Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

SummaryThe plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighboring healthy cells. However, we know very little about the pathways that can trigger apoptosis upon tissue deformation, and the contribution of compaction-driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show first that tissue compaction induces cell elimination through the downregulation of epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Second, we assessed in vivo the contribution of compaction-driven death to pretumoral cell expansion. We found that the activation of the oncogene Ras in clones can downregulate ERK and activate apoptosis in the neighboring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumor progression.

In order to study the Salmonella typhimurium cobalamin biosynthetic pathway, the S. typhimurium cob operon was isolated and cloned into Escherichia coli. This approach has given the new host of the cob operon the ability to make cobalamins de novo, an ability that had probably been lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombinant strains have been shown to produce up to 100 times more corrin than the parent S. typhimurium strain. These measurements have been performed with a quantitative cobalamin microbiological assay which is detailed in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are grown under anaerobic conditions. Derivatives of the cobalamin-producing E. coli strains were constructed in which genes of the cob operon were inactivated. These strains, together with S. typhimurium cob mutants, have permitted the determination of the genes necessary for cobalamin production and classification of cbiD and cbiG as cobI genes. When grown in the absence of endogenous cobalt, the oxidized forms of precorrin-2 and precorrin-3, factor II and factor III, respectively, were found to accumulate in the cytosol of the corrinproducing E. coli. Together with the finding that S. typhimurium cbiL mutants are not complemented with the homologous Pseudomonas denitrificans gene, these results lend further credence to the theory that cobalt is required at an early stage in the biosynthesis of cobalamins in S. typhimurium.

H epatitis B virus (HBV) infection is a major health problem.There are more than 350 million chronic carriers worldwide, and they are at high risk of developing liver cirrhosis and hepatocellular carcinoma (1). Chronic HBV infection is the result of impaired HBV-specific immune responses such that the infected hepatocytes cannot be eliminated or cured efficiently, but many of the associated issues remain unclear (2, 3).Due to the paucity of in vitro and in vivo models for HBV infection, HBV-transgenic mice are the most widely used model. These mice have the viral genome integrated into the chromosome and produce infectious HBV particles or viral antigens in the liver; however, the main limitation of HBV-transgenic mouse models is that they are immunologically tolerant to viral antigens (4, 5). Various routes have been exploited to introduce the HBV genome into the hepatocytes of adult mice. One is to introduce a replication-competent HBV genome into the mouse liver by hydrodynamic injection (HDI) through the tail vein (6); although HBV replicates in the mouse liver, the virus is rapidly cleared by immune responses against HBV proteins (7). Recently, Huang and colleagues used HDI to create a nontransgenic model of persistent HBV replication (8). The virus persisted in 40% of mice or was eliminated according to the genetic background. These mice rapidly develop anti-hepatitis B virus core (HBc) antibody, which is the first serological marker of acute HBV infection in humans. An alternative method uses adenoviral vectors to transfer 1.3 copies of the HBV genome into immunocompetent mice (9, 10), and acute or chronic HBV infection was obtained depending on the dose of adenoviral vector injected.Here, we describe an alternative murine model for the study of HBV persistence based on the liver-targeted transduction of adeno-associated virus serotype 2/8 (AAV2/8). We produced an AAV2/8 construct carrying a replication-competent HBV DNA genome and by intravenous injection established a model of HBV persistence in humanized HLA-A2/DR1 immunocompetent mice. Hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), and HBV DNA persisted for at least 1 year in sera of all AAV2/8-injected mice, and viral replication intermediates and transcripts were detected in their livers. HBcAg was expressed in 60% of hepatocytes without significant inflammation in the liver. The persistence of infection was associated with the presence of regulatory T cells (Tregs) in the liver. This mouse model of HBV persistence recapitulates viral and histological characteristics of human chronic HBV infection in the immunetolerant stage of the disease (11,12).In HLA-A2/DR1 mice, cellular immune responses were completely restricted to HLA molecules. Antibody, T-helper, and cytotoxic-T-lymphocyte responses to vaccination with recombinant HBsAg or HBsAg-expressing DNA were similar to those in vaccinated humans (13,14) or in HBV-infected individuals (15). Therefore, this AAV2/8-HBV-transduced HLA-A2/DR1 murine model may be useful fo...

Summary What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo . Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

The LIM-only protein FHL2 acts as a transcriptional modulator that positively or negatively regulates multiple signaling pathways. We recently reported that FHL2 cooperates with CREB-binding protein/p300 in the activation of ␤-catenin/T cell factor target gene cyclin D1. In this paper, we demonstrate that FHL2 is associated with the cyclin D1 promoter at the T cell factor/CRE site, providing evidence that cyclin D1 is a direct target of FHL2. We show that deficiency of FHL2 greatly reduces the proliferative capacity of spontaneously immortalized mouse fibroblasts, which is associated with decreased expression of cyclin D1 and p16INK4a , and hypophosphorylation of Rb. Reexpression of FHL2 in FHL2-null fibroblasts efficiently restores cyclin D1 levels and cell proliferative capacity, indicating that FHL2 is critical for cyclin D1 activation and cell growth. Moreover, ectopic cyclin D1 expression is sufficient to override growth inhibition of immortalized FHL2-null fibroblasts. Gene expression profiling revealed that FHL2 deficiency triggers a broad change of the cell cycle program that is associated with down-regulation of several G 1 /S and G 2 /M cyclins, E2F transcription factors, and DNA replication machinery, thus correlating with reduced cell proliferation. This change also involves down-regulation of the negative cell cycle regulators, particularly INK4 inhibitors, which could counteract the decreased expression of cyclins, allowing cells to grow. Our study illustrates that FHL2 can act on different aspects of the cell cycle program to finely regulate cell proliferation.The LIM-only protein FHL2 is a member of the four-and-ahalf LIM (FHL) family (1). Individual LIM domains consist of two zinc finger motifs rich in cysteine and histidine that serve as protein-binding interface for the assembly of multiprotein complexes. The zinc fingers of some transcriptional regulators can interact with DNA, but there is no evidence for DNA binding activity of a LIM domain. FHL2 interacts with multiple transcription factors, including the androgen receptor, AP1, CREB, PLZF, SKI, and ␤-catenin (2-8). It functions as either a coactivator or a corepressor, depending on cell type and promoter contexts (7, 9). Moreover, FHL2 can bind several transcription factors simultaneously and participates in the assembly of multiprotein complexes (10, 11).FHL2 is found in both the cytoplasm and the nucleus (8, 12). In the cytoplasm, FHL2 interacts with integrins and focal adhesion kinase at focal adhesions (13,14). Integrins bind extracellular matrix proteins and certain cell surface receptors, serving as sensors for both chemical and mechanical cues (15). FHL2 shuttles between focal adhesions and nuclei to relay the flow of genetic information at different execution points. Serum response factor, which regulates the expression of immediate early genes, directly controls FHL2 expression in a RhoA-dependent manner (16), and the timing of FHL2 induction is coordinated with that of the early response proteins Fos and Jun (3). Following stimu...

The regulatory protein HBx is essential for hepatitis B virus (HBV) replication in vivo and for transcription of the episomal HBV genome. We previously reported that in infected cells HBx activates genes targeted by the transcription factor CREB [cyclic adenosine monophosphate (cAMP) response element-binding protein]. cAMP induces phosphorylation and activation of CREB, and CREB inactivation is promoted by protein phosphatase 1 (PP1), which binds to CREB through histone deacetylase 1 (HDAC1). We showed that CREB was recruited to HBV DNA. Phosphorylation induced by cAMP had a longer half-life when CREB was bound to the episomal HBV genome compared to when it was bound to the promoter of a host target gene not regulated by HBx, suggesting that the virus has developed a mechanism to favor its own transcription. This mechanism required HBx, which interacted with and inhibited PP1 to extend the half-life of CREB phosphorylation. Silencing of PP1 rescued replication of an HBx-deficient HBV genome, suggesting that HBx enhances viral transcription in part by neutralizing PP1 activity. Our results illustrate a previously unknown mechanism of HBV transcriptional activation by HBx in which HBx interferes with the inactivation of CREB by the PP1 and HDAC1 complex.

Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor B (NF-B) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-B targets by stabilizing the protein.