

BACKGROUND & AIMS: Epigenetic regulation of gene expression plays a critical role in the development of liver cancer; however, the molecular mechanisms of epigenetic-driven liver cancers are not well understood. The aims of this study were to examine molecular mechanisms that cause the dedifferentiation of hepatocytes into cancer cells in aggressive hepatoblastoma and test if the inhibition of these mechanisms inhibits tumor growth. METHODS: We have analyzed CCAAT/Enhancer Binding Protein alpha (C/EBPα), Transcription factor Sp5, and histone deacetylase (HDAC)1 pathways from a large biobank of fresh hepatoblastoma (HBL) samples using high-pressure liquid chromatography-based examination of protein-protein complexes and have examined chromatin remodeling on the promoters of markers of hepatocytes and p21. The HDAC1 activity was inhibited in patient-derived xenograft models of HBL and in cultured hepatoblastoma cells and expression of HDAC1-dependent markers of hepatocytes was examined. RESULTS: Analyses of a biobank showed that a significant portion of HBL patients have increased levels of an oncogenic de-phosphorylated-S190-C/EBPα, Sp5, and HDAC1 compared with amounts of these proteins in adjacent regions. We found that the oncogenic de-phosphorylated-S190-C/EBPα is created in aggressive HBL by protein phosphatase 2A, which is increased within the nucleus and dephosphorylates C/EBPα at Ser190. C/EBPα-HDAC1 and Sp5-HDAC1 complexes are abundant in hepatocytes, which dedifferentiate into cancer cells. Studies in HBL cells have shown that C/EBPα-HDAC1 and Sp5-HDAC1 complexes reduce markers of hepatocytes and p21 via repression of their promoters. Pharmacologic inhibition of C/EBPα-HDAC1 and Sp5-HDAC1 complexes by Suberoylanilide hydroxamic acid (SAHA) and small interfering RNA-mediated inhibition of HDAC1 increase expression of hepatocyte markers, p21, and inhibit proliferation of cancer cells. CONCLUSIONS: HDAC1-mediated repression of markers of hepatocytes is an essential step for the development of HBL, providing background for generation of therapies for aggressive HBL by targeting HDAC1 activities.

Adeno-Associated Virus Serotype 8-Mediated Genetic Labeling of Cholangiocytes in the Neonatal Murine Liver.

Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. It has been widely accepted that transduction of hepatocytes using adeno-associated virus serotype 8 (AAV8) is a promising approach to correct inborn errors in neonates, but the type of neonatal hepatic cells transduced by AAV8 has not been thoroughly investigated. To address this question, we used a reporter mouse that carries Cre recombinase (Cre)-inducible yellow fluorescent protein (YFP). Our analysis primarily focused on cholangiocytes, given their pivotal roles in normal liver function and disease. We treated RosaYFP/+ mice at postnatal day 2 (P2) with AAV8-cytomegalovirus (CMV) promoter-Cre and analyzed livers at P10 and P56. The vast majority of HNF4α+ hepatocytes were labeled with YFP at both time points, and 11.6% and 24.4% of CK19+ cholangiocytes were marked at P10 and P56, respectively. We also detected YFP+ cells devoid of hepatocyte and cholangiocyte markers, and a subset of these cells expressed the endothelial and fibroblast marker CD34. Next, we used the hepatocyte-specific thyroxine-binding globulin (TBG) promoter. Surprisingly, AAV8-TBG-Cre marked 6.8% and 30.9% of cholangiocytes at P10 and P56, respectively. These results suggest that AAV8 can be a useful tool for targeting cholangiocytes in neonatal livers.

Reactive Ductules Are Associated With Angiogenesis and Tumor Cell Proliferation in Pediatric Liver Cancer.

While reactive ductules (RDs) have been observed in viral hepatitis, biliary atresia, nonalcoholic fatty liver disease, and adult hepatocellular carcinoma (HCC), RDs in pediatric liver cancer remain uncharacterized. This study investigated the relationship of RDs with angiogenic paracrine factors, the extent of angiogenesis, and tumor cell proliferation in pediatric hepatoblastoma (HBL)/HCC livers. We quantified the extent of RDs and their expression of paracrine factors that include vascular endothelial growth factor (VEGF), vascular endothelial growth factor D (VEGFD), platelet-derived growth factor C, and angiopoietin 1 (ANGPT1). In addition, we performed immunohistochemical detection of the endothelial marker clusters of differentiation (CD)34 and the proliferation marker Ki67 followed by correlation analyses. In HBL, we found the percentage of RDs with Ki67 expression (% Ki67+ RDs) significantly correlated with intratumoral Ki67+ areas (r = 0.5138, P = 0.0349) and % ANGPT1+ RDs positively correlated with % Ki67+ RDs (r = 0.5851, P = 0.0136). In HCC, the high ANGPT1+ RDs group (i.e., cases with % ANGPT1+ RDs ≥50) exhibited high intratumoral Ki67+ areas compared to the low ANGPT1+ RDs group. In the combined HBL and HCC liver tumor group, there was a positive association between % platelet-derived growth factor C+ RDs and intratumoral Ki67+ areas (r = 0.4712, P = 0.0099) and the high VEGFD+ RDs group (≥50%) exhibited a high number of peritumoral CD34+ vessels compared to the low VEGFD+ RDs group. Conclusion: Paracrine factor-expressing RDs are associated with angiogenesis and proliferation of pediatric liver tumors.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation.

Background & Aims: Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods: We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results: Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions: These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

Sleeve Gastrectomy Improves Glycemia Independent of Weight Loss by Restoring Hepatic Insulin Sensitivity.

Bariatric surgery dramatically improves glycemic control, yet the underlying molecular mechanisms remain controversial because of confounding weight loss. We performed sleeve gastrectomy (SG) on obese and diabetic leptin receptor-deficient mice (db/db). One week postsurgery, mice weighed 5% less and displayed improved glycemia compared with sham-operated controls, and islets from SG mice displayed reduced expression of diabetes markers. One month postsurgery SG mice weighed more than preoperatively but remained near-euglycemic and displayed reduced hepatic lipid droplets. Pair feeding of SG and sham db/db mice showed that surgery rather than weight loss was responsible for reduced glycemia after SG. Although insulin secretion profiles from islets of sham and SG mice were indistinguishable, clamp studies revealed that SG causes a dramatic improvement in muscle and hepatic insulin sensitivity accompanied by hepatic regulation of hepatocyte nuclear factor-α and peroxisome proliferator-activated receptor-α targets. We conclude that long-term weight loss after SG requires leptin signaling. Nevertheless, SG elicits a remarkable improvement in glycemia through insulin sensitization independent of reduced feeding and weight loss.

C/EBPα-dependent preneoplastic tumor foci are the origin of hepatocellular carcinoma and aggressive pediatric liver cancer.

Recent publications show that classic hepatoblastoma (HBL) is the result of failure of hepatic stem cells to differentiate into hepatocytes, while hepatocellular carcinoma (HCC) is caused by the dedifferentiation of hepatocytes into cancer stem cells. However, the mechanisms of aggressive HBL and the mechanisms that cause dedifferentiation of hepatocytes into cancer stem cells are unknown. We found that, similar to HCC but opposite to classic HBL, aggressive HBL is the result of dedifferentiation of hepatocytes into cancer stem cells. In both cases of liver cancer, the dephosphorylation of tumor suppressor protein CCAAT/enhancer binding protein α (C/EBPα) at Ser193 (Ser190 in human protein) or mutation of Ser193 to Ala results in a modified protein with oncogenic activities. We have investigated liver cancer in a mouse model C/EBPα-S193A, in a large cohort of human HBL samples, and in Pten/p53 double knockout mice and found that these cancers are characterized by elevation of C/EBPα that is dephosphorylated at Ser190/193. We found that dephosphorylated C/EBPα creates preneoplastic foci with cancer stem cells that give rise to HCC and aggressive HBL. C/EBPα-dependent dedifferentiation of hepatocytes into cancer stem cells includes increased proliferation of hepatocytes, followed by generation of multinucleated hepatocytes and subsequent appearance of hepatocytes with delta-like 1 homolog-positive intranuclear inclusions. We further isolated C/EBPα-dependent multinucleated hepatocytes and found that they possess characteristics of tumor-initiating cells, including elevation of stem cell markers. C/EBPα-dependent cancer stem cells are observed in patients with aggressive HBL and in patients with a predisposition for liver cancer. CONCLUSION: The earliest steps of adult HCC and aggressive pediatric liver cancer have identical features that include conversion of the tumor suppressor C/EBPα into an oncogenic isoform, which further creates preneoplastic foci where hepatocytes dedifferentiate into cancer cells, giving rise to liver cancer. (Hepatology 2018;67:1857-1871).

Genetic lineage tracing analysis of the cell of origin of hepatotoxin-induced liver tumors in mice.

UNLABELLED: The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell-like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage-labeled hepatocytes by injecting adeno-associated virus containing thyroxine-binding globulin promoter-driven causes recombination (AAV-TBG-Cre) into Rosa(YFP) mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV-TBG-Cre; Rosa(YFP) mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP(+) lineage-labeled; positive for osteopontin, SRY (sex determining region Y)-box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta-like 1 homolog. Next, we performed the converse experiment and lineage-labeled forkhead box protein L1(Foxl1)-positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1-expressing cells are not the origin for hepatotoxin-induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1-Cre-marked cells in a second model of toxin-induced hepatic neoplasia, using DEN and 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP). CONCLUSION: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163-1177).

Foxl1-expressing mesenchymal cells constitute the intestinal stem cell niche.

BACKGROUND & AIMS: Intestinal epithelial stem cells that express Lgr5 and/or Bmi1 continuously replicate and generate differentiated cells throughout life1. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells2. However, ablating Paneth cells has no effect on maintenance of functional stem cells3-5. Here, we demonstrate definitively that a small subset of mesenchymal, subepithelial cells expressing the winged-helix transcription factor Foxl1 are a critical component of the intestinal stem cell niche. METHODS: We genetically ablated Foxl1+ mesenchymal cells in adult mice using two separate models by expressing either the human or simian diphtheria toxin receptor (DTR) under Foxl1 promoter control. CONCLUSIONS: Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor-cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells.

Ablation of Foxl1-Cre-labeled hepatic progenitor cells and their descendants impairs recovery of mice from liver injury.

BACKGROUND & AIMS: Foxl1(+) hepatic progenitor cells (HPCs) differentiate into cholangiocytes and hepatocytes after liver injury. We investigated the requirement for Foxl1(+) HPCs in recovery from liver injury in mice. METHODS: We developed mice in which we could trace and delete Foxl1-expressing HPCs and their descendants (Foxl1-Cre;Rosa(YFP/iDTR)-inducible diphtheria toxin receptor [iDTR] mice). Foxl1-Cre-negative mice were used as controls. Liver damage was induced in male mice by placing them on choline-deficient, ethionine-supplemented (CDE) diets for 15 days; mice then were placed on normal diets and allowed to recover. Liver damage was induced in female mice by placing them on 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diets, followed by a recovery period. Some mice were given injections of diphtheria toxin during the recovery phase to delete Foxl1-Cre-marked HPCs and their descendants. Livers were collected from all mice and analyzed by immunofluorescence, quantitative reverse-transcription polymerase chain reaction, flow cytometry, and histologic analyses. RESULTS: Foxl1-Cre-marked HPCs were required for the development of cholangiocytes and hepatocytes in livers after CDE diet-induced injury. A smaller percentage of yellow fluorescent protein-positive (YFP(+)) hepatocytes contained markers of oxidative stress, DNA damage, or cell death than YFP-negative hepatocytes, indicating that YFP(+) hepatocytes are newly formed cells. Injection of diphtheria toxin deleted YFP(+) cells from Foxl1-Cre;Rosa(YFP/iDTR) mice and prevented the resolution of hepatic steatosis. In mice recovering from DDC diet-induced injury, most cholangiocytes arose from Foxl1-Cre-marked HPCs. Deletion of YFP(+) cells did not alter levels of markers of liver injury or liver function. CONCLUSIONS: Based on studies of Foxl1-Cre;Rosa(YFP/iDTR) mice, Foxl1(+) HPCs and/or their descendants are required for the development of cholangiocytes and hepatocytes in liver after CDE diet-induced injury.

CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse ß-cells.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) plays a major role in pancreatic ß-cell function and survival by increasing cytoplasmic cAMP levels, which are thought to affect transcription through activation of the basic leucine zipper (bZIP) transcription factor CREB. Here, we test CREB function in the adult ß-cell through inducible gene deletion. METHODS: We employed cell type-specific and inducible gene ablation to determine CREB function in pancreatic ß-cells in mice. RESULTS: By ablating CREB acutely in mature ß-cells in tamoxifen-treated Creb (loxP/loxP);Pdx1-CreERT2 mice, we show that CREB has little impact on ß-cell turnover, in contrast to what had been postulated previously. Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis. Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in ß-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling. CONCLUSIONS: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating ß-cell function and mass. However, we reveal an important role for CREB in the ß-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

The origin, biology, and therapeutic potential of facultative adult hepatic progenitor cells.

The liver plays an essential role in glucose and lipid metabolism, synthesis of plasma proteins, and detoxification of xenobiotics and other toxins. Chronic disease of this important organ is one of the leading causes of death in the United States. Following loss of tissue, liver mass can be restored by two mechanisms. Under normal conditions, or after massive loss of parenchyma by surgical resection, liver mass is maintained by division of hepatocytes. After chronic injury, or when proliferation of hepatocytes is impaired, facultative adult hepatic progenitor cells (HPCs) proliferate and differentiate into hepatocytes and cholangiocytes (biliary epithelial cells). HPCs are attractive candidates for cell transplantation because of their potential contribution to liver regeneration. However, until recently, the lack of highly specific markers has hampered efforts to better understand the origin and physiology of HPCs. Recent advances in cell isolation methods and genetic lineage tracing have enabled investigators to explore multiple aspects of HPC biology. In this review, we describe the potential origins of HPCs, the markers used to detect them, the contribution of HPCs to recovery, and the signaling pathways that regulate their biology. We end with an examination of the therapeutic potential of HPCs and their derivatives.

Bile acid-induced inflammatory signaling in mice lacking Foxa2 in the liver leads to activation of mTOR and age-onset obesity.

Cytokine signaling has been connected to regulation of metabolism and energy balance. Numerous cytokine gene expression changes are stimulated by accumulation of bile acids in livers of young Foxa2 liver-conditional null mice. We hypothesized that bile acid-induced inflammation in young Foxa2 mutants, once chronic, affects metabolic homeostasis. We found that loss of Foxa2 in the liver results in a premature aging phenotype, including significant weight gain, reduced food intake, and decreased energy expenditure. We show that Foxa2 antagonizes the mammalian target of rapamycin (mTOR) pathway, resulting in increased hepatic lipogenesis and adiposity. While much prior work has focused on adipose tissue in obesity, we discovered a novel age-onset obesity phenotype in a model where gene deletion occurs only in the liver, underscoring the importance of the role hepatic lipogenesis plays in the development of obesity.

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.