Wataru Shibata

Deletion of Apoptosis Signal-Regulating Kinase 1 Attenuates Acetaminophen-Induced Liver Injury by Inhibiting c-Jun N-Terminal Kinase Activation

BACKGROUND & AIMS Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure. C-jun N-terminal kinase (JNK) is thought to play a central role in APAP-induced liver injury, although its upstream activator has not yet been identified. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family and is important for stress-induced JNK activation. We tested the hypothesis that ASK1 was involved in APAP-induced JNK activation and liver injury. METHODS ASK1-deficient (ASK1(-/-)) mice and wild-type (WT) mice were given 300 mg/kg of APAP. Serum alanine aminotransferase levels and liver histology were assessed. To investigate the involvement of ASK1 in direct hepatocellular damage and the subsequent inflammatory response, we used primary hepatocytes and splenocytes from WT and ASK1(-/-) mice. RESULTS In ASK1(-/-) mouse liver, APAP toxicity was attenuated significantly and the prolonged activation of JNK was inhibited. In addition, thioredoxin, a direct ASK1 inhibitor, dissociated from ASK1 after APAP overdose with concomitant ASK1 activation. Although the prolonged activation of p38 also was attenuated in ASK1(-/-) mice, the p38 signaling pathway was not likely to be involved in APAP-induced liver injury. Primary hepatocyte culture also revealed that ASK1 and JNK, but not p38, contributed to direct APAP-induced cellular damage. CONCLUSIONS Our data suggest that ASK1 is activated by APAP overdose, most likely via a mechanism involving thioredoxin-ASK1 dissociation, and that it plays a role in APAP-induced liver injury through JNK activation.

Constitutive NF-κB Activation in Colorectal Carcinoma Plays a Key Role in Angiogenesis, Promoting Tumor Growth

Purpose: Nuclear factor κB (NF-κB) is an important transcription factor in various biological processes. Constitutive NF-κB activation has been noted in many tumors, including colorectal cancers. However, the precise role of this activation in colorectal cancer is unclear. Experimental Design: Constitutive NF-κB activation was evaluated in colorectal cancer tissues and cell lines. To inhibit NF-κB activation, we established cancer cells with stable knockdown of IκB kinase γ (NF-κB essential modulator), which is the regulatory subunit of the IκB kinase complex, by RNA interference. Cell growth and apoptosis were evaluated in wild-type cells (WT) and knocked-down cells (KD). Microarray and protein array analysis were also done. To determine involvement of angiogenesis, human umbilical vein endothelial cells were used. By s.c. transplantation of the cells into nude mice, tumor sizes, vascularity, and chemodrug sensitivity were analyzed. Results: Constitutive NF-κB activation was observed in 40% of colorectal cancer tissues and 67% of cell lines. Cell proliferation was not different between WT and KD in vitro, whereas apoptosis mediated by tumor necrosis factor-α and 5-fluorouracil were increased in KD. Several angiogenic chemokines were decreased in KD. Human umbilical vein endothelial cells incubated in WT supernatant showed more branch points than in KD, suggesting that constitutive NF-κB activation was involved in angiogenesis. Subcutaneous tumor expansion was suppressed to 23% in KD, and vessels were also decreased. By 5-fluoruracil treatment, tumor expansion was suppressed to a greater extent in KD (to 6%) than in WT (to 50%). Conclusion: NF-κB inhibition may represent a potent treatment modality in colorectal cancer, especially in cases with constitutive NF-κB activation.

Cutting Edge: The IκB Kinase (IKK) Inhibitor, NEMO-Binding Domain Peptide, Blocks Inflammatory Injury in Murine Colitis

Inflammatory mediators such as TNF-α, IL-6, and IL-1 are important in the pathogenesis of inflammatory bowel diseases and are regulated by the activation of NF-κB. The aim of the present study was to investigate whether the NF-κB essential modulator (NEMO)-binding domain (NBD) peptide, which has been shown to block the association of NEMO with the IκB kinaseβ subunit (IKKβ) and inhibit NF-κB activity, reduces inflammatory injury in mice with colitis. Two colitis models were established by the following: 1) inclusion of dextran sulfate sodium salt (DSS) in the drinking water of the mice; and 2) a trinitrobenzene sulfonic acid enema. Marked NF-κB activation and expression of proinflammatory cytokines were observed in colonic tissues. The NBD peptide ameliorated colonic inflammatory injury through the down-regulation of proinflammatory cytokines mediated by NF-κB inhibition in both models. These results indicate that an IKKβ-targeted NF-κB blockade using the NBD peptide could be an attractive therapeutic approach for inflammatory bowel disease.

The effect of Helicobacter pylori eradication on reducing the incidence of gastric cancer.

Epidemiologically, the association between chronic Helicobacter pylori infection and development of gastric cancer is well established. Although the possibility of preventing gastric cancer by eradicating H. pylori infection was recently investigated by several research groups, the results remain controversial. The aim of this study was to determine whether the eradication of H. pylori infection would reduce the incidence of gastric cancer. In total, 304 patients with persistent H. pylori infection and 404 patients with H. pylori infection eradicated were examined annually for gastric cancer by endoscopy. Over an average of 3.1 years for the first group and 3.2 years for the second group, 13 and 6 patients, respectively, were diagnosed as having new gastric cancer. The cumulative incidence of gastric cancer was statistically different between the groups (P=0.019; log-rank test). The hazard ratio of H. pylori eradication was 0.335 by Cox proportional hazards model (P=0.047). Differentiated gastric cancer was found in 11 patients in the persistent infection group and 3 patients in the eradicated group. The incidence of differentiated cancer was significantly different (P=0.017) between the groups, but not for undifferentiated cancer (P=0.847). The results of the current study suggest that the eradication of H. pylori infection reduces the incidence of gastric cancer.

C-Jun NH2-Terminal Kinase 1 Is a Critical Regulator for the Development of Gastric Cancer in Mice

c-Jun NH(2)-terminal kinase (JNK) links several cellular processes, including proliferation and survival, and is believed to be involved in carcinogenesis. However, the role of JNK in gastric tumorigenesis is unknown. Immunohistochemical analysis reveals that JNK is frequently activated in human gastric cancer tissue. We investigated whether JNK1, a major JNK isozyme, is involved in chemically induced gastric cancer development. Mice lacking JNK1 exhibited a marked decrease in gastric carcinogenesis induced by N-methyl-N-nitrosourea, relative to their wild-type counterparts. Impaired tumor development correlated with decreased tumor initiation, which is associated with the production of reactive oxygen species. We also found that lower levels of tumorigenesis were correlated with the decreased expression of cyclin D and CDK as well as decreased cell proliferation. Taken together, JNK seems to be involved in both tumor initiation and promotion and may be an attractive target for the prevention of gastric carcinogenesis.

MyD88 and TNF Receptor-Associated Factor 6 Are Critical Signal Transducers in Helicobacter pylori-Infected Human Epithelial Cells

Helicobacter pylori induces NF-κB activation, leading to mucosal inflammation via cag pathogenicity island. Although recent studies have implicated several candidate proteins of both H. pylori and host, the molecular mechanism by which H. pylori activates NF-κB remains unclear. The aim of this study was to analyze the mechanism of cag pathogenicity island-mediated NF-κB activation in epithelial cells. The responses of human cell lines and mouse embryonic fibroblasts to infection with wild-type H. pylori or cagE mutant were investigated. The effect of small interfering RNAs (siRNAs) for several NF-κB signaling intermediate molecules was evaluated in H. pylori-induced IκBα phosphorylation and IL-8 production. Protein interactions of exogenously expressed TNFR-associated factor 6 (TRAF6) and MyD88 or receptor-interacting protein 2 and nucleotide-binding oligomerization domain 1 or those of endogenous IκB kinase, TGF-β-activated kinase 1 (TAK1), and TRAF6 were assessed by immunoprecipitation. Cag pathogenicity island-dependent NF-κB activation was observed in human cell lines, but not in mouse fibroblasts. In human epithelial cells, H. pylori-induced IκBα phosphorylation and IL-8 production were severely inhibited by siRNAs directed against TAK1, TRAF6, and MyD88. In contrast, siRNAs for TRAF2, IL-1R-associated kinases 1 and 4, and cell surface receptor proteins did not affect these responses. H. pylori infection greatly enhanced MyD88 and TRAF6 complex formation in a cag-dependent manner, but did not enhance Nod1 and receptor-interacting protein 2 complex formation. H. pylori also induced TAK1 and TRAF6 complexes. These results suggest that the cag pathogenicity island of H. pylori is a cell type-specific NF-κB activator. TAK1, TRAF6, and MyD88 are important signal transducers in H. pylori-infected human epithelial cells.

K-ras Mutation Targeted to Gastric Tissue Progenitor Cells Results in Chronic Inflammation, an Altered Microenvironment, and Progression to Intraepithelial Neoplasia

Chronic infectious diseases, such as Helicobacter pylori infection, can promote cancer in a large part through induction of chronic inflammation. Oncogenic K-ras mutation in epithelial cells activates inflammatory pathways, which could compensate for a lack of infectious stimulus. Gastric histopathology and putative progenitor markers [doublecortin and calcium/calmodulin-dependent protein kinase-like 1 (Dcamkl1) and keratin 19 (K19)] in K19-K-ras-V12 (K19-kras) transgenic mice were assessed at 3, 6, 12, and 18 months of age, in comparison with Helicobacter felis-infected wild-type littermates. Inflammation was evaluated by reverse transcription-PCR of proinflammatory cytokines, and K19-kras mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow. Both H. felis infection and K-ras mutation induced upregulation of proinflammatory cytokines, expansion of Dcamkl1(+) cells, and progression to oxyntic atrophy, metaplasia, hyperplasia, and high-grade dysplasia. K19-kras transgenic mice uniquely displayed mucous metaplasia as early as 3 months and progressed to high-grade dysplasia and invasive intramucosal carcinoma by 20 months. In bone marrow-transplanted K19-kras mice that progressed to dysplasia, a large proportion of stromal cells were GFP(+) and bone marrow-derived, but only rare GFP(+) epithelial cells were observed. GFP(+) bone marrow-derived cells included leukocytes and CD45(-) stromal cells that expressed vimentin or α smooth muscle actin and were often found surrounding clusters of Dcamkl1(+) cells at the base of gastric glands. In conclusion, the expression of mutant K-ras in K19(+) gastric epithelial cells can induce chronic inflammation and promote the development of dysplasia.

CagA protein secreted by the intact type IV secretion system leads to gastric epithelial inflammation in the Mongolian gerbil model.

Helicobacter pylori causes various gastro‐duodenal diseases, including gastric cancer. The CagA protein, an H. pylori virulence factor, induces morphological changes in host cells and may be associated with the development of peptic ulcer and gastric carcinoma. The present study has analysed the role of CagA protein in the pathogenesis of H. pylori infection in the Mongolian gerbil model. Mongolian gerbils were challenged with wild‐type H. pylori strain TN2, which has a functional cag pathogenicity island or isogenic mutants with disrupted cagA (ΔcagA) or cagE (ΔcagE) genes. They were sacrificed at 7, 13, and 25 weeks after inoculation. Pathological changes of the gastric mucosa were determined and apoptosis was assessed by the TUNEL assay. Immunohistochemistry for PCNA, phospho‐IκBα, and phospho‐Erk was also performed. All of the bacterial strains colonized the gerbil stomach at similar densities; however, the ΔcagA mutant induced milder gastritis than did the wild type. The extent of apoptosis and lymphoid follicle formation in the epithelium appeared to depend on intact cagA. The ΔcagA mutant induced less phosphorylation of IκBα and Erk, and less expression of interferon‐γ and interleukin‐1β mRNA in the epithelium than did the wild type. It is concluded that CagA protein may be essential for the induction of severe gastritis in the Mongolian gerbil model. Copyright

Helicobacter pylori Induces Antiapoptosis through Nuclear Factor–κB Activation

Although Helicobacter pylori is classified as a definite carcinogen, the mechanism underlying gastric carcinogenesis is not yet clear. We previously have shown that H. pylori activates an antiapoptotic gene, the cellular inhibitor of apoptosis protein 2 (c-IAP2), the underlying mechanism of which was investigated in the present study. cDNA array and real-time PCR analyses indicated that H. pylori showed a stimulatory effect on the expression of c-IAP2. Isogenic mutant strains with impaired cag pathogenicity island (cagPAI) expression showed weaker induction. Analyses that used the in situ terminal deoxynucleotide transferase-mediated dUTP nick end-labeling method indicated suppression of antiapoptosis by the antisense c-IAP2 oligonucleotide. Reporter assays with deletion and mutation constructs for the c-IAP2 promoter showed that nuclear factor-kappaB (NF-kappaB) binding sites are indispensable for transactivation. Super-repressor IkappaBalpha or NF-kappaB inhibitor reduced c-IAP2 transactivation by H. pylori, and exogenous expression of c-IAP2 inhibited apoptosis seen with H. pylori. In conclusion, H. pylori induces antiapoptosis through c-IAP2 transactivation following cagPAI-dependent NF-kappaB activation. The interaction of these stimuli may play a role in gastric carcinogenesis.

Helicobacter pylori induces IkappaB kinase alpha nuclear translocation and chemokine production in gastric epithelial cells.

ABSTRACT NF-κB is an important transcriptional factor that is involved in multiple cellular responses, such as inflammation and antiapoptosis. IκB kinase α (IKKα) and IKKβ, which are critical regulators of NF-κB activity, possess various mechanisms for NF-κB activation. This variability in NF-κB signaling may be associated with distinct inflammatory responses in specific cell types. The gastric pathogen Helicobacter pylori is known to activate NF-κB. However, the role of IKK in H. pylori infection remains unclear. In this report, we show that H. pylori activates both IKKα and IKKβ in gastric cancer cells and enhances NF-κB signaling in distinct manners. We found that IKKβ acted as an IκBα kinase during H. pylori infection, whereas IKKα did not. H. pylori induced IKKα nuclear translocation in time-, multiplicity of infection-, and cag pathogenicity island-dependent manners. In contrast, p100 processing, which is a known IKKα activity induced by several cytokines, was not induced by H. pylori. Both IKKs were responsible for chemokine secretion by infected cells. However, the antiapoptotic effect of H. pylori was merely transduced by IKKβ. Microarray analysis and real-time PCR indicated that both IKKs were involved in the transcriptional activation of genes associated with inflammation, antiapoptosis, and signal transduction. Our results indicate that H. pylori activates NF-κB via both IKKα and IKKβ using distinct mechanisms. IKKα nuclear translocation induced by H. pylori is indispensable for appropriate inflammatory responses but not for antiapoptosis, which suggests a critical role for IKKα in gastritis development.