Deficiency of multidrug resistance 2 contributes to cell transformation through oxidative stress.

Multidrug resistance 2 (Mdr2), also called adenosine triphosphate-binding cassette B4 (ABCB4), is the transporter of phosphatidylcholine (PC) at the canalicular membrane of mouse hepatocytes, which plays an essential role for bile formation. Mutations in human homologue MDR3 are associated with several liver diseases. Knockout of Mdr2 results in hepatic inflammation, liver fibrosis and hepatocellular carcinoma (HCC). Whereas the pathogenesis in Mdr2 (-/-) mice has been largely attributed to the toxicity of bile acids due to the absence of PC in the bile, the question of whether Mdr2 deficiency per se perturbs biological functions in the cell has been poorly addressed. As Mdr2 is expressed in many cell types, we used mouse embryonic fibroblasts (MEF) derived from Mdr2 (-/-) embryos to show that deficiency of Mdr2 increases reactive oxygen species accumulation, lipid peroxidation and DNA damage. We found that Mdr2 (-/-) MEFs undergo spontaneous transformation and that Mdr2 (-/-) mice are more susceptible to chemical carcinogen-induced intestinal tumorigenesis. Microarray analysis in Mdr2-/- MEFs and cap analysis of gene expression in Mdr2 (-/-) HCCs revealed extensively deregulated genes involved in oxidation reduction, fatty acid metabolism and lipid biosynthesis. Our findings imply a close link between Mdr2 (-/-) -associated tumorigenesis and perturbation of these biological processes and suggest potential extrahepatic functions of Mdr2/MDR3.

The four and a half LIM-only protein 2 (FHL2) activates transforming growth factor ß (TGF-ß) signaling by regulating ubiquitination of the E3 ligase Arkadia.

Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-ß signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-ß target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-ß signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-ß signal transduction.

Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules.

Hepatitis B virus (HBV) persistence may be due to impaired HBV-specific immune responses being unable to eliminate efficiently or cure infected hepatocytes. The immune mechanisms that lead to HBV persistence have not been completely identified, and no appropriate animal model is available for such studies. Therefore, we established a chronic HBV infection model in a mouse strain with human leukocyte antigen A2/DR1 (HLA-A2/DR1) transgenes and an H-2 class I/class II knockout. The liver of these mice was transduced with adeno-associated virus serotype 2/8 (AAV2/8) carrying a replication-competent HBV DNA genome. In all AAV2/8-transduced mice, hepatitis B virus surface antigen, hepatitis B virus e antigen, and HBV DNA persisted in serum for at least 1 year. Viral replication intermediates and transcripts were detected in the livers of the AAV-injected mice. The hepatitis B core antigen was expressed in 60% of hepatocytes. No significant inflammation was observed in the liver. This was linked to a higher number of regulatory T cells in liver than in controls and a defect in HBV-specific functional T-cell responses. Despite the substantial tolerance resulting from expression of HBV antigens in hepatocytes, we succeeded in priming functional HBV-specific T-cell responses in peripheral tissues, which subsequently reached the liver. This AAV2/8-HBV-transduced HLA-A2/DR1 murine model recapitulates virological and immunological characteristics of chronic HBV infection, and it could be useful for the development of new treatments and immune-based therapies or therapeutic vaccines for chronic HBV infections.

Patterned apoptosis has an instructive role for local growth and tissue shape regulation in a fast-growing epithelium.

What regulates organ size and shape remains one fundamental mystery of modern biology. Research in this area has primarily focused on deciphering the regulation in time and space of growth and cell division, while the contribution of cell death has been overall neglected. This includes studies of the Drosophila wing, one of the best-characterized systems for the study of growth and patterning, undergoing massive growth during larval stage and important morphogenetic remodeling during pupal stage. So far, it has been assumed that cell death was relatively neglectable in this tissue both during larval stage and pupal stage, and as a result, the pattern of growth was usually attributed to the distribution of cell division. Here, using systematic mapping and registration combined with quantitative assessment of clone size and disappearance as well as live imaging, we outline a persistent pattern of cell death and clone elimination emerging in the larval wing disc and persisting during pupal wing morphogenesis. Local variation of cell death is associated with local variation of clone size, pointing to an impact of cell death on local growth that is not fully compensated by proliferation. Using morphometric analyses of adult wing shape and genetic perturbations, we provide evidence that patterned death locally and globally affects adult wing shape and size. This study describes a roadmap for precise assessment of the contribution of cell death to tissue shape and outlines an important instructive role of cell death in modulating quantitatively local growth and morphogenesis of a fast-growing tissue.

NDP52 mediates an antiviral response to hepatitis B virus infection through Rab9-dependent lysosomal degradation pathway.

Autophagy receptor NDP52 triggers bacterial autophagy against infection. However, the ability of NDP52 to protect against viral infection has not been established. We show that NDP52 binds to envelope proteins of hepatitis B virus (HBV) and triggers a degradation process that promotes HBV clearance. Inactivating NDP52 in hepatocytes results in decreased targeting of viral envelopes in the lysosome and increased levels of viral replication. NDP52 inhibits HBV at both viral entry and late replication stages. In contrast to NDP52-mediated bacterial autophagy, lysosomal degradation of HBV envelopes is independent of galectin 8 and ATG5. NDP52 forms complex with Rab9 and viral envelope proteins and links HBV to Rab9-dependent lysosomal degradation pathway. These findings reveal that NDP52 acts as a sensor for HBV infection, which mediates a unique antiviral response to eliminate the virus. This work also suggests direct roles for autophagy receptors in other lysosomal degradation pathways than canonical autophagy.

The four and a half LIM-only protein 2 regulates liver homeostasis and contributes to carcinogenesis.

BACKGROUND & AIMS: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/ß-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Microtubule disassembly by caspases is an important rate-limiting step of cell extrusion.

The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, we showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.

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.

Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation.

The 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.

LIM-Only Protein FHL2 Is a Negative Regulator of Transforming Growth Factor ß1 Expression.

Transforming growth factor ß1 (TGF-ß1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-ß1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-ß1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-ß1 promoter, whereas the upregulation of TGF-ß1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-ß1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-ß1 gene transcription. Enhanced expression of TGF-ß1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-ß1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-ß1 expression and contribute to the understanding of the TGF-ß-mediated fibrogenic response.

LIM-only protein FHL2 activates NF-κB signaling in the control of liver regeneration and hepatocarcinogenesis.

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 κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Inhibition of PP1 phosphatase activity by HBx: a mechanism for the activation of hepatitis B virus transcription.

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.

Deficiency of the LIM-only protein FHL2 reduces intestinal tumorigenesis in Apc mutant mice.

BACKGROUND: The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including beta-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in beta-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc(Delta14/+) mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.

The LIM-only protein FHL2 mediates ras-induced transformation through cyclin D1 and p53 pathways.

BACKGROUND: Four and a half LIM-only protein 2 (FHL2) has been implicated in multiple signaling pathways that regulate cell growth and tissue homeostasis. We reported previously that FHL2 regulates cyclin D1 expression and that immortalized FHL2-null mouse embryo fibroblasts (MEFs) display reduced levels of cyclin D1 and low proliferative activity. METHODOLOGY/PRINCIPAL FINDINGS: Here we address the contribution of FHL2 in cell transformation by investigating the effects of oncogenic Ras in FHL2-null context. We show that H-RasV12 provokes cell cycle arrest accompanied by accumulation of p53 and p16(INK4a) in immortalized FHL2(-/-) MEFs. These features contrast sharply with Ras transforming activity in wild type cell lines. We further show that establishment of FHL2-null cell lines differs from conventional immortalization scheme by retaining functional p19(ARF)/p53 checkpoint that is required for cell cycle arrest imposed by Ras. However, after serial passages of Ras-expressing FHL2(-/-) cells, dramatic increase in the levels of D-type cyclins and Rb phosphorylation correlates with the onset of cell proliferation and transformation without disrupting the p19(ARF)/p53 pathway. Interestingly, primary FHL2-null cells overexpressing cyclin D1 undergo a classical immortalization process leading to loss of the p19(ARF)/p53 checkpoint and susceptibility to Ras transformation. CONCLUSIONS/SIGNIFICANCE: Our findings uncover a novel aspect of cellular responses to mitogenic stimulation and illustrate a critical role of FHL2 in the signalling network that implicates Ras, cyclin D1 and p53.

The LIM-only protein FHL2 regulates cyclin D1 expression and cell proliferation.

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 beta-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 p16(INK4a), 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.

Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer.

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.