Inflammatory disease and cancer with a decrease in Kupffer cell numbers in Nucling-knockout mice

Abstract

Nucling is a stress-inducible protein associated with apoptosomes. The cytochrome c-triggered formation of apoptosomes represents a key-initiating event in apoptosis. We have recently reported that Nucling regulates the apoptotic pathway by controlling the activation of NF-κB as well. Here we show that hepatocellular carcinoma (HCC) arising spontaneously against a background of hepatitis occurred more frequently in Nucling-knockout (KO) mice than wild-type (WT) mice. Biochemical serum testing revealed potential liver dysfunction with hypercholesterolemia in Nucling-KO males. In the background of Nucling-KO mice, we observed the up-regulation of TNFα, spontaneous NF-κB-activation and the induction of galectin-3 expression in liver. In addition, we observed a decrease in the number of Kupffer cells (KCs) in the KO mice. KCs are important for the hepatic immune system, acting as phagocytes or antigen-presenting cells (APCs). We found that KCs in Nucling-KO mice were apoptotic possibly through the up-regulation of TNFα. These observations indicate that Nucling is important for the regulation of NF-κB signals in liver. We propose that Nucling deficiency could be a powerful tool to reveal the NF-κB-related molecular networks leading to hepatitis and HCC development.

Nucling, a stress-inducible protein, is associated with the mitochondrial apoptotic pathway regulated by apoptosomes composed of cytochrome c, apoptosis activating factor (Apaf)-1 and caspase-9.1 Nucling was found to reside in an Apaf-1/procaspase-9 complex and regulate Apaf-1 expression following cytotoxic stress. Nucling also regulates the expression of galectin-3 (Gal-3) via the suppression of nuclear factor (NF)-κB signaling.2 Galectins are members of a newly defined and growing family of animal lectins of which Gal-3 is the most extensively studied.3, 4 This lectin of approximately Mr 30,000 is composed of 2 domains: a carboxylterminal domain that contains the carbohydrate-binding region and an amino-terminal domain consisting primarily of tandem repeats of 9 amino acids.5 Various studies suggest roles of Gal-3 in a variety of biological processes. In particular, Gal-3 is reported as an antiapoptotic protein whose production is triggered by NF-κB. Gal-3, which contains the NWGR anti-death motif of the Bcl-2 family, distributes in the cytoplasm and perinuclear mitochondrial membranes,6, 7 where it is involved in the control of apoptosis, possibly through interaction with the Bcl-2 protein.8 In the Nucling-KO mouse, we observed a high incidence of inflammatory lesions in the preputial gland where the expression of Gal-3 was up-regulated.2 In addition, the strictly regulated intracellular localization of NF-κB protein during its signal transduction was disturbed in Nucling-KO mice.9 The activation of NF-κB, indicated by its DNA-binding activity and the nuclear expression of RelA (p65), was increased in Nucling^−/−-MEFs compared with Nucling^+/+-MEFs.2

Several lines of evidence were reported indicating a connection between Gal-3 expression and hepatocellular carcinoma (HCC) as described below. Relevance of this lectin in cell growth and neoplastic transformation in liver has been suggested in several studies. Gal-3 is expressed in various tissues and organs but is significantly absent in normal hepatocytes. However, HCC frequently expresses significant levels of this lectin.10 On the other hand, high levels of Gal-3 promote tumor growth. Furthermore, patients with strong Gal-3 expression in HCCs show a markedly poor prognosis.11 It was also reported that Gal-3 overexpression inhibits immune response by inducing apoptosis in lymphocytes and by promoting tumor growth.12, 13 Taken together, these results indicate that the expression of Gal-3 plays an important role in the development of HCCs.

Ab: antibody; ACAT: acyl-coenzyme A:cholesterol acyltransferase; Apaf-1: apoptosis activating factor-1; CCl₄: carbon tetrachloride; DC: dendritic cell; DEN: diethyl-nitrosamine; FITC: fluorescein isothiocyanate; Gal-3: galectin-3; H&E: hematoxylin-eosin; HBSS: Hanks' balanced salt solution; HCC: hepatocellular carcinoma; HPL: hepatoportal lymph node; KC: Kupffer cell; KO: knockout; MEF: mouse embryonal fibroblast; NF-κB: nuclear factor-kappa B; NK: natural killer cell; NKT: natural killer T cell; NPC: non-parenchymal cell; Nucling^−/−: Nucling gene deficient; PBS: phosphate buffered saline; RT-PCR: reverse-transcription-polymerase chain reaction; SNP: single-nucleotide polymorphism; TNFα: tumor necrosis factor-alpha; WT: wild-type

Recent studies have revealed that the development of HCC is regulated by the balance of the differential effects of NF-κB's activation between hepatocytes and liver non-parenchymal cells (NPCs), including Kupffer cells (KCs).14 A key conclusion was that NF-κB activity in KCs contributes to cancer and that NF-κB activity in hepatocytes acts as a cancer suppressor. However, the story is not quite so simple. KCs also have multifunctional roles against cancers. KCs showed cytotoxicity in colon cancer,15, 16 which was related to their expression of tumor-necrosis factor (TNF)-α.17, 18 Nitric oxide produced by KCs after stimulation with endotoxin, TNFα and prostaglandin E₂ may also be effective against tumor cells.19 In addition, it was recently reported that estrogen-mediated reduction of interleukin-6 production by KCs reduces the risk of HCC in female mice.20, 21 Osteopontin, a highly modified integrin-binding extracellular matrix glycophosphoprotein secreted from KCs in the liver, has also been a focus of attention for its pathophysiological role in hepatic inflammation and cancer.22 On the other hand, recent studies have shown the presence of pathways for the apoptosis of KCs either via TNFα stimulation,23 or via Fas/FasL signaling mediated by NF-κB.24 These results suggest the importance of novel machinery for the maintenance of KCs, preventing their apoptosis while they fight inflammatory disease or cancer.

Defects in apoptotic mechanisms are known to contribute to cancer, autoimmune diseases and neurodegenerative disorders. Understanding these mechanisms is expected to aid in the diagnosis or treatment of diseases. One of the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis has recently been clarified by studies showing that NF-κB is important for the promotion of inflammation-associated cancer in mice.25–27

In this report, we show that a high incidence of hepatitis and HCC in Nucling-KO males. As a candidate for the machinery of HCC development in mice, we observed the up-regulation of TNFα with the spontaneous activation of NF-κB in liver. In addition, KCs were apoptotic, and decreased in number in Nucling-KO males. Furthermore, we detected the expression of Gal-3 apparently in normal liver of Nucling-KO males, and found the up-regulation of Gal-3 in HCC of Nucling-KO males. Hypercholesterolemia observed in the KO mice also can be a risk factor for the hepatic diseases as well. These findings suggest the physiological importance of Nucling for the maintenance of KCs, cholesterol level, and Gal-3 expression through the regulation of NF-κB, linking to the development of hepatic inflammation and tumorigenesis.

Material and Methods

Mice

The background of the Nucling-KO and wild-type mice is B6;129 originating from a study to produce Nucling null mice.1 E14K embryonic stem cells (derived from 129/Ola mice) were used to prepare Nucling null cells, which were subsequently injected into C57BL/6J blastocysts. The resultant male chimeras were mated with female C57BL/6J mice. The lines are referred to as B6;129.28 The male and female B6;129 Nucling^+/− mice were bred by brother x sister matings. B6;129 Nucling-KO and wild-type mice were generated by intercrossing at least six times for use in this study. Only for the diethyl-nitrosamine (DEN)-treated experiment, B6-background mice generated by backcrossing with B6 at least six times were used. Mice were maintained at 5 per polycarbonate cage on hardwood chips bedding. The mice were pathogen-free. They were sacrificed when moribund or on showing signs of chronic progressive disease.

Tumor induction and analysis

Fifteen-day-old mice and littermates on a C57BL/6 background (Nucling^+/− mice were backcrossed at least six times) were injected intraperitoneally (i.p.) with 25 mg/kg DEN (Sigma). After 8 months, they were sacrificed and their livers removed and separated into individual lobes. Externally visible tumors (≥0.5 mm) were counted and measured by stereomicroscopy. Large lobes were fixed in 4% paraformaldehyde overnight and paraffin embedded. Sections (5 μm) were H&E stained, and tumor-occupied areas were measured using Scion Image for Windows. Remaining lobes were microdissected into tumor and non-tumor tissue and stored at −80°C until analyzed.

Antibodies

Antibodies used in this study were: p65 (clone F-6) from Santa Cruz Biotechnologies; TNFα from R&D systems, Inc.; β-actin (clone AC-15) from Sigma; and Nucling (anti-Nucl. mid1) and HRP-conjugated secondary antibodies from Amersham Biosciences. Rat anti-mouse F4/80 (Serotec) was used as the marker of KCs for the immunohistochemistry of mouse liver tissues. Antibodies used for the flow cytometric analysis are described in the corresponding section.

Isolation of KCs and NPCs from liver

KCs were isolated from the mouse liver by collagenase perfusion and centrifugation using Percoll (Pharmacia). The liver was perfused in situ through the portal vein with Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution (HBSS) containing 0.5 mM EGTA at 37°C for 5 min at a flow rate of 26 ml/min. Subsequently, it was perfused with HBSS containing 0.025% collagenase IV (Sigma-Aldrich, Japan) at 37°C for 5 min (collagenase perfusion). After digestion, the liver was excised and cut into small pieces in the collagenase buffer described above. The suspension was filtered through nylon gauze, and the filtrate was centrifuged twice at 50 g for 3 min at 4°C to remove parenchymal cells (PCs) as a pellet. The non-parenchymal cell (NPC) fraction was washed with buffer and centrifuged on a density cushion of Percoll at 1,000 g for 15 min to obtain the Kupffer cell fraction.

Experimental model of liver injury

Approximately 100-day-old male mice received a single intraperitoneal injection of 10 ml/kg of a 10% (v/v) mixture of CCl₄ in mineral oil and were killed at 6 h after the injection. At least 4 mice were prepared for this experiment.

Animal experiments and tissue preparation

All animal experiments were performed in accordance with the guidelines of the institutional committee for the use of animals for research. Mice were killed with a lethal dose of anesthetic. In some animals, liver samples were removed and snap-frozen for protein analyses. The animal was then perfused through the left ventricle with 10 ml of cold PBS, followed by 25 ml of 4% buffered formalin. The liver was removed, weighed and fixed in formalin overnight. The next day the entire liver was embedded in paraffin in three to five cassettes. Sections 4-μm thick were stained with H&E and evaluated by a pathologist to whom the genetic makeup or treatment group was not known.

Histological analysis

Hepatocellular necrosis in the liver sections was assessed by a registered pathologist unaware of the treatments and scored by examining randomly chosen fields of view per tissue section as follows: grade 0 (absent), grade I (spotty necrosis; one or few necrotic hepatocytes), and grade II (bridging necrosis).

Depletion of NK1.1⁺ cells

NKT/NK cells were depleted by using mouse anti-mouse NK1.1 monoclonal Ab, PK136 (Serotec, Oxford, UK). Mice were injected with 50 μg i.p. The depletion of NK1.1⁺ cells was assessed by FACS analysis as described in the flow cytometric analysis section. Cells were isolated from spleen, hepatoportal lymph node (HPL) and liver of the mice 7 days after the Ab injection.

Immunohistochemistry

For RelA (p65) immunostaining, 4-μm thick sections, cut on the same day, were de-waxed and hydrated through graded ethanol. After 5 min of treatment with 3% H₂O₂, slides were incubated with rabbit polyclonal p65 Ab diluted 1:100 in PBS with 3% goat serum for 2 h at room temperature, washed three times with PBS, and treated with biotinylated anti-mouse (1:500, Vectastain ABC kit) for 1 h and avidin-biotin complex (Vectastain ABC kit) for 1 h. Detection was performed with 0.05% DAB and 0.03% hydrogen peroxide in 0.1M Tris-HCl, pH 7.4. Sections were counterstained with methyl green to visualize the nuclei.

Real-time RT-PCR (reverse-transcription-polymerase chain reaction)

Total RNA was extracted from parenchymal or non-parenchymal cells of liver in Nucling-KO or WT mice using ISOGEN (Wako Pure Chemical, Osaka, Japan), and reverse transcribed. Transcript levels of NF-κB and β-actin were examined using PTC-200 DNA Engine Cycler (MJ Research Incorporated, Waltham, MA) with SYBR Premix Ex Taq (Takara, Kyoto, Japan). Results were calculated with the software of the DNA Engine Opicon System (Roche Molecular System, Inc., Alameda, CA). In addition, conventional RT-PCR was performed for the transcript levels of Fas, TNFα, and G3PDH. Primer sequences were as follows: NF-κB: forward, 5′-ATGGCAGACGATGATCCCTA-3′ and reverse, 5′-TAGG CAAGGTCAGAATGCAC-3′, β-actin: forward, 5′-GTG GGCCGCTCTAGGCACCA-3′ and reverse, 5′-CGGTTGGCC TTAGGGTTCAGGGGGG-3′, Fas: forward, 5′-CAGACATGC TGTGGATCTG-3′ and reverse, 5′-GGTTCTTGTCCATGTAC TCC-3′, TNFα: forward, 5′-ATGAGCACAGAAAGCATGAT CC-3′ and reverse, 5′-CCAAAGTAGACCTGCCCGGACTC-3′, G3PDH: forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′.

Flow cytometric analysis

Using monoclonal Abs in conjunction with 2- or 3-color immunofluorescence tests, surface phenotypes of the cells were identified. The monoclonal Abs used included fluorescein isothiocyanate (FITC)-conjugated anti-NK1.1 Ab (eBioscience, San Diego, CA), phycoerythrin (PE)-or FITC-conjugated anti-F4/80 Ab (eBioscience), and FITC-conjugated anti-CD11c Ab (eBioscience). Other reagents used were PE- or Cy5-conjugated streptavidin (eBioscience), and an Annexin V-PE or FITC Apoptosis Detection Kit (BioVision, Inc., Mountain View, CA). Lymphocytes were gated by forward scatter, side scatter and propidium iodide gating, and were analyzed by EPICS flow cytometer (Beckman Coulter, Inc., Miami FL).

Western blotting

Western blotting was performed according to the standard procedures. All antibodies were used at recommended dilutions. Briefly, whole liver cells or NPCs prepared as described above were lysed in RIPA buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride), and 50 μg of protein was fractionated by SDS-PAGE. The membrane was blocked for 1 h with PBS containing 5% nonfat dry milk, and 0.1% Tween-20, followed by incubation with appropriate antibodies in blocking buffer for 2 h. Bound primary antibodies were detected by incubating with horseradish peroxidase-conjugated goat secondary antibodies. The membrane was developed using ECL reagent (Amersham). Bands were quantified by densitometry.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts from mouse whole liver cells were prepared using the cell fractionation procedure described previously.29 EMSA was performed according to the standard procedures as described previously.2 A NF-κB-specific consensus oligonucleotide described previously2 used as a cognate DNA-binding sequence was end-labeled with γ-P32 ATP (Amersham). Samples of 10 μg of nuclear protein extract were incubated in binding buffer (10mM Tris, pH 7.5, 1 M NaCl, 1 mM EDTA, 4% glycerol, and sonicated sperm DNA) with or without excess unlabeled NF-κB-specific oligonucleotide for 15 min on ice. End-labeled NF-κB (1.5 × 10⁶ cpm) was added and samples were incubated for an additional 20 min at room temperature. Free oligonucleotide and oligonucleotide-bound protein were separated by electrophoresis on a non-denatured 6% polyacrylamide gel. Gels were dried and exposed on X-ray film (BioMax MS, Kodak). An absence of binding in the presence of excess unlabeled NF-κB-specific oligonucleotide confirmed NF-κB-binding specificity.

Results

Hepatitis and HCC in Nucling-KO mice

HCC, one of the leading causes of cancer deaths in the world, commonly develops against a background of chronic hepatitis.30 We observed the spontaneous development of hepatic diseases in aged Nucling-KO mice, mimicking human hepatocarcinogenesis. Mice were allowed to live out their life, until moribund, or until 104–105 weeks of age, at which time all surviving mice were sacrificed. We found that the rate of morbidity from hepatitis was significantly higher in moribund or naturally deceased Nucling-KO males than WT mice (Fisher's exact test; p < 0.01). In addition, tumor development in KO males progressed with inflammation in the liver as is commonly observed in human HCC: 17.9% (5/28) of KO males, 8.3% (1/12) of heterozygotes, and 8.3% (1/12) of WT males developed HCC with hepatitis (Table 1). HCCs were present in 8 of the 29 Nucling-KO males, 4 of the 31 KO females, 1 of the 15 WT males, and 1 of the 13 females (Table 1). Hepatocellular neoplasms are conventionally classified as hepatocellular adenomas, and well-differentiated, moderately-well differentiated and poorly differentiated HCCs.31 In the case of Nucling-KO mice, none of the hepatocellular neoplasms were adenomas, but all were diagnosed as HCC. The diagnosis of HCC was based on the presence of a distinct trabecular or pseudoglandular pattern as well as of cytologic features characteristic of malignancy. Cytologic features suggestive of malignancy included combinations of an increased nuclear to cytoplasmic ratio, an increased mitotic index, bizarre mitotic figures and pleomorphism. Of the total of 12 HCCs in Nucling-KO mice in the study, 8 were classified as well-differentiated, 4 as moderately-well differentiated, and none as poorly differentiated. On the other hand, of the total of 2 HCCs in WT mice, 1 was classified as moderately-well differentiated and the other as poorly differentiated. The well-differentiated HCCs were composed of fairly uniform cells with a moderate amount of eosinophilic granular cytoplasm. The tumor cells formed a thin trabecular (arrowhead) or pseudoglandular (arrow) pattern (Fig. 1a). The moderately-well differentiated HCCs (Fig. 1b) were composed of larger cells that varied more in size and shape, and frequently formed cords (arrowhead) or solid sheets (arrow). The poorly differentiated foci, which are composed of large anaplastic cells with bizarre nuclei, were rarely observed in HCCs of our experiment. They presented either a prominent thick trabecular or a solid pattern (Fig. 1c). HCCs often appeared to invade adjacent hepatic parenchyma but metastasized infrequently. In conclusion, the tumorigenesis in the Nucling-KO mouse, as observed in human HCCs,30 presented against a background of chronic inflammation in the portal areas (Fig. 1d), and showed multiple carcinogenic steps, preneoplastic foci, hyperplastic nodules and carcinomas. Macro-vesicular fatty changes in HCC were often observed in Nucling-KO mice (37.5% in males (n = 16) and 37.5% in females (n = 8) but not in WT mice (0% in males (n = 4)). These phenotypes were found only in the aged mice older than 70 weeks. As we previously reported, no obvious phenotypic abnormalities or developmental defects had been observed in Nucling-KO mice younger than 1 year old, except frequent inflammatory lesions of preputial glands in some of the males.2 Therefore, these hepatocellular abnormalities were the alternative unique feature found in aged Nucling-KO mice.

Figure 1. H&E-stained sections from the liver of a Nucling-KO mouse with spontaneous HCC. Many of the pathological features of human HCC were found in the Nucling-KO mouse with HCC. (a) As observed in human HCCs, trabecular (arrowhead) and pseudoglandular arrangements (arrow) of the tumor cells are evident in many of the Nucling-KO mice with HCC. (b) The moderately-well differentiated HCCs were composed of larger cells which varied more in size and shape, and frequently formed cords (arrowhead) or solid sheets (arrow). Fatty change of the tumor cells is occasionally found in moderately-well differentiated HCCs (right panel). (c) The poorly differentiated foci, which were rarely observed in HCCs of our experiment, presented either a prominent thick trabecular or a solid pattern. (d) Necroinflammatory processes are seen in the portal and parenchymal areas of non-tumorous regions of tumor-bearing animals. Magnification of the area indicated in the left panel clearly shows that lymphocytic inflammatory infiltrates widened the portal areas and focal necrosis occurred in the parenchymal areas (right panel). (e) Livers of 10-month-old DEN-treated male WT and Nucling-KO mice. Arrows, neovascularization. (f) Typical liver histology (2.5 × magnification; H&E stain) in 10-month-old DEN-treated male WT and Nucling-KO mice. (g) Numbers of tumors (≥0.5 mm) in livers of WT (male, n = 13; female, n = 11) and Nucling-KO (male, n = 11; female, n = 9) mice 10 months after DEN (5 mg/kg) injection. (h) Maximum tumor size (diameters). (i) Incidence of HCCs (≥0.5 mm) in WT (male, n = 13; female, n = 11) and Nucling-KO (male, n = 11; female, n = 9) mice 10 months after DEN injection. (j) Incidence of fatty changes in livers of WT and Nucling-KO mice 10 months after DEN (5 mg/kg) injection. (k) Weight of livers of WT (male, n = 7 for DEN (−), n = 6 for DEN (+); female, n = 11 for DEN (−), n = 6 for DEN (+)) and Nucling-KO (male, n = 9 for DEN (−), n = 6 for DEN (+); female, n = 7 for DEN (−), n = 7 for DEN (+)) mice 10 months after DEN (5 mg/kg) (+) or PBS (−) injection. Results in g, h and k are averages ±S.E. Asterisks, p* < 0.05, p** < 0.01 by Student's t test.

Table 1. Prevalence of hepatocellular carcinoma (HCC) and/or hepatitis in Nucling-knockout (KO), Nucling-heterozygote (Hetero), and wild-type (WT) mice

Nucling-KO mice exhibit increased susceptibility to chemical hepatocarcinogenesis

To exclude the effect of mixed background (B6;129), we generated Nucling-KO mice with B6 background. Then, we investigated the susceptibility to chemical hepatocarcinogenesis in the mice. Nucling-KO or WT mice were injected with DEN (5 mg/kg) on day 15 postnatally. Ninety percent of Nucling-KO males given DEN developed typical HCCs within 10 months (Fig. 1e and f). Strikingly, the number of detectable HCCs was 5-fold higher in Nucling-KO males than in WT controls (Fig. 1g). The maximal tumor diameter was also 2-fold larger in Nucling-KO males (Fig. 1h), and so were tumor incidence (Fig. 1i). Notably, many HCCs in Nucling-KO males, but less in the controls, exhibited signs of neovascularization (Fig. 1e). Macro-vesicular fatty change in liver was often observed in DEN-injected Nucling-KO males (50%, n = 6), but not in KO females (n = 4) or WT mice (n = 6 for males, n = 4 for females) (Fig. 1j). DEN-injected Nucling-KO mice also exhibited significantly larger livers than similarly treated WT mice (Fig. 1k).

These observations strongly indicate that Nucling gene-deficiency increases susceptibility to DEN-induced carcinogenesis. This supports the direct effect of Nucling gene disruption toward the spontaneous development of HCCs in mice.

Nucling-KO mice tend to suffer from liver dysfunction with concomitant decrease of KCs

Since we observed a high prevalence of spontaneous HCC and/or hepatitis in Nucling-KO males, we investigated whether the KO males potentially suffer from liver dysfunction. Biochemical assays for liver function revealed that the activities of AST, ALT, LDH (supplementary Fig. 1) and serum alkaline phosphatase (data not shown) tended to increase significantly in apparently healthy Nucling-KO males. Therefore, we focused on the origin of the potential liver dysfunction in Nucling-KO males. In addition to having abnormal levels of liver enzymes, Nucling-KO mice strongly tended to suffer from hypercholesterolemia (≥180 mg/dL) (11.9% for males, and 11.1% for females) in comparison to WT mice (0% for both genders). Total cholesterol levels were significantly increased in Nucling-KO mice (144.9 mg/dL on average for males, and 136.4 for females) compared to WT mice (103.1 for males, and 99.3 for females) (supplementary Fig. 1).

Since KCs are a major site for the uptake and removal of LDL from plasma, and also play a crucial role in systemic immune defense, we investigated whether Nucling-KO mice have dysfunctional KCs. First, we analyzed the population of KCs. A Percoll gradient preparation method and immunohistochemistry using antibody against F4/80, which is a specific marker for mature macrophages and used as a marker for KCs in the liver, clearly showed that the number of KCs was smaller in Nucling-KO males than WT mice (Fig. 2a and b). Flow cytometric analyses revealed that the population of KCs in the liver of Nucling-KO males was approximately 3.1%, while that in the WT liver was about 13.6% (Fig. 2c). Statistic analyses also showed a significant difference in the population of KCs between the KO and WT livers of males (Fig. 2c). We also confirmed that the decrease in number of KCs was not observed in the livers of Nucling-KO females (Supplementary Fig. 2). Thus we hypothesized that the HCC development in Nucling-KO males is closely related with the decrease in the number of KCs.

Figure 2. The populations of liver Kupffer cells and dendritic cells were decreased in Nucling-KO mice. (a) A percoll gradient was used to separate KCs from NPCs prepared from WT and Nucling-KO mice. The asterisk (*) indicates the layer containing cell debris and clumped red blood cells. (b) An immunohistochemical analysis using F4/80 Ab was performed to investigate the expression profile of KCs in WT and Nucling-KO liver. Many of the liver cells of WT mice were stained brown. Few cells were stained in the livers of Nucling-KO mice. Scale bars, 30 μm. (c) Flow cytometric analysis revealed that the population of F4/80-positive cells (=KCs) among liver NPCs was decreased in Nucling-KO mice compared with WT mice. (d) KCs and DCs are absent in the hepatoportal lymph node of Nucling-KO mice. DCs (left panel) or KCs (right panel) from Nucling-WT (black line) and KO (red line) mice were incubated with FITC-conjugated CD11c (for DCs) or FITC-conjugated F4/80 Ab (for KCs) and analyzed by flow cytometry.

The KC/DC liver defense system is impaired in Nucling-KO mice

Since DCs are selectively trapped by KCs in the sinusoid and migrate to the lymphoid tissue of the portal area with KCs, the population of DCs and KCs must have shrunk in the hepatoportal lymph nodes (HPL) of Nucling-KO mice if the number of KCs in the Nucling-KO liver did indeed decrease. Therefore, we analyzed the population of DCs with the use of flow cytometry in order to check whether the KCs were functionally impaired in Nucling-KO mice. We observed that the populations of CD11c⁺ DCs and F4/80⁺ cells, regarded as KCs, decreased in size in HPL in Nucling-KO mice (Fig. 2d). We also confirmed that the populations of CD11c⁺ DCs and F4/80⁺ cells in spleen and mesenteric lymph nodes did not differ significantly between WT and Nucling-KO mice (supplementary Fig. 3). These results indicate that the decrease in numbers of DCs and KCs was liver specific and was not due to the impaired functioning of hematopoietic stem cells. We also verified the normal production and maintenance of hematopoietic cells in Nucling-KO mice by confirming normal cell counts in the peripheral blood of Nucling-KO mice (data not shown). Thus we concluded that the reduction in numbers of KCs and DCs was a posteriori phenomenon in the liver of Nucling-KO mice.

Figure 3. Reaction against CCl₄ treatment in the liver of Nucling-KO mice. (a) Western blotting was performed to investigate the expression of TNFα in the liver of Nucling-KO (ko) and WT (wt) mice following the i.p. injection of PBS (-) or CCl₄. A monoclonal Ab against β-actin was used as an internal control. (b) Nucling-KO liver is resistant to hepatocellular necrosis in CCl₄-treated mice. Representative photomicrographs of liver sections stained with H&E from 6-h PBS-treated (control) or CCl₄-treated mice of WT (left panels) and Nucling-KO (right panels) mice.

We then suspected that the liver dysfunction and inflammatory reactions in Nucling-KO mice were associated with a decreased number of CD11c⁺ DCs and a decreased number of KCs, because inflammatory reactions would be expected if the liver KC/DC defense system was impaired. Actually, we have observed a high prevalence of several inflammatory disorders including preputial gland abscess, pneumonia (unpublished data), and hepatitis in Nucling-KO mice. In order to test our hypothesis, we investigated whether the production of pro-inflammatory cytokines was up-regulated or not in Nucling-KO liver. Western blotting was performed to check the level of TNFα in the liver of Nucling-KO and WT mice (Fig. 3a). We observed an increase in the cytokine in the liver of Nucling-KO mice. We also observed that the liver of Nucling-KO mice was much more sensitive to stimulation with carbon tetrachloride (CCl₄) for the induction of TNFα expression than was that of WT mice (Fig. 3a), when CCl₄ was used as an inflammatory stress inducer. Thus, we concluded that Nucling-KO males tend to suffer from inflammatory disorders with the production of pro-inflammatory cytokines even under slight stress.

KCs in Nucling-KO mice were impaired by apoptosis

Since Nucling is an apoptosis-associated protein, we next investigated whether the decrease in the number of KCs in the liver of Nucling-KO mice results from apoptosis. Annexin V staining was performed to check the population of apoptotic cells among NPCs including KCs prepared from the liver of Nucling-KO mice. Flow cytometry revealed that a considerable number of NPCs prepared from Nucling-KO mice were apoptotic (supplementary Fig. 4a). The flow cytometric analysis using F4/80 antibody with propidium iodine staining (supplementary Fig. 4b) or Annexin V staining (data not shown) indicated that more than 8% of F4/80-positive cells (=KCs) were apoptotic in Nucling-KO mice, and less than 4% in WT mice.

Figure 4. NF-κB was spontaneously activated in the normal liver of Nucling-KO mice. (a) Nuclear extracts from 8-month-old Nucling-WT and KO livers were isolated and analyzed for activated NF-κB by EMSA. (b) Liver tissue sections from 4-month-old WT and KO mice were immunostained with Ab against RelA (p65) (right panels). RelA-positive nuclei were ubiquitous in normal KO livers. H&E-stained sections of livers from WT and KO mice (left panels) showed no differences. Scale bars, 50 μm. (c) Transcriptional expression of NF-κB was investigated in parenchymal (PC) or non-parenchymal (NPC) cells prepared from livers of Nucling-KO and WT mice. The mRNA of NF-κB was detected by real-time RT-PCR. Data are shown relative to β-actin mRNA, and represent means SE for triplicate wells. In order to confirm the result of real-time RT-PCR, Western blotting was performed. 10 μg of whole cell lysates prepared from PCs and NPCs of WT or Nucling-KO males were loaded on the 10/20% gradient SDS-PAGE gel followed by the detection using ECL detection kit with appropriate antibodies. (d) Expression of Fas and TNFα decreased in the NPCs of Nucling-KO mice. Fas and TNFα levels were monitored by RT-PCR of polyA⁺ RNA prepared from WT-NPC (NPC^+/+) and Nucling-KO-NPC (NPC^−/−) mice with appropriate primers. G3PDH levels were checked as an internal control. Western blotting was also performed to check the protein levels of TNFα in NPCs of WT and Nucling-KO males.

To confirm the apoptotic impairment of KCs in Nucling-KO liver, we examined WT and mutant mice with CCl₄-induced liver injury. Previous reports have indicated that KCs participate in the toxicity of CCl₄, based on observations of hepatic depletion of KCs by apoptosis induced by reagents such as gadolinium chloride32–35 and Bay-X-1005, a potent 5-lipoxygenase-activating protein inhibitor.36 Histological examination of livers from WT mice treated with CCl₄ for 6 h revealed, as expected, acute inflammatory reactions including massive and severe necrosis of hepatocytes and bridging necrosis, and a severely disrupted lobular architecture of the liver (Fig. 3b, left panels). All WT mice treated with CCl₄ had grade II hepatocellular necrosis. In contrast, CCl₄-treated Nucling-KO mice showed remarkable protection from hepatocellular necrosis with no evidence of bridging necrosis or ballooning hepatocytes (Fig. 3b, right panels). In fact, all Nucling-KO mice treated with CCl₄ for 6 h had grade 0 or I necrosis. These results are quite similar to those for CCl₄-treated animals receiving Bay-X-1005,36 suggesting that the absence of KCs provided the protection. These observations clearly indicate that KCs are functionally impaired in Nucling-KO liver.

KCs in Nucling-KO mice were rendered apoptotic possibly through the up-regulation of TNFα

Since KC numbers were considered to be decreased with apoptosis in the liver of Nucling-KO mice, we speculated that some apoptotic pathway operating in KCs is up-regulated in the liver of Nucling-KO mice. It was suggested that NF-κB was capable of inducing the expression of p53, Fas, FasL, Bax, c-myc, DR4, DR5 and Bcl-xS, which are actively involved in the promotion of apoptosis.37 Therefore, we subsequently investigated the activation of NF-κB in the liver of Nucling-KO mice. An electro-mobility-shift assay (EMSA) revealed that NF-κB was activated to a much greater extent in aged Nucling-KO liver than WT liver (Fig. 4a). RelA immunostaining revealed that nuclear labeling, indicative of NF-κB activation, was evident in 83.3% of hepatocytes from KO mice (n = 6), but only 14.3% of those of age-matched WT mice (n = 7) (Fig. 4b). We also confirmed that the expression of NF-κB in parenchymal cells (PCs) was significantly higher in Nucling-KO liver than WT liver at both transcription and protein levels (Fig. 4c). On the other hand, the expression level of NF-κB in NPCs was lower in KO liver than WT liver (Fig. 4c), which was consistent with the decreases in numbers of KCs and DCs, major populations of NPCs. Indeed, RT-PCR revealed that the expression level of Fas, the NF-κB regulated gene, was significantly decreased in the NPCs from Nucling-KO mice (Fig. 4d).

In order to reveal the mechanism of apoptosis in KCs, we checked several apoptosis-related genes in liver of Nucling-KO mice, in which NF-κB was spontaneously activated. Thus we performed real-time RT-PCR to investigate whether or not such downstream signal pathway of NF-κB leading to apoptosis is abnormally activated (Fig. 5a). We checked six NF-κB target genes, Fas,37 c-IAP1,38 A1,39 GADD45β,40 IL-6, and TNFα. As expected from the previous results, the expression of TNFα in PCs was significantly increased, while that in NPCs was decreased. Among others, only c-IAP1 showed similar expression patterns. Fas, A1 and GADD45β showed no significant difference between WT and KO both in PC and NPC. Only the expression of IL-6 was increased in the NPC of Nucling-KO mice. These data suggest that Nucling is selectively important for some of the NF-κB-related apoptosis pathways in liver. Especially, TNFα or cIAP seem to be more crucial than Fas for KCs apoptosis. In addition, it has been reported that TNFα mediated apoptosis in KCs.41 Thus we next focused on TNFα as a candidate for the key molecule of KCs apoptosis.

Figure 5. Expression profiles of NF-κB regulate genes in Nucling-KO liver before and after the treatment with PK136. (a) Relative expression levels of six NF-κB regulated genes against β-actin were checked by real-time RT-PCR. Five micrograms of total RNA prepared from PCs or NPCs of WT or Nucling-KO males was used to investigate the transcriptional levels of TNFα, Fas, cIAP1, A1, GADD45β, and IL-6. Results are averages ± S.E. of at least three independent trials. (b) Western blotting was performed to examine the changes in expression of F4/80, β-actin and TNFα in the lysates of NPCs from the liver of WT or Nucling-KO mice injected i.p. with PK136 or saline. In both genotypes, TNFα expression was strongly up-regulated with the injection of PK136. (c), Flow cytometric analysis was performed to check the population of KCs in NPCs of WT or Nucling-KO males after the injection of PK136 or PBS. Arrows indicate KCs populations.

TNFα is one of the candidates, which induce cell death via multiple mechanisms.42, 43 In order to investigate whether or not the decrease of KCs can be promoted by the up-regulation of TNFα in our system, we used the antibody against NK1.1 (PK136). PK136 is known to induce in vivo depletion of NK1.1 positive cells with the induction of TNFα but not of IL-12 or IFN-γ44 At first, we investigated whether or not the induction of TNFα was promoted in NPCs after the injection of PK136. As expected, PK136 injection highly increased the levels of TNFα in both NPCs from Nucling-KO and WT mice (Fig. 5b). Next, flow cytometric analysis revealed that PK136 rendered the depletion of KCs in both WT and Nucling-KO liver (Fig. 5c). In conclusion, KCs depletion with the injection of PK136 can be explained by the induction of TNFα. This result strongly suggests that the decrease in number of KCs in Nucling-KO mice is led by the spontaneous up-regulation of TNFα.

Gal-3 is up-regulated in the liver of Nucling-KO male during the development of HCC

Gal-3 is also a strong candidate for the key molecule in the development of tumors.11, 12 Thus we checked the expression pattern of Gal-3 in livers of Nucling-KO HCCs by Western blotting. As expected from the previous reports,10, 45, 46 Gal-3 expression was not observed in normal hepatocytes in WT livers (Fig. 6a, lane 1 and 2). On the other hand, weak but clear Gal-3 expression was observed in normal hepatocytes in livers of Nucling-KO males (Fig. 6a, lane 3). No expression of Gal-3 was observed in normal hepatocytes of Nucling-KO females (Fig. 6a, lane 4). In human and rat HCC, the over-expression of Gal-3 has been reported.10, 47 Thus we checked the expression of Gal-3 in the chemically induced HCC of mice by immunoblot analysis. HCCs were prepared from males and females of WT and Nucling-KO mice of 10 months after the injection of DEN at 15 days of age. In HCCs of WT mice, we could observed the induction of Gal-3 only in males (n = 3) but not in females (n = 2) (Fig. 6b, lane 1, 2 and 5). On the other hand, much stronger up-regulation of Gal-3 was observed in HCCs of Nucling-KO males (n = 3) (Fig. 6b, lane 3 and 4). We could also see the induction of Gal-3 in HCCs of Nucling-KO females (n = 2) (Fig. 6b, lane 6 and 7). These observations suggest the close correlation between Gal-3 and the initiation of HCC in Nucling-KO mice.

Figure 6. Gal-3 is expressed in liver of Nucling-KO male, and is upregulated by DEN injection. (a) Western blotting was performed to check the expression of Gal-3 in PC of WT male, WT female, Nucling-KO male, and KO female. Endogenous Gal-3 expression was observed only in PC of Nucling-KO male. (b) Gal-3 was induced in PC of WT male and Nucling-KO male and female 10 months after DEN (5 mg/kg) injection. Among them, induction level of Gal-3 in Nucling-KO male was much higher than others. At least three individual preparations were investigated, and one (lane 5 for WT female) or two (lanes 1, 2 for WT male, lanes 3, 4 for Nucling-KO male and lanes 6, 7 for WT female) representative results were shown.

Discussion

A high rate of morbidity from hepatic neoplasia was observed only in male Nucling-KO mice over a period of more than two years. The background of the mice used in the study for the spontaneous development of HCC was B6;129 as described in the METHODS section. There was no significant difference among aged WT male, WT female, and hetero mice concerning the prevalence of HCC (6.7%, 7.7%, and 8.3%, respectively) (Table 1). These results are consistent with those for aged B6;129 mice.48, 49 On the other hand, the prevalence of hepatitis varied among aged WT males, WT female and hetero mice (14.3%, 36.4%, and 8.3%, respectively) (Table 1). In a previous report, the incidence of inflammatory findings in aged B6;129 liver was 56% in males and 74% in females.48 It should be emphasized that the incidences of tumor and inflammation in our study are based on single groups of males and females. In animal experiments, there is a binomial or nonbinomial distribution of the incidence of tumor or inflammation probably depending, in part, on etiology, genetics, environmental conditions, diet and unknown factors.50 Group size also plays a role in the variation and range of incidences. In addition, HCC usually occurs in human individuals with chronic liver disease, such as hepatitis, with a clear disadvantage for the male sex.51 In Nucling-KO mice, not only HCC but also hepatitis was observed more frequently in males than in females (Table 1). Thus we consider the Nucling-KO mouse to be a good model for human HCC. On the other hand, it still remains to be clarified the contribution of Nucling's dysfunction in human HCC tissues, and its correlation to HCC patient survival.

Hepatocarcinogenesis is a multistep process with a multifactorial etiology. In the current study, we found several molecular or biochemical aspects in Nucling-KO mice to elucidate the pathways for the initiation or development of HCC. Among them, we categorize the activation of NF-κB, the induction of Gal-3, and hypercholesterolemia as the basal background leading to hepatitis and HCC (Fig. 7). These background components were observed in both males and females of Nucling-KO mice. Previous studies by several laboratories have shown the crosstalk among these three backgrounds. NF-κB signal can be involved in the regulation of Gal-3 expression (Fig. 7a).52 Mechanism for the hypercholesterolemia in Nucling-KO mice remains to be clarified. Previous works have shown the function of Gal-3 for lipid loading (Fig. 7b). Gal-3 mediates the endocytotic uptake of modified low-density lipoproteins resulting in intracellular accumulation of cholesteryl esters.53 Among the NF-κB-regulated genes, we focused on TNFα (Fig. 7c). In this study, we found the elevation of TNFα level in the liver of Nucling-KO males. Previous studies have also reported that TNFα itself is tumorigenic and acts as a tumor promoter.54, 55 It has been found that circulating TNFα levels were elevated in patients with HCC.56, 57 Recent study has shown that there was a strong association between the single-nucleotide polymorphism (SNP) of TNFα gene (TNF308.2 SNP) and risk for HCC.58 TNF308.2 SNP has been shown to increase the constitutive and inducible expression of TNFα protein.59, 60 It is reasonable to speculate that the high circulating TNFα levels may be attributed to the initiation of HCC (Fig. 7d).

Figure 7. Hypothetical pathways for the development of HCC and hepatitis. NF-κB activation and Gal-3 expression in hepatocyte are abnormally observed with hypercholesterolemia in Nucling-KO mice. These background components are closely related with each other with no gender disparity. In brief, Gal-3 (a) or TNFα (c) is transcriptionally regulated by NF-κB signaling. Gal-3 is also reported to be a regulator for lipid loading. Up-regulation of Gal-3 may lead to hypercholesterolemia (b), followed by the onset of steatosis (e). Up-regulation of TNFα in hepatocyte and decrease in number of KCs in Nucling-KO males can be simply explained as downstream components of NF-κB signaling (c and f). On the other hand, abnormal up-regulation of Gal-3 or TNFα can directly link to inflammatory disorders or tumorigenesis (d). (g) Decrease of KCs can directly influence the defect of immune system in the liver. (h) Apoptotic stimulation for KCs may induce the release of mitogenic factors including TNFα and IL-6 from the cells before their final death. Bold arrows indicate links that we clarified or suggested from our results. Narrow arrows indicate links shown or suggested in previous reports.

Previously, we showed that Gal-3 expression is inhibited by Nucling leading to resistance to proapoptotic stress.2 In addition, some recent reports showed a connection between Gal-3 expression and HCC. High levels of Gal-3 promote tumor growth. Furthermore, patients with strong Gal-3 expression in HCCs showed a markedly poor prognosis.11 It was also reported that Gal-3 overexpression inhibited immune response by inducing apoptosis in lymphocytes and by promoting tumor growth.12, 13 Taken together, these results indicate that the expression of Gal-3 plays an important role in the development of HCCs (Fig. 7d). Actually, we could observe the induction of Gal-3 in liver of Nucling-KO mice and the up-regulation during the development of chemically induced HCC.

In the biochemical serum testing, cholesterol levels of females are not well correlated with the incidence of HCC. Hypercholesterolemia was observed in both genders of Nucling-KO mice. This may suggest that Nucling gene deficiency leads dyslipidemia independent from liver dysfunction. In the experiment using DEN treated mice, we noticed that pathological fatty changes were significantly introduced in the liver of Nucling-KO males. The similar fatty changes were often observed in the spontaneous HCC livers of Nucling-KO mice, too. From these observations, we think that Nucling-KO males have a strong tendency to exhibit steatosis (Fig. 7e). In addition, DEN-injected females are likely to be prevented from liver fatty changes, underlying mechanism of which remains to be elucidated.

In conclusion for gender disparity in Nucling-KO mice, we observed no significant difference of NF-κB activation, up-regulation of TNFα, and hypercholesterolemia between males and females (data not shown and supplementary Fig. 1). On the other hand, the decrease in number of KCs in the females was not evident (supplementary Fig. 2), and the expression profiles of NF-κB regulated genes showed disparity between males and females of Nucling-KO mice (Fig. 5a and supplementary Fig. 5). These observations suggest that the gender disparity of development of hepatitis and HCC in Nucling-KO mice is closely related with the gender specific events for the steps d–f in Figure 7. Recent study has revealed that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females.20 In our system, IL-6 production was increased in NPCs of Nucling-KO males (Fig. 5a), and decreased in that of KO females (Supplementary Fig. 5). IL-6 production from TNFα-stimulated KCs in Nucling-KO males may be important for the HCC development (Fig. 7h).26

In this report, we showed that the maintenance of the population of KCs is impaired in Nucling-KO mice. As potential factors in the regulation of KC population, NK1.1-positive cells and the activation of NF-κB were examined for a correlation with KCs. Flow cytometric analysis revealed that the decrease in the population of KCs was correlated with the stimulation of NK1.1⁺ cells with PK136 in the NPCs of Nucling-KO mice (Fig. 5c). These results suggest that the induction of the apoptosis of KCs is closely related with the activation of NK/NKT cells. Actually, PK136 is not only useful for the depletion of NK1.1⁺ cells,61 but also an antibody for the activation of these cells.62 Thus, some factors like cytokines produced with the activation of NK1.1⁺ cells by PK136 possibly acted to induce apoptosis of KCs. As evidence in support of this hypothesis, we observed the constitutive activation of NF-κB in Nucling-KO liver. In addition, we observed strong expression of TNFα in liver following the depletion of NK/NKT cells on the injection of PK136 (Fig. 5b). These observations suggest that the apoptosis of KCs is induced by the up-regulation of NF-κB-regulated genes in Nucling-KO liver. Among the NF-κB-regulated genes, TNFα is a pleotropic cytokine that can induce both cell death and cell proliferation. TNFα-induced cell death is normally blocked by the simultaneously activated NF-κB pathway.63 In Nucling-KO mice, NF-κB pathway has some defects.9 Thus, the cell death-inducing effect by TNFα may result in the decrease in number of KCs in Nucling-KO mice (Fig. 7f).

The high prevalence of hepatitis and HCC in Nucling-KO mice can be closely related to the decrease in number of KCs, because KCs are required for the maintenance of the immune system in the liver (Fig. 7g). It has been shown that KCs are attracted to tumor cells in the hepatic circulation and have the ability to phagocytose these cells.64 The immune system plays an important role in the elimination of tumors in mammals. KCs, the resident liver macrophages lining the hepatic sinusoidal walls, represent an important component of the innate liver immune system, as blood-borne tumor cells encounter these sinusoids via the portal system. The liver actively screens and captures antigens in the blood via KCs, which directly face the bloodstream. Also, minute amounts of microorganisms in the gut may frequently encounter this organ via the portal vein, altering the liver's defense system. KCs selectively trap bloodborne dendritic cells (DCs) and help them to function as antigen-presenting cells in the liver. The DC system is generally the initiator and modulator of immune responses. The recruited DCs extravasate into Disse's space and migrate to the lymphoid tissue of portal areas. Thus, the liver is considered to play essential roles in the defense against bloodborne antigens through a specific immune system composed of KCs and DCs. These findings strongly suggest the importance of the KC machinery in combating tumor development.

Several studies have revealed that KCs play an important role in the development of hepatocarcinogenesis by releasing mitogenic factors.26, 65 This seems to contradict our results showing the high prevalence of HCC in Nucling-KO mice with decrease in number of KCs. In addition, there is no report showing the responsibility of the decrease of KCs for the tumor development. However, we also confirmed that the release of TNFα, a key factor in the tumorigenesis of HCC, from stimulated NPCs was promoted in the liver of Nucling-KO mice compared with WT mice (Fig. 5b). In fact, this is not the direct evidence showing the relationship between the decrease of KCs and the development of HCC. We have no data showing how the decrease of KCs contributes to the tumor development in Nucling-KO mice. In addition, release of cytokines from KCs is not required to explain the machinery of HCC development even in our model. The treatment of Nucling-KO mice with CCl₄ induced the expression of TNFα (Fig. 3a), indicating that hepatocytes themselves can produce TNFα for tumorigenesis. The decrease of KCs and the high prevalence ratio of HCC in Nucling-KO mice may be just a correlation. Thus, we propose that apoptotic signaling might stimulate KCs to release mitogenic factors before their final death (Fig. 7h) as one of the possibilities. In addition, KCs are a major site for the uptake and removal from plasma of oxidatively modified forms of low-density lipoprotein (LDL).66 The homeostatic intracellular cholesterol content is regulated by cholesterol synthesis, influx and efflux. Cholesterol is stored either as free cholesterol (FC) in the membrane or as cholesterol ester in cytoplasmic vesicles. The dynamic equilibrium between FC and cholesterol ester in the cell is tightly controlled by acyl-coenzyme A: cholesterol acyltransferase (ACAT).67–70 ACAT1 is most abundant in KCs.71 Moreover, KCs respond in a highly adaptive fashion by stimulating the expression of genes involved in cholesterol metabolism and transport compared to hepatic endothelial cells or parenchymal cells.72 These observations suggest that the decrease of KCs may participate in leading to hypercholesterolemia in Nucling-KO mice (Fig. 7i).

One of the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis has recently been clarified by studies showing that NF-κB is important for the promotion of inflammation-associated cancer in mice.25–27 Concerning hepatic tumorigenesis, NF-κB is often activated in human HCCs, particularly those arising against a background of chronic hepatitis.30 It has also been reported that the inhibition of NF-κB has a tumor-suppressive effect by promoting the apoptosis of transformed hepatocytes.27 Nucling is important for the regulation of the TNFα-induced activation of NF-κB.2 We, therefore, postulated that Nucling constitutes a missing link between inflammation and cancer via the NF-κB pathway. In this study, we also showed an involvement of TNFα in the tumor-development in the Nucling-KO mice. We are now interested in the tumorigenesis in Nucling-KO mice with TNF-null or TNF receptor-null background for further investigation. Taken together, our findings may indicate that Nucling plays crucial roles in the promotion of inflammation-associated cancer by regulating the activation of related apoptotic pathways.

Acknowledgements

The authors thank M. Shono for assistance with confocal microscopy. We also thank J. Yamada and C. Takai for technical support and Y. Manabe for conducing biochemical analyses.