JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

NF-κB/RelA and Nrf2 cooperate to maintain hepatocyte integrity and to prevent development of hepatocellular adenoma.

BACKGROUND & AIMS: The liver is frequently challenged by toxins and reactive oxygen species. Therefore, hepatocytes require cytoprotective strategies to cope with these insults. Since the transcription factors Nrf2 and NF-κB regulate the cellular antioxidant defense system and important survival pathways, we determined their individual and overlapping functions in the liver. METHODS: We generated mice lacking Nrf2 and the NF-κB RelA/p65 subunit in hepatocytes and we analyzed their liver by using histopathology, immunohistochemistry, quantitative RT-PCR, Western blot and Oxyblot analysis. Human inflammatory hepatocellular adenomas (iHCA) were analyzed by immunohistochemistry. RESULTS: Loss of either Nrf2 or NF-κB/RelA had only a minor effect on liver homeostasis, but the double knockout mice spontaneously developed liver inflammation and fibrosis. Upon aging, more than one-third of the female double mutant mice developed tumors, which histologically resemble human iHCA, a tumor that predominantly occurs in women. The mouse tumors also recapitulated the immunohistochemical marker profile characteristic for human iHCA. Moreover, pNRF2 and NF-κB RelA/p65 was not detectable in the nuclei of iHCA tumor cells. The mouse phenotype was not due to a synergistic effect of both transcription factors on cytoprotective Nrf2 target genes. Rather, loss of Nrf2 or NF-κB/RelA altered the expression of different genes, and the combination of these alterations likely affects liver homeostasis in the double mutant mice. CONCLUSIONS: Our results provide genetic evidence for a functional cross-talk of Nrf2 and NF-κB/RelA in hepatocytes, which protects the liver from necrosis, inflammation and fibrosis. Furthermore, the double mutant mice represent a valuable animal model for iHCA.

Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

OBJECTIVE: Fibroblast growth factors (Fgfs) are key orchestrators of development, and a role of Fgfs in tissue repair is emerging. Here we studied the consequences of inducible loss of Fgf receptor (Fgfr) 4, the major Fgf receptor (Fgfr) on hepatocytes, alone or in combination with Fgfr1 and Fgfr2, for liver regeneration after PH. DESIGN: We used siRNA delivered via nanoparticles combined with liver-specific gene knockout to study Fgfr function in liver regeneration. Liver or blood samples were analysed using histology, immunohistochemistry,real-time RT-PCR, western blotting and ELISA. RESULTS: siRNA-mediated knockdown of Fgfr4 severely affected liver regeneration due to impairment of hepatocyte proliferation combined with liver necrosis.Mechanistically, the proliferation defect resulted from inhibition of an Fgf15-Fgfr4-Stat3 signalling pathway,which is required for injury-induced expression of the Foxm1 transcription factor and subsequent cell cycle progression, while elevated levels of intrahepatic toxicbile acids were identified as the likely cause of the necrotic damage. Failure of liver mass restoration in Fgfr4 knockdown mice was prevented at least in part by compensatory hypertrophy of hepatocytes. Most importantly, our data revealed partially redundant functions of Fgf receptors in the liver, since knock down of Fgfr4 in mice lacking Fgfr1 and Fgfr2 in hepatocytes caused liver failure after PH due to severe liver necrosis and a defect in regeneration. CONCLUSIONS: These results demonstrate that Fgfr signalling in hepatocytes is essential for liver regeneration and suggest activation of Fgfr signalling asa promising approach for the improvement of the liver's regenerative capacity.

Molecular detection of hepatitis E virus (HEV) in liver biopsies after liver transplantation.

We aimed to determine the rate of hepatitis E virus (HEV) infection, a recently increasingly recognized disease in the Western world, in liver transplant patients by direct molecular testing of liver tissue. A RT-PCR assay was designed for detecting the HEV open reading frame (ORF) 2/3 gene region in formalin-fixed, paraffin-embedded tissues, and applied to all liver biopsies (n=683) taken 4 weeks or later from all patients (n=282) after liver transplantation of two large academic centers. HEV-RNA was detected in ten biopsies from four different patients (rate: 1%). Histology in early HEV infection was variable including cases with only few hepatocellular apoptoses, no or only minute inflammation. Hepatitis lasted for at least 6 months in 3/4 patients. Serologic testing for HEV-RNA in a subcohort (159 patients) was positive in five patients (rate: 3%), resulting in an overall HEV detection rate of 3% (8/282). In case both liver tissue and sera of a patient were available from the same time period, all cases tested positive in one material were also tested positive in the other material, respectively. All patients had de novo autochthonous infection with HEV genotype 3. Our data confirm that HEV infection is a relevant cause of liver injury after liver transplantation. Molecular testing for HEV in routinely processed transplant liver biopsies is powerful for evaluating patients with elevated transaminases of unknown origin. Histology of HEV infection under immunosuppression in the early phase is distinct from HEV infection in immunocompetent individuals.

FGF receptors 1 and 2 control chemically induced injury and compound detoxification in regenerating livers of mice.

BACKGROUND & AIMS: Fibroblast growth factor receptor 4 (FGFR4) controls bile acid metabolism and protects the liver from fibrosis, but the roles of FGFR1 and FGFR2 in the adult liver are largely unknown. We investigated the functions and mechanisms of action of these receptors in liver homeostasis, regeneration, and fibrosis. METHODS: We generated mice with hepatocytes that lack FGFR1 and FGFR2 and subjected them to acute and chronic carbon tetrachloride-induced liver injury and partial hepatectomy; mice were also injected with FGF7. We performed histology, histomorphometry, real-time reverse transcription polymerase chain reaction, and immunoblot analyses. RESULTS: In hepatocytes, loss of FGFR1 and FGFR2 eliminated responsiveness to FGF7 and related FGF family members but did not affect toxin-induced liver injury and fibrosis. However, mortality after partial hepatectomy increased because of severe hepatocyte necrosis. These effects appeared to be mediated by a failure of hepatocytes to induce the expression of the transcriptional regulators Dbp and Tef upon liver surgery; this affected expression of their target genes, which encode detoxifying cytochrome P450 enzymes. We found that Dbp and Tef expression was directly controlled by FGFR signaling in hepatocytes. As a consequence of the reduced expression of genes that control detoxification, the liver tissue that remained after partial hepatectomy failed to efficiently metabolize endogenous compounds and the drugs applied for anesthesia/analgesia. CONCLUSIONS: We identified a new, cytoprotective effect of FGFR1 and FGFR2 in the regenerating liver and suggest the use of recombinant FGF7 to increase survival of patients after surgical resection of large amounts of liver tissue.

Hepatocyte apoptosis is tumor promoting in murine nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease is the most common chronic liver disease and may progress to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The molecular determinants of this pathogenic progression, however, remain largely undefined. Since liver tumorigenesis is driven by apoptosis, we examined the effect of overt hepatocyte apoptosis in a mouse model of NASH using mice lacking myeloid cell leukemia 1 (Mcl1), a pro-survival member of the BCL-2 protein family. Hepatocyte-specific Mcl1 knockout (Mcl1∆hep) mice and control littermates were fed chow or FFC (high saturated fat, fructose, and cholesterol) diet, which induces NASH, for 4 and 10 months. Thereafter, liver injury, inflammation, fibrosis, and tumor development were evaluated biochemically and histologically. Mcl1∆hep mice fed with the FFC diet for 4 months displayed a marked increase in liver injury, hepatocyte apoptosis, hepatocyte proliferation, macrophage-associated liver inflammation, and pericellular fibrosis in contrast to chow-fed Mcl1∆hep and FFC diet-fed Mcl1-expressing littermates. After 10 months of feeding, 78% of FFC diet-fed Mcl1∆hep mice developed liver tumors compared to 38% of chow-fed mice of the same genotype. Tumors in FFC diet-fed Mcl1∆hep mice were characterized by cytologic atypia, altered liver architecture, immunopositivity for glutamine synthetase, and histologically qualified as HCC. In conclusion, this study provides evidence that excessive hepatocyte apoptosis exacerbates the NASH phenotype with enhancement of tumorigenesis in mice.

Potentiation of fibroblast growth factor activity by synthetic heparin oligosaccharide glycodendrimers.

Heparin is a highly sulfated polysaccharide that regulates a variety of cellular processes by interaction with a host of proteins. We report the preparation of synthetic heparin oligosaccharide glycodendrimers and their use as heparin mimetics to regulate heparin-protein interactions. The multivalent display of sugar epitopes mimics the naturally occurring glycans found on cell surfaces and enhances their binding capacity. Binding of the heparin dendrimers to basic fibroblast growth factor (FGF-2) was analyzed using heparin microarray experiments and surface plasmon resonance measurements on gold chips. Heparin-coated dendrimers bind FGF-2 significantly more effectively than monovalent heparin oligosaccharides. Dendrimer 1, which displays multiple copies of the sulfated hexasaccharide (GlcNSO(3)[6-OSO(3)]-IdoA[2-OSO(3)])3, was employed to promote FGF-2-mediated mitogen-activated kinase activation, demonstrating the utility of glycodendrimers to modulate heparin-protein interactions.

The SAF-box domain of chromatin protein DEK.

DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number of nuclear proteins. Isolated SAF-box peptides (amino acids 137-187) bind weakly to DNA in solution, but when many SAF-box peptides are brought into close proximity on the surface of Sephadex beads, cooperative effects lead to a high affinity to DNA. Furthermore, a peptide (amino acids 87-187) that includes a sequence on the N-terminal side of the SAF-box binds efficiently to DNA. This peptide prefers four-way junction DNA over straight DNA and induces supercoils in relaxed circular DNA just like the full-length DEK. Interestingly, however, the 87-187 amino acid peptide introduces negative supercoils in contrast to the full-length DEK, which is known to introduce positive supercoils. We found that two adjacent regions (amino acids 68-87 and 187-250) are necessary for the formation of positive supercoils. Our data contribute to the ongoing characterization of the abundant and ubiquitous DEK chromatin protein.

Large-Scale Quantitative Proteomics Identifies the Ubiquitin Ligase Nedd4-1 as an Essential Regulator of Liver Regeneration.

The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Intratumor heterogeneity in hepatocellular carcinoma.

PURPOSE: Morphologic intratumor heterogeneity is well known to exist in hepatocellular carcinoma (HCC), but very few systematic analyses of this phenomenon have been performed. The aim of this study was to comprehensively characterize morphologic intratumor heterogeneity in HCC. Also, taken into account were well-known immunohistochemical markers and molecular changes in liver cells that are considered in proposed classifications of liver cell neoplasms or discussed as molecular therapeutic targets. EXPERIMENTAL DESIGN: In HCC of 23 patients without medical pretreatment, a total of 120 tumor areas were defined. Analyzed were cell and tissue morphology, expression of the liver cell markers cytokeratin (CK)7, CD44, α-fetoprotein (AFP), epithelial cell adhesion molecule (EpCAM), and glutamine synthetase (GS) along with mutations of TP53 and CTNNB1, assayed by both Sanger and next-generation sequencing. RESULTS: Overall, intratumor heterogeneity was detectable in the majority of HCC cases (20 of 23, 87%). Heterogeneity solely on the level of morphology was found in 6 of 23 cases (26%), morphologic heterogeneity combined with immunohistochemical heterogeneity in 9 of 23 cases (39%), and heterogeneity with respect to morphologic, immunohistochemical, and mutational status of TP53 and CTNNB1 in 5 of 23 cases (22%). CONCLUSIONS: Our findings demonstrate that intratumor heterogeneity represents a challenge for the establishment of a robust HCC classification and may contribute to treatment failure and drug resistance in many cases of HCC.

Nrf2 Activation Promotes Keratinocyte Survival during Early Skin Carcinogenesis via Metabolic Alterations.

Pharmacologic activation of the transcription factor NRF2 has been suggested to offer a strategy for cancer prevention. In this study, we present evidence from murine tumorigenesis experiments suggesting there may be limitations to this possibility, based on tumorigenic effects of Nrf2 in murine keratinocytes that have not been described previously. In this setting, Nrf2 expression conferred metabolic alterations in keratinocytes that were protumorigenic in nature, affecting enzymes involved in glutathione biosynthesis or in the oxidative pentose phosphate pathway and other NADPH-producing enzymes. Under stress conditions, coordinate increases in NADPH, purine, and glutathione levels promoted the survival of keratinocytes harboring oncogenic mutations, thereby promoting tumor development. The protumorigenic activity of Nrf2 in keratinocytes was particularly significant in a mouse model of skin tumorigenesis that did not rely upon chemical carcinogenesis. In exploring the clinical relevance of our findings, we confirm that NRF2 and protumorigenic NRF2 target genes were activated in some actinic keratoses, the major precancerous lesion in human skin. Overall, our results reveal an unexpected tumor-promoting activity of activated NRF2 during early phases of skin tumorigenesis.

Dual role of the antioxidant enzyme peroxiredoxin 6 in skin carcinogenesis.

The antioxidant enzyme peroxiredoxin 6 (Prdx6) is a key regulator of the cellular redox balance, particularly under stress conditions. We identified Prdx6 as an important player in different phases of skin carcinogenesis. Loss of Prdx6 in mice enhanced the susceptibility to skin tumorigenesis, whereas overexpression of Prdx6 in keratinocytes of transgenic mice had the opposite effect. The tumor-preventive effect of Prdx6, which was observed in a human papilloma virus 8-induced and a chemically induced tumor model, was not due to alterations in keratinocyte proliferation, apoptosis, or in the inflammatory response. Rather, endogenous and overexpressed Prdx6 reduced oxidative stress as reflected by the lower levels of oxidized phospholipids in the protumorigenic skin of Prdx6 transgenic mice and the higher levels in Prdx6-knockout mice than in control animals. In contrast to its beneficial effect in tumor prevention, overexpression of Prdx6 led to an acceleration of malignant progression of existing tumors, revealing a dual function of this enzyme in the pathogenesis of skin cancer. Finally, we found strong expression of PRDX6 in keratinocytes of normal human skin and in the tumor cells of squamous cell carcinomas, indicating a role of Prdx6 in human skin carcinogenesis. Taken together, our data point to the potential usefulness of Prdx6 activators or inhibitors for controlling different stages of skin carcinogenesis.

Regulation of liver regeneration by growth factors and cytokines.

The capability of the liver to fully regenerate after injury is a unique phenomenon essential for the maintenance of its important functions in the control of metabolism and xenobiotic detoxification. The regeneration process is histologically well described, but the genes that orchestrate liver regeneration have been only partially characterized. Of particular interest are cytokines and growth factors, which control different phases of liver regeneration. Historically, their potential functions in this process were addressed by analyzing their expression in the regenerating liver of rodents. Some of the predicted roles were confirmed using functional studies, including systemic delivery of recombinant growth factors, neutralizing antibodies or siRNAs prior to liver injury or during liver regeneration. In particular, the availability of genetically modified mice and their use in liver regeneration studies has unraveled novel and often unexpected functions of growth factors, cytokines and their downstream signalling targets in liver regeneration. This review summarizes the results obtained by functional studies that have addressed the roles and mechanisms of action of growth factors and cytokines in liver regeneration after acute injury to this organ.

Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis.

Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal gammadelta T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal-epithelial interactions in skin homeostasis and disease.