Hanne Cathrine Bisgaard

Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.

Hepatic regeneration from toxic or surgical injury to the adult mammalian liver, endorses different cellular responses within the hepatic lineage. The molecular mechanisms determining commitment of a cell population at a specific lineage level to participate in liver repair as well as the fate of its progeny in the hostile environment created by the injury are not well defined. Based on the role of the Notch/Delta/Jagged system in cell fate specification and recent reports linking Notch signaling with normal bile duct formation in mouse and human liver, we examined the expression of Notch1, Notch2, Notch3, Delta1, Delta3, Jagged1, and Jagged2, and delta-like protein/preadipocyte factor 1/fetal antigen 1 (dlk) in four well-defined experimental rat models of liver injury and regeneration. Although Delta3 and Jagged2 were undetectable by reverse transcriptase-polymerase chain reaction and Northern blot, we observed the most significant up-regulation of all other transcripts in the 2-acetylaminofluorene-70% hepatectomy (AAF/PHx) model, in which liver mass is restored by proliferation and differentiation of transit-amplifying ductular (oval) cells. The most profound change was observed for dlk. Accordingly, immunohistochemical analyses in the AAF/PHx model showed a specific expression of dlk in atypical ductular structures composed of oval cells. Delta-like protein was not observed in proliferating hepatocytes or bile duct cells after partial hepatectomy or ligation of the common bile duct whereas clusters of dlk immunoreactive oval cells were found in both the retrorsine and the AAF/PHx models. Finally, we used dlk to isolate alpha-fetoprotein-positive cells from fetal and adult regenerating rat liver by a novel antibody panning technique.

Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion

Although normally quiescent, the adult mammalian liver possesses a great capacity to regenerate after different types of injuries in order to restore the lost liver mass and ensure maintenance of the multiple liver functions. Major players in the regeneration process are mature residual cells, including hepatocytes, cholangiocytes and stromal cells. However, if the regenerative capacity of mature cells is impaired by liver‐damaging agents, hepatic progenitor cells are activated and expand into the liver parenchyma. Upon transit amplification, the progenitor cells may generate new hepatocytes and biliary cells to restore liver homeostasis. In recent years, hepatic progenitor cells have been the subject of increasing interest due to their therapeutic potential in numerous liver diseases as alternative or supportive/complementary tools to liver transplantation. While the first investigations on hepatic progenitor cells have focused on their origin and phenotypic characterization, recent attention has focused on the influence of the hepatic microenvironment on their activation and proliferation. This microenvironment comprises the extracellular matrix, epithelial and non‐epithelial resident liver cells, and recruited inflammatory cells as well as the variety of growth‐modulating molecules produced and/or harboured by these elements. The cellular and molecular responses to different regenerative stimuli seem to depend on the injury inflicted and consequently on the molecular microenvironment created in the liver by a certain insult. This review will focus on molecular responses controlling activation and expansion of the hepatic progenitor cell niche, emphasizing similarities and differences in the microenvironments orchestrating regeneration by recruitment of progenitor cell populations or by replication of mature cells.

Hepatic Stem Cell Compartment: Activation and Lineage Commitment

There is increasingly robust experimental evidence in support of the presence of a pluripotent cell compartment in the liver 1 , 2 , 3 , 4 , 5 , 6 , 7 . This compartment can under certain conditions function as a stem cell compartment and provide the needed progeny for regeneration of the hepatic parenchyma 8 , 9 . In the adult rat, specific conditions can be utilized to induce proliferation of a distinct population of small epithelial cells in the ductal structures of the liver 10 , 11 . These cells, conventionally described as oval cells, are characterized by ovoid nuclei and basophilic cytoplasma 10 , and display features of both bile duct cells and fetal hepatocytes 11 , 12 , 13 . There are three experimental systems, two in the rat and one in the mouse, in which it has been conclusively demonstrated that oval cells are capable of differentiation into hepatocytes 8 , 11 , 14 . The developmental potential of oval cells is, however, not restricted to hepatic lineages. Oval cells can differentiate into intestinal-type epithelia, and have been implicated in the development of pancreatic tissues 8 , 11 , 15 , 16 , 17 ; Fig. 1). The observations that sub-populations of proliferating oval cells phenotypically similar to early hepatoblasts, and that oval cells originate in or around the ductular structures in the portal area, strongly support the notion that the hepatic stem cell compartment resides in these structures 2 , 7 , 9 . Furthermore, present evidence clearly indicates that the hepatic stem cell compartment functions as a facultative stem cell compartment that is activated when the parenchymal cells are unable to proliferate in response to growth stimuli 2 , 8 , 18 , 19 .

Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration.

The experimental protocols used in the investigation of stem cell–mediated liver regeneration in rodents are characterized by activation of the hepatic stem cell compartment in the canals of Hering followed by transit amplification of oval cells and their subsequent differentiation along hepatic lineages. Although the protocols are numerous and often used interchangeably across species, a thorough comparative phenotypic analysis of oval cells in rats and mice using well‐established and generally acknowledged molecular markers has not been provided. In the present study, we evaluated and compared the molecular phenotypes of oval cells in several of the most commonly used protocols of stem cell–mediated liver regeneration—namely, treatment with 2‐acetylaminofluorene and partial (70%) hepatectomy (AAF/PHx); a choline‐deficient, ethionine‐supplemented (CDE) diet; a 3,5‐diethoxycarbonyl‐1,4‐dihydro‐collidin (DDC) diet; and N‐acetyl‐paraaminophen (APAP). Reproducibly, oval cells showing reactivity for cytokeratins (CKs), muscle pyruvate kinase (MPK), the adenosine triphosphate–binding cassette transporter ABCG2/BCRP1 (ABCG2), alpha‐fetoprotein (AFP), and delta‐like protein 1/preadipocyte factor 1 (Dlk/Pref‐1) were induced in rat liver treated according to the AAF/PHx and CDE but not the DDC protocol. In mouse liver, the CDE, DDC, and APAP protocols all induced CKs and ABCG2‐positive oval cells. However, AFP and Dlk/Pref‐1 expression was rarely detected in oval cells. Conclusion: Our results delineate remarkable phenotypic discrepancies exhibited by oval cells in stem cell–mediated liver regeneration between rats and mice and underline the importance of careful extrapolation between individual species. (HEPATOLOGY 2007;45:1462–1470.)

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLα and hMLH1–hEXO1 complexes

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC–hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC–hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1–hEXO1 complex formation and hMutLα heterodimer assembly essential for MMR activity.

Heterogeneity of Ductular Reactions in Adult Rat and Human Liver Revealed by Novel Expression of Deleted in Malignant Brain Tumor 1

The regenerative capacity of mammalian adult liver reflects the ability of a number of cell populations within the hepatic lineage to take action. Limited information is available regarding factors and mechanisms that determine the specific lineage level at which liver cells contribute to liver repair as well as the fate of their progeny in the hostile environment created by liver injury. In the present study, we attempted to identify novel molecules preferentially involved in liver regeneration by recruitment of transit-amplifying, ductular (oval) cell populations. With a subtractive cDNA library screening approach, we identified 48 enriched, nonredundant gene products associated with liver injury and oval cell proliferation in the adult rat liver. Of these, only two, namely alpha-fetoprotein and a novel transcript with high homology to human DMBT1 (deleted in malignant brain tumor 1), were specifically associated with the emergence of ductular (oval) cell populations in injured liver. Subsequent cloning and characterization of the rat DMBT1 homologue revealed a highly inducible expression in ductular reactions composed of transit-amplifying ductular (oval) cells, but not in ductular reactions after ligation of the common bile duct. In human liver diseases, DMBT1 was expressed in ductular reactions after infection with hepatitis B and acetaminophen intoxication, but not in primary biliary cirrhosis, primary sclerosing cholangitis, and obstruction of the large bile duct. The expression heterogeneity in ductular reactions and multiple functions of DMBT1 homologues point to intriguing roles in regulating not only tissue repair but also fate decision and differentiation paths of specific cell populations in the hepatic lineage.

UPF2 Is a Critical Regulator of Liver Development, Function and Regeneration

Background Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full regulatory potential of the NMD pathway in mammals will require the functional assessment of NMD in different tissues. Methodology/Principal Findings Here we use mouse genetics to address the role of UPF2, a core NMD component, in the development, function and regeneration of the liver. We find that loss of NMD during fetal liver development is incompatible with postnatal life due to failure of terminal differentiation. Moreover, deletion of Upf2 in the adult liver results in hepatosteatosis and disruption of liver homeostasis. Finally, NMD was found to be absolutely required for liver regeneration. Conclusion/Significance Collectively, our data demonstrate the critical role of the NMD pathway in liver development, function and regeneration and highlights the importance of NMD for mammalian biology.

Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries

Dynamic shifts in transcription factor binding are central to the regulation of biological processes by allowing rapid changes in gene transcription. However, very few genome-wide studies have examined how transcription factor occupancy is coordinated temporally in vivo in higher animals. Here, we quantified the genome-wide binding patterns of two key hepatocyte transcription factors, CEBPA and CEBPB (also known as C/EBPalpha and C/EBPbeta), at multiple time points during the highly dynamic process of liver regeneration elicited by partial hepatectomy in mouse. Combining these profiles with RNA polymerase II binding data, we find three temporal classes of transcription factor binding to be associated with distinct sets of regulated genes involved in the acute phase response, metabolic/homeostatic functions, or cell cycle progression. Moreover, we demonstrate a previously unrecognized early phase of homeostatic gene expression prior to S-phase entry. By analyzing the three classes of CEBP bound regions, we uncovered mutually exclusive sets of sequence motifs, suggesting temporal codes of CEBP recruitment by differential cobinding with other factors. These findings were validated by sequential ChIP experiments involving a panel of central transcription factors and/or by comparison to external ChIP-seq data. Our quantitative investigation not only provides in vivo evidence for the involvement of many new factors in liver regeneration but also points to similarities in the circuitries regulating self-renewal of differentiated cells. Taken together, our work emphasizes the power of global temporal analyses of transcription factor occupancy to elucidate mechanisms regulating dynamic biological processes in complex higher organisms.

Expression of prostasin and its inhibitors during colorectal cancer carcinogenesis

BackgroundClinical trials where cancer patients were treated with protease inhibitors have suggested that the serine protease, prostasin, may act as a tumour suppressor. Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of hepatocyte growth factor activator inhibitor-1 (HAI-1), HAI-1A, and HAI-1B.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels for prostasin and PN-1 in colorectal cancer tissue (n = 116), severe dysplasia (n = 13), mild/moderate dysplasia (n = 93), and in normal tissue from the same individuals. In addition, corresponding tissues were examined from healthy volunteers (n = 23). A part of the cohort was further analysed for the mRNA levels of the two variants of HAI-1, here denoted HAI-1A and HAI-1B. mRNA levels were normalised to β-actin. Immunohistochemical analysis of prostasin and HAI-1 was performed on normal and cancer tissue.ResultsThe mRNA level of prostasin was slightly but significantly decreased in both mild/moderate dysplasia (p < 0.001) and severe dysplasia (p < 0.01) and in carcinomas (p < 0.05) compared to normal tissue from the same individual. The mRNA level of PN-1 was more that two-fold elevated in colorectal cancer tissue as compared to healthy individuals (p < 0.001) and elevated in both mild/moderate dysplasia (p < 0.01), severe dysplasia (p < 0.05) and in colorectal cancer tissue (p < 0.001) as compared to normal tissue from the same individual. The mRNA levels of HAI-1A and HAI-1B mRNAs showed the same patterns of expression. Immunohistochemistry showed that prostasin is located mainly on the apical plasma membrane in normal colorectal tissue. A large variation was found in the degree of polarization of prostasin in colorectal cancer tissue.ConclusionThese results show that the mRNA level of PN-1 is significantly elevated in colorectal cancer tissue. Future studies are required to clarify whether down-regulation of prostasin activity via up regulation of PN-1 is causing the malignant progression or if it is a consequence of it.

Delta-like protein (DLK) is a novel immunohistochemical marker for human hepatoblastomas

Delta-like protein (DLK) is a membrane protein with mostly unknown function. It is expressed by several embryonic tissues among others by the hepatoblasts of rodent and human fetal livers. We have investigated in the present study if this protein is expressed in human hepatoblastomas. The presence of DLK has been studied by standard immunohistochemistry in 31 hepatoblastomas and in several differential diagnostically related tumours: hepatocellular carcinomas and in undifferentiated childhood neoplasms. All the hepatoblastomas were positive for DLK; the surrounding liver tissue remained negative. The reaction was present in the epithelial component of the tumours. The staining pattern was mostly membranous, occasionally cytoplasmic. The other studied tumours were negative for DLK, except one hepatocellular carcinoma and the differentiating cells of two ganglioneuroblastomas. Therefore, DLK seems to be a highly sensitive and specific marker for hepatoblastomas.