Search

The Epstein-Barr virus (EBV) infection status in patients with inflammatory bowel disease (IBD), particularly those using thiopurines, may be associated with the risk of lymphoproliferative disorder and hemophagocytic lymphohistiocytosis. This was the first multicenter survey of EBV infection in Japanese patients with IBD. Factors related to the EBV infection status were also investigated.Five tertiary institutions in Japan participated in this study to examine pediatric and adult patients with IBD. Serum EBV anti-viral capsid antigen (VCA) IgG, EBV anti-VCA IgM, and anti-EBV nuclear antigen-antibody were measured in 495 patients with IBD. The patients' information was obtained from their medical records. Prior EBV infection was defined as anti-VCA IgM negativity and anti-VCA IgG positivity (UMIN000033004).The patients' median age was 25 years (range 0-92 years). Of the 495 patients, nine were anti-VCA IgM-positive and 354 were anti-VCA IgG-positive (seroprevalence: 72.8%). The proportion of patients with prior EBV infection was 0% for those aged 5 years, 60% for those aged 30 years, and 90% for those aged 30 years. The proportion of EBV-uninfected patients using thiopurines was 28.4% (52/183) for all patients and 51.8% (44/85) for pediatric patients. Age was significantly associated with anti-VCA IgG seropositivity (p 0.01, odds ratio: 0.902, 95% confidence interval: 0.880-0.925). No cases of lymphoproliferative disorder, hemophagocytic lymphohistiocytosis, or chronic active EBV infection were reported.Approximately 30% of Japanese patients with IBD were EBV-uninfected, including those using thiopurines. Age was a significant factor for anti-VCA IgG seropositivity.

Background Gastric cancer after Helicobacter pylori (HP) eradication is a crucial clinical challenge today as HP eradication therapy is widely performed. Detecting gastric cancer after HP eradication tends to be difficult with normal white-light endoscopy. In the present study, we aimed to identify easily-evaluated endoscopic findings that indicate the presence of gastric cancer after HP eradication so that endoscopists can consider additional detailed examinations at the site. Methods We analyzed the endoscopic images of 43 patients who underwent endoscopic submucosal dissection for early gastric cancer after HP eradication and 119 patients with an HP eradication history who underwent esophagogastroduodenoscopy for a medical checkup. Endoscopic findings were evaluated according to the Kyoto classification of gastritis (atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness) and map-like redness. Results Patients with gastric cancer had significantly higher total Kyoto risk scores; more atrophy, intestinal metaplasia, and diffuse redness; and a significantly higher prevalence of map-like redness compared with those without gastric cancer, in the univariate analyses. We used logistic regression analysis with forward selection based on the likelihood ratio to develop a model using atrophy and diffuse redness. Receiver operating characteristic analysis showed that a score of A2 in the Kyoto classification of gastritis (open-type atrophic pattern in the Kimura–Takemoto classification) was an endoscopic marker for the presence of post-HP-eradication gastric cancer. Conclusions Endoscopic severe gastric mucosal atrophy is useful to screen patients for gastric cancer after HP eradication.

Background and aims Small intestinal lesions in patients with Behcet disease (BD) have a risk of perforation and hemorrhage requiring surgery. However, no screening strategy for such lesions has been established. We investigated small intestinal lesions in BD patients with video capsule endoscopy (VCE) and analyzed clinical characteristics to identify noninvasive biomarkers of such lesions. Methods This study included 33 BD patients who underwent VCE (PillCam® SB3) at our institution from June 2016 to January 2019. Clinical characteristics, including age, sex, disease duration, body mass index, gastrointestinal symptoms, eye involvement, and blood examinations, were obtained from the medical records of 27 of the 33 patients. Fecal immunochemical tests for hemoglobin, fecal calprotectin (FC), and fecal lactoferrin (FL) were measured. VCE findings of 145 healthy Japanese individuals from a previous report were used as controls. Results Two intestinal BD patients were included in the 27 patients. We observed that BD patients exhibit more small intestinal lesions compared with healthy individuals, including erosions, ulcers, and total lesions (erosions or ulcers). FC and FL levels were significantly higher in patients with versus without small intestinal lesions (P = 0.034 and P = 0.046, respectively). Receiver operating characteristic analyses demonstrated that FC (cutoff value = 119 μg/g) and FL (cutoff value = 17 μg/g) were biomarkers for small intestinal lesions in patients with BD. Conclusion The present study using VCE showed that patients with BD had more small intestinal lesions than healthy individuals. FC and FL could be useful for screening BD patients who may have small intestinal lesions.

Background & Aims Perturbations in the early-life gut microbiome are associated with increased risk for complex immune disorders like inflammatory bowel diseases. We previously showed that maternal antibiotic-induced gut dysbiosis vertically transmitted to offspring increases experimental colitis risk in interleukin (IL) 10 gene deficient (IL10–/–) mice, a finding that may result from the loss/lack of essential microbes needed for appropriate immunologic education early in life. Here, we aimed to identify key microbes required for proper development of the early-life gut microbiome that decrease colitis risk in genetically susceptible animals. Methods Metagenomic sequencing followed by reconstruction of metagenome-assembled genomes was performed on fecal samples of IL10–/– mice with and without antibiotic-induced dysbiosis to identify potential missing microbial members needed for immunologic education. One high-value target strain was then engrafted early and/or late into the gut microbiomes of IL10–/– mice with antibiotic-induced dysbiosis. Results Early-, but not late-, life engraftment of a single dominant Bacteroides strain of non–antibiotic-treated IL10–/– mice was sufficient to restore the development of the gut microbiome, promote immune tolerance, and prevent colitis in IL10–/– mice that had antibiotic-induced dysbiosis. Conclusions Restitution of a keystone microbial strain missing in the early-life antibiotic-induced gut dysbiosis results in recovery of the microbiome, proper development of immune tolerance, and reduced risk for colitis in genetically prone hosts.

SummaryGut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. Here, we report the host C-type lectin antimicrobial peptideReg3γworks with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic hostReg3γexpression and increased abundance and oscillation ofReg3γ-independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiota’s capacity to sense and transduce dietary signals mediated by specific host factors such asReg3γcould be harnessed to improve metabolic dysfunction.

Background & AimsInflammatory bowel diseases (IBD) are chronic inflammatory disorders where predictive biomarkers for the disease development and clinical course are sorely needed for development of prevention and early intervention strategies that can be implemented to improve clinical outcomes. Since gut microbiome alterations can reflect and/or contribute to impending host health changes, we examined whether gut microbiota metagenomic profiles would provide more robust measures for predicting disease outcomes in colitis-prone hosts.MethodsUsing the IL-10 gene-deficient (IL-10 KO) murine model where early life dysbiosis from antibiotic (cefoperozone, CPZ) treated dams vertically-transferred to pups increases risk for colitis later in life, we investigated temporal metagenomic profiles in the gut microbiota of post-weaning offspring and determined their relationship to eventual clinical outcomes.ResultsCompared to controls, offspring acquiring maternal CPZ-induced dysbiosis exhibited a restructuring of intestinal microbial membership both in bacteriome and mycobiome that were associated with alterations in specific functional subsystems. Furthermore, among IL-10 KO offspring from CPZ-treated dams, several functional subsystems, particularly nitrogen metabolism, diverged between mice that developed spontaneous colitis (CPZ-colitis) versus those that did not (CPZ-no-colitis) at a time point prior to eventual clinical outcome.ConclusionsOur findings provide support that functional metagenomic profiling of gut microbes has potential and promise meriting further study for development of tools to assess risk and manage human IBD.SynopsisCurrently, predictive markers for the development and course of inflammatory bowel diseases (IBD) are not available. This study supports the notion that gut microbiome metagenomic profiles could be developed into a useful tool to assess risk and manage human IBD.

Several experimental models demonstrate a role for gut microbiota in the progression of type 1 diabetes (T1D) in genetically prone hosts. While the association between disturbances in gut microbiota, or microbial dysbiosis, and complex immune diseases such as inflammatory bowel diseases (IBD) are well established, less is known about its role in T1D pathogenesis. In IBD-prone interleukin-10 deficient (IL-10 KO) mice, the absence of gut microbiota under germ-free (GF) conditions prevents IBD development. However, in aged GF IL-10 KO mice (>6-months of age), polyuria and pancreatic lymphocytic infiltration resembling T1D lesions was observed. Approximately 50% of male and female mice above 6-months of age develop pancreatic immune cell infiltration, as compared to none in conventionally-raised and fecal microbiota transplanted (FMT) IL-10 KO counterparts. Immunofluorescence staining of islet infiltrates was positive for adaptive and innate immunological markers, including lymphoid and myeloid cell markers, which typically characterize autoimmune T1D lesions. A subset of GF IL-10 KO mice was also positive for insulin autoantibodies (IAA), but the majority of mice did not become diabetic. Our findings of early stage lymphocytic infiltrates in the pancreas and IAA in the absence of overt diabetes in GF IL-10 KO mice embody the early stages of T1D pathogenesis. As such, we propose that the presence of gut microbiota play a protective role against immune infiltration in the pancreas of genetically prone hosts. Moreover, our model provides an opportunity to better understand the role of the microbiota in the early stages of immune pathogenesis and perhaps conceive the development of microbe-mediated prophylactic strategies to treat or even prevent T1D.

Background and Aim. Half-elemental diet (ED) (900 kcal/day of ED) has clinical efficacy to treat Crohn’s disease (CD). However, the underlying mechanisms of how the ED exerts its efficacy remain unclear. Alterations of the gut microbiota, known as dysbiosis, have been reported to play a role in CD pathogenesis. Many variables including diet affect the gut microbiota. We hypothesized that half-ED has the potential to change the gut microbiota composition and functions leading to anti-inflammatory actions. Given that inflammation can be a confounding factor affecting the intestinal microbiota, we aimed to test our hypothesis among healthy individuals in this pilot study. Methods. This prospective study included four healthy volunteers. The subjects continued their dietary habits for 2 weeks after the registration of the study and then started half-ED replacing 900 kcal of the regular diet with ED (time point 1, T1). The subjects continued half-ED for 2 weeks (T2). After the withdrawal of ED, subjects resumed their original dietary habits for 2 weeks (T3). Fecal samples were collected from all subjects at all time points, T1-3. Fecal DNA and metabolites were extracted from the samples. We performed 16S rRNA gene amplicon sequencing and metabolomic analysis to examine the bacterial compositions and intestinal metabolites. Results. There were differences in the gut bacterial compositions and metabolites at each time point as well as overtime changing patterns between subjects. Several bacteria and metabolites including short-chain fatty acids and bile acids altered significantly across the subjects. The bacterial membership and intestinal metabolites at T3 were different from T1 in all subjects. Conclusions. Half-ED shifts the gut bacterial compositions and metabolites. The changes varied with each individual, while some microbes and metabolites change commonly across individuals. The impact of half-ED may persist even after the withdrawal. This trial is registered with UMIN ID: 000031920.

SummaryPerturbations in the early life gut microbiome are associated with increased risk to complex immune disorder like inflammatory bowel diseases. We previously showed maternal antibiotic-induced gut dysbiosis vertically passed to offspring increases experimental colitis risk in IL-10 gene deficient (IL-10−/−) mice. While this could arise from emergence of pathobionts or loss/lack of essential microbes needed for appropriate immunological education, our findings suggest the latter. A dominant Bacteroides strain absent following antibiotic-induced perturbation was cultivated from murine fecal samples. Addition of this strain into mice with antibiotic-induced dysbiosis significantly promoted immune tolerance and reduced incidence of colitis in IL-10−/− mice, but only if engrafted early in life, and not during adulthood. Thus, key members of the gut microbiome are essential for development of immune tolerance to commensal microbes in early life and their addition in presence of gut dysbiosis during this period can reduce colitis risk in genetically prone hosts.HighlightsSpecific gut microbes promote early life immune tolerance to key commensal microbesLoss of early life keystone microbes increases colitis risk in genetically prone hostsEmergence of absent commensal microbes late in life worsened colitis outcomeEarly life exposure to a missing keystone Bacteroides strain reduced colitis risk

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. We report the host C-type lectin antimicrobial peptide REG3γ works with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic host REG3γ expression and increased abundance and oscillation of REG3γ-independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiota’s capacity to sense and transduce dietary signals mediated by specific host factors such as REG3γ could be harnessed to improve metabolic dysfunction.

These data support the findings that dietary micronutrients influence the inflammatory responses and intestinal microbial community structure and function in a model of pouchitis-like small bowel inflammation reported in “Dietary Antioxidant Micronutrients Alter Mucosal Inflammatory Risk in a Murine Model of Genetic and Microbial Susceptibility” (Pierre et al., 2018) [1]. Briefly, wild-type and IL-10 deficient mice underwent surgical placement of small intestinal self-filling loops (SFL) and were subsequently fed purified control diet (CONT) or control diet supplemented with 4 micronutrients (AOX), retinoic acid, Vitamin C, Vitamin E, and selenium, for 14 days. These data include changes in host markers, such as body weight, mucosal levels of myeloperoxidase and syndecan-1, and luminal IgA and IgG levels. These data also include changes in the microbial compartment, including 16S community structure in the self-filling loop, conventionalized germ-free mice, and microbial substrate preference performed through anaerobic bacterial culturing of SLF CONT and AOX microbiota.

Summary Factors affecting the developing neonatal gut microbiome and immune networks may increase the risk of developing complex immune disorders such as inflammatory bowel diseases (IBD). In particular, peripartum antibiotics have been suggested as risk factors for human IBD, although direct evidence is lacking. Therefore, we examined the temporal impact of the commonly used antibiotic cefoperazone on both maternal and offspring microbiota when administered to dams during the peripartum period in the IL-10-deficient murine colitis model. By rigorously controlling for cage, gender, generational, and murine pathobiont confounders, we observed that offspring from cefoperazone-exposed dams develop a persistent gut dysbiosis into adulthood associated with skewing of the host immune system and increased susceptibility to spontaneous and chemically dextran sodium sulfate (DSS)-induced colitis. Thus, early life exposure to antibiotic-induced maternal dysbiosis during a critical developmental window for gut microbial assemblage and immune programming elicits a lasting impact of increased IBD risk on genetically susceptible offspring.

Background The incidence and prevalence of Crohn’s disease (CD) are increasing globally. The efficacy of elemental nutrition (EN) for CD has been reported for decades. Recent studies demonstrated that elemental diet (ED), one of the types of EN, which contains individual amino acids, sugars (mono- or polysaccharides), and low fat, improves the anti-TNFa antibody therapy outcome by reducing the loss of response and has the preventive effect against the postoperative recurrence. Given its excellent safety, ED can be a promising concomitant therapeutic option for CD. However, the underlying mechanisms of ED remain unclear. Imbalance of the gut microbiota, dysbiosis, is thought to be involved in CD pathogenesis. Meanwhile, diet influences gut microbiota. We hypothesised that ED exerts clinical efficacy by shifting the gut microbiota to the anti-inflammatory environment. We also believe that investigating human samples is crucial to obtain mechanistic insights into ED therapy, considering species-specific differences in interactions between the host and microbiota. In this study, we examined the impact of ED on the intestinal bacterial compositions as well as metabolites in human subjects. Methods Four healthy individuals participated in the study. The subjects continued their dietary habits for 2 weeks after the registration of the study and then replaced 900 kcal/day of their regular diet with 900 kcal/day of ED (time point 1, T1). ED was continued for 2 weeks (T2). After the withdrawal of ED, subjects resumed their original dietary habits for 2 weeks until the endpoint of this study (T3). Faecal samples were collected from all subjects at all time points, T1-3. Faecal DNA and metabolites were extracted and analysed with 16S rRNA gene amplicon sequencing and metabolomic analysis, respectively. This study was approved by the Institutional Review Board of Kyorin University School of Medicine (IRB No. 720) and performed in accordance with the principles of the Declaration of Helsinki. Results Interindividual differences were observed in the gut bacterial compositions (Figure 1) as well as metabolites (Figure 2) at all time points. Changes over time varied among subjects. On the other hand, some bacteria (e.g. [Ruminococcus] gnavus group and Parabacteroides) and metabolites including short-chain fatty acids and bile acids changed significantly between time points among the 4 subjects. The bacterial and metabolite profiles at T3 were different from those T1 in all subjects. Conclusion The alterations of the gut microbiota induced by ED vary with each individual. Some microbes and metabolites change commonly across the subjects. ED may have a lasting effect on the gut environment even after the withdrawal.

Chemopreventative properties of traditional medicines and underlying mechanisms of action are incompletely investigated. This study demonstrates that dietary daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper effectively suppresses intestinal tumor development and progression in the azoxymethane (AOM) and APCmin/+ mouse models. For the AOM model, TU-100 was provided after the first of six biweekly AOM injections. Mice were sacrificed at 30 weeks. APCmin/+ mice were fed diet without or with TU-100 starting at 6 weeks, and sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In APC min/+ mice, the number of small intestinal tumors was significantly decreased. In the AOM model, both TU-100 and Japanese ginseng decreased colon tumor numbers. Decreased Ki-67 and β-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression were observed. EGF receptor expression and stimulation/phosphorylation in vitro were investigated in C2BBe1 cells. TU-100, ginger, and 6-gingerol suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol inhibited EGF ERK1/2 activation. TU-100 and some of its components and metabolites of these components inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation. Copyright © 2016 John Wiley & Sons, Ltd.

Murine models are widely used to explore host-microbe interactions because of the challenges and limitations inherent to human studies. However, microbiome studies in murine models are not without their nuances. Inter-individual variations in gut microbiota are frequent even in animals housed within the same room. We therefore sought to find an efficient and effective standard operating procedure (SOP) to minimize these effects to improve consistency and reproducibility in murine microbiota studies. Mice were housed in a single room under specific-pathogen free conditions. Soiled cage bedding was routinely mixed weekly and distributed among all cages from weaning (three weeks old) until the onset of the study. Females and males were separated by sex and group-housed (up to five mice/cage) at weaning. 16S rRNA gene analyses of fecal samples showed that this protocol significantly reduced pre-study variability of gut microbiota amongst animals compared to other conventional measures used to normalize microbiota when large experimental cohorts have been required. A significant and consistent effect size was observed in gut microbiota when mice were switched from regular chow to purified diet in both sexes. However, sex and aging appeared to be independent drivers of gut microbial assemblage and should be taken into account in studies of this nature. In summary, we report a practical and effective pre-study SOP for normalizing the gut microbiome of murine cohorts that minimizes inter-individual variability and resolves co-housing problems inherent to male mice. This SOP may increase quality, rigor, and reproducibility of data acquisition and analysis.

Medications or dietary components can affect both the host and the host’s gut microbiota. Changes in the microbiota may influence medication efficacy and interactions. Daikenchuto (TU-100), a herbal medication, comprised of ginger, ginseng, and Japanese pepper, is widely used in Japanese traditional Kampo medicine for intestinal motility and postoperative paralytic ileus. We previously showed in mice that consumption of TU-100 for 4 weeks changed the gut microbiota and increased bioavailability of bacterial ginsenoside metabolites. Since TU-100 is prescribed in humans for months to years, we examined the time- and sex-dependent effects of TU-100 on mouse gut microbiota. Oral administration of 1.5% TU-100 for 24 weeks caused more pronounced changes in gut microbiota in female than in male mice. Changes in both sexes largely reverted to baseline upon TU-100 withdrawal. Effects were time and dose dependent. The microbial profiles reverted to baseline within 4 weeks after withdrawal of 0.75% TU-100 but were sustained after withdrawal of 3% TU-100. In summary, dietary TU-100 changed mouse microbiota in a time-, sex-, and dose-dependent manner. These findings may be taken into consideration when determining optimizing dose for conditions of human health and disease with the consideration of differences in composition and response of the human intestinal microbiota.

Inflammatory bowel diseases (IBD) are caused by the convergence of microbial, environmental, and genetic factors. Diet significantly alters these interactions by affecting both the host and microbiome. Using a mucosal inflammatory model that resembles the human condition of ileal pouchitis, we investigated the effects of Control (CONT) or Antioxidant (AOX) diet, containing pharmacologically relevant levels of 4 micronutrients, on disease risk in wild-type and IL-10(−/−) animals following surgical self-filling (SF) ileal blind loop placement. Although no differences were found in body weight change or survival, IL-10(−/−) CONT animals had significantly larger lymphoid organs compared with IL-10(−/−) AOX or with WT. SF loops from IL-10(−/−) CONT loop mucosa demonstrated histological inflammation, characterized by goblet cell depletion, increased mucosal myeloperoxidase (MPO), and elevated IFNγ, TNFα, and IL-17α gene expression, which AOX attenuated. AOX elevated luminal IgA in IL-10(−/−) animals, but not significantly in WT. In IL-10(−/−) animals, AOX significantly decreased the percentage of CD4+T-bet and CD4+RORγ T-cells compared with CONT, with no changes in CD4+Foxp3+ Treg cells. 16S rRNA gene sequencing demonstrated AOX increased microbial alpha diversity compared with CONT in both genotypes. Notably, colonizing germ-free IL-10(−/−) hosts with CONT bacterial communities, but not AOX, recapitulated the inflammatory phenotype. Collectively, these findings highlight that common dietary antioxidant micronutrients reshape the gut microbial community to mitigate intestinal inflammatory profiles in genetically susceptible hosts. Insights into the dietary-immune-microbial nexus may improve understanding for recurrent inflammatory episodes in susceptible patient populations and opportunities for practical therapeutics to restore immune and microbial homeostasis.

Few reports have analyzed the clinical importance of sporadic fundic gland polyps (FGPs). The aim of this study was to investigate the relationship between sporadic FGPs and condition of the gastric mucosa stratified by serum pepsinogen levels and Helicobacter pylori antibody level.Three hundred and seventy-five subjects undergoing gastrointestinal endoscopy were enrolled. Subjects on proton pump inhibitors were excluded. Pathologically proven FGPs, and other endoscopic findings (reflux esophagitis, gastric and duodenal ulcer) were examined and serum pepsinogen levels, H. pylori antibody concentration and gastric juice pH were measured simultaneously. Subjects with normal serum pepsinogen and negative H. pylori antibodies were defined as having "low risk" stomachs, suggesting low risk of gastric carcinogenesis.Of the 375 subjects, 44 showed FGPs. The prevalence of "low risk" stomach in subjects with and without FGPs was 98% and 48%, respectively. Multivariable logistic regression analysis indicated three variables as independent factors positively associated with "low risk" stomachs: FGPs (odds ratio [OR] 38.6), reflux esophagitis (OR 4.8), and age60 years (OR 1.89). Gastric juice pH, which is associated with mucosal atrophy grade and low pH indicates less mucosal atrophy, was significantly lower in subjects with (1.64 ± 0.64) than without FGPs in "low risk" (1.94 ± 1.12) and "high risk" stomachs (3.99 ± 2.31).Sporadic FGPs tend to be related to the least atrophic mucosa among non-gastric atrophy subjects without H. pylori infection, and can be used as predictors of a low risk of gastric carcinogenesis.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b+Gr-1low macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b+/Gr-1− and CD11b+/Gr-1high cells, but not CD11b+/Gr-1low cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b+-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

Aims/hypothesis The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study. Methods We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp). Results Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription. Conclusions/interpretation Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp. Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-2879-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Inflammatory bowel diseases (IBD) are chronic diseases of unclear etiology that affect over 1 million individuals in the United States and over 2.5 million people in Europe(1). However, they are also expanding globally, affecting populations in Asia, South America, and the Middle East as they become more industrialized. These diseases are believed to arise from the convergence of genetic, environmental, and microbial factors that trigger aberrant immune and tissue responses, resulting in intestinal inflammation. Advances in cultivation-independent investigations, experimental models, and bioinformatics approaches have improved our understanding of the role of gut microbiota in IBD. However, determining and understanding the functional consequences of gut dysbiosis and altered host-microbiota interactions in IBD remain a challenge due to the limits of current experimental models and difficulty in establishing causal links in human-based investigations. Continued development of new methodologies and improvements in clinical study design are needed to better understand the interplay of genetic, microbial, and immunological factors in IBD. This knowledge can then be applied clinically to improve therapeutic strategies and outcomes for IBD.

In palatogenesis, bilateral palatal shelves grow and fuse with each other to establish mesenchyme continuity across the horizontal palate. The palatal shelves are covered with the medial edge epithelium (MEE) in which most apical cells are periderm cells. We investigated localization and roles of tight junction (TJ) and adherens junction (AJ) components and an apical membrane marker in the MEE in palatogenesis. Immunofluorescence and immunoelectron microscopy analyses revealed that TJs were located at the boundary between neighboring periderm cells, whereas AJ components were localized at the boundary between all epithelial cells in the MEE. Specifically, typical AJs were observed at the boundaries between neighboring periderm cells and between periderm cells and underlying epithelial cells where the signal for nectin-1 was observed. The TGF-β-induced desquamation of periderm cells reduced the polarity of remaining epithelial cells as estimated by changes of epithelial cell morphology and the staining of the polarity marker and the AJ components. These less polarized epithelial cells then intermingled and finally disappeared at least partly by apoptosis. These results indicate that periderm cells covering growing palatal shelves have bona fide TJs and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis.

Stress response gene ATF3 is one of the p53 target genes and has a tumor suppressor role in cancer. However, the biological role of p53-ATF3 pathway is not well understood. Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers cell death upon binding to its ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and a combination of TRAIL and agents that increase the expression of DR5 is expected as a novel anticancer therapy. In this report, we demonstrate that ATF3 is required for efficient DR5 induction upon DNA damage by camptothecin (CPT) in colorectal cancer cells. In the absence of ATF3, induction of DR5 messenger RNA and protein is remarkably abrogated, and this is associated with reduced cell death by TRAIL and CPT. By contrast, exogenous expression of ATF3 causes more rapid and elevated expression of DR5, resulting in enhanced sensitivity to apoptotic cell death by TRAIL/CPT. Reporter assay and DNA affinity precipitation assay demonstrate that at least three ATF/CRE motifs at the proximal promoter of the human DR5 gene are involved in the activation of DNA damage-induced DR5 gene transcription. Furthermore, ATF3 is shown to interact with p53 to form a complex on the DR5 gene by Re-chromatin immunoprecipitation assay. Taken together, our results provide a novel insight into the role of ATF3 as an essential co-transcription factor for p53 upon DNA damage, and this may represent a useful biomarker for TRAIL-based anticancer therapy.

In the auditory epithelium of the cochlea, the sensory hair cells and supporting cells are arranged in a checkerboard-like fashion, but the mechanism underlying this cellular patterning is unclear. We found that mouse hair cells and supporting cells express the immunoglobulin-like adhesion molecules nectin-1 and -3, respectively, and that their interaction mediates the heterotypic adhesion between these two cell types. Genetic removal of nectin-1 or -3 disrupted the checkerboard-like pattern, inducing aberrant attachment between hair cells. When cells expressing either nectin-1 or -3 were cocultured, they arranged themselves into a mosaic pattern. Thus, nectin-1 and -3 promote the formation of the checkerboard-like pattern of the auditory epithelia.

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP–dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Afadin interacts with the cytoplasmic region of nectins, which are immunoglobulin-like cell adhesion molecules at adherens junctions, and links them to the actin cytoskeleton. Afadin regulates activities of cells in culture such as directional motility, proliferation and survival. We used Cre-loxP technology to generate mice conditionally lacking afadin specifically in the intestinal epithelia after birth. The loss of afadin caused increased paracellular permeability in the intestinal mucosa and enhanced susceptibility to the tissue destruction induced by dextran sulfate sodium. The junctional architecture of the intestinal epithelia appeared to be preserved, whereas the deficiency of afadin caused the mislocalization of nectin-2 and nectin-3 from adherens junctions to basolateral membrane domains but not that of other components of apical junctions. By contrast, such phenotypic changes were undetected in mice lacking nectin-2, nectin-3 or both. These findings suggest that afadin plays crucial roles, independently of the role as the nectin–afadin module, in barrier function and homeostasis of the intestinal epithelia once the epithelial structure has been established.

LIM-domain only protein 7 (LMO7) has been suggested to act as a tumor suppressor for murine lung adenocarcinoma, while its splice variant p100 LMO7/#16 is associated with invasion and metastasis of rat AH130W1 cells. However, the importance of LMO7 in human lung cancer is unknown. We investigated LMO7 protein expression by immunohistochemistry in tumor tissues obtained from 57 patients with adenocarcinoma of the lung using a rabbit anti-LMO7 antibody. Signals for LMO7 were localized to the apical surface of the bronchial epithelium and to the cell membranes of pneumocytes in non-cancerous pulmonary tissues, but were noted circumferentially around the plasma membrane of cancer cells in all 57 patients with adenocarcinoma. The LMO7-positive group (24 patients, 42%) showed equivocal to strong expression of LMO7 in more than 50% cancer cells, while the remaining 33 patients (58%) showed LMO7 expression in less than 50% of their cancer cells. The latter group had significantly more advanced disease than the LMO7-positive group with regard to T factor (p=0.011), nodal involvement (p=0.026) and p-stage (p=0.010; χ(2) test). Multivariate analysis using a logistic regression model showed that LMO7 expression was independently associated with the T factor (p=0.041). Kaplan-Meier analysis showed that a poor prognosis was associated with low expression of LMO7 (p=0.036; log-rank test). Our findings are consistent with earlier observations and demonstrate that LMO7 is inversely correlated with the development and prognosis of human lung adenocarcinoma.

During mammalian oogenesis, intercellular communication between oocytes and the surrounding follicle cells through gap junction channels is crucial for oocyte development and maturation. The channel properties of gap junctions may be affected by the composition or combination of connexins, the expression of which is regulated by gonadotropins and other factors. Thus, identification and expression analysis of connexin genes in oocytes and follicle cells will help us to better understand how oogenesis and folliculogenesis are regulated in a species-specific manner in mammals. We previously reported the spatiotemporal expression of multiple connexin genes in porcine follicle cells. Here, we searched for connexin genes specifically expressed in porcine oocytes that may be involved in the formation of gap junctions between oocytes and follicle cells. To achieve this, we constructed an oocyte-specific cDNA library to identify which connexin genes are expressed in these cells and found that gap junction protein, alpha 10, which encodes connexin-60, and a porcine ortholog of mouse gap junction protein, gamma 1 encoding connexin-45, are the major connexins expressed in porcine oocytes during folliculogenesis. Immunostaining and in situ hybridization of sectioned porcine ovaries confirmed oocyte expression of these genes at 3 different stages of ovary development. Furthermore, their gap junction channel activity was assessed using a heterologous cell system. However, gap junction protein, alpha 4, which encodes connexin-37 and is expressed in the oocytes of several other mammals, was undetectable. We demonstrate that there is diversity in the connexin genes expressed in mammalian oocytes, and hence in the gap junctions connecting oocytes and cumulus cells.

Nectins are immunoglobulin-like cell adhesion proteins and their interactions recruit various cell-cell junctions. Mutations in human NECTIN-1 cause an ectodermal dysplasia syndrome, but Nectin-1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin-3 with nectin-1 and enamel abnormality in the nectin-3 mutant. We, therefore, generated nectin-1;nectin-3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin-1 was expressed in ameloblasts whereas nectin-3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin-1 and nectin-3 recruits desmosomal junctions, and that these are required for proper enamel formation.

Rationale : Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. Objective : We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. Methods and Results : Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell–cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin–PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K–Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. Conclusions : These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Differential expression of cell adhesion molecules regulates stem cell location, self-renewal and lineage selection under steady state conditions and during tissue repair. We show that the intercellular adhesion protein nectin-like molecule 2 (Necl2) is highly expressed in bulge stem cells of adult human and mouse hair follicles. Overexpression of Necl2 in cultured human keratinocytes led to upregulation of calcium/calmodulin-associated Ser/Thr kinase (CASK), increased calcium-independent intercellular adhesion, and inhibition of cell motility and in vitro wound healing. Although the rate of cell proliferation was reduced, terminal differentiation was unaffected. To assess the role of Necl2 in vivo, we examined the epidermis of Necl2-null mice and developed transgenic mice that expressed Necl2 in the basal layer of murine epidermis. Necl2 overexpression led to a reduction in S-phase cells and an increase in quiescent cells retaining DNA label in the bulge. Although epidermal homeostasis appeared normal in both transgenic and knockout mice, wound healing was markedly delayed. Necl2 overexpression resulted in reduced proliferation and increased levels of CASK and E-cadherin at the leading edge of healing wounds, consistent with its effects in culture. Our results demonstrate that Necl2 is involved in regulating epidermal stem cell quiescence and location.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM−/− mice. CRTAM−/− mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM−/− CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

In the hippocampus, synapses are formed between mossy fiber terminals and CA3 pyramidal cell dendrites and comprise highly developed synaptic junctions (SJs) and puncta adherentia junctions (PAJs). Dynamic remodeling of synapses in the hippocampus is implicated in learning and memory. Components of both the nectin–afadin and cadherin–catenin cell adhesion systems exclusively accumulate at PAJs. We investigated the role of afadin at synapses in mice in which the afadin gene was conditionally inactivated in hippocampal neurons. In these mutant mice, the signals for not only nectins, but also N-cadherin and β-catenin, were hardly detected in the CA3 area, in addition to loss of the signal for afadin, resulting in disruption of PAJs. Ultrastructural analysis revealed an increase in the number of perforated synapses, suggesting the instability of SJs. These results indicate that afadin is involved not only in the assembly of nectins and cadherins at synapses, but also in synaptic remodeling.

Necl-5 is an immunoglobulin-like molecule that was originally identified as a poliovirus receptor. Although Necl-5 expression is often up-regulated in cancer cells, its pathophysiological significance in the development of cancer remains unclear. We investigated the roles of Necl-5 in the development of colitis-associated neoplasia. Necl-5-deficient mice were generated and treated with dimethylhydrazine (DMH) and/or dextran sodium sulphate (DSS) to induce colitis and its associated neoplasias. Colon tissues were examined for histology, Ki-67 expression by immunohistochemistry and K-ras gene mutation. Colon tumours occurred significantly less frequently in heterozygous (Necl-5(+/-)) or homozygous Necl-5-deficient (Necl-5(-/-)) mice than in wild-type (WT) mice with DMH/DSS treatment. Total ulcer index and inflammatory cell infiltration were significantly lower in Necl-5(-/-) mice than in WT mice with DSS alone or DMH/DSS treatment. Colon tumours in both WT and Necl-5(-/-) mice showed high cell proliferation ability but lacked K-ras mutation. The total Ki-67 labelling index in non-neoplastic colon epithelium was significantly higher in WT (45.9 +/- 0.94) than in Necl-5(+/-) (34.3 +/- 1.40) or Necl-5(-/-) (27.7 +/- 1.15) mice with DMH/DSS treatment (p < 0.001). Necl-5 plays a role in the development of colitis-associated cancer by up-regulating colonic mucosal cell proliferation.

Blockade of mineralocorticoid receptor has been shown to improve the clinical outcomes of proteinuric kidney diseases. However, little is known about the regulation of mineralocorticoid receptor-dependent transcriptional activity in renal disease. Here we identify a new role for Rac1, a member of the Rho family GTPases, as a potent activator of mineralocorticoid receptor signal transduction both in vitro and in vivo. Transient transfection assays in HEK 293 cells revealed that constitutively active Rac1 (CA-Rac1) enhanced mineralocorticoid receptor-dependent reporter activity, which was accompanied by increased nuclear translocation of mineralocorticoid receptor. CA-Rac1 facilitated mineralocorticoid receptor nuclear accumulation also in podocytes via p21-activated kinase phosphorylation. In mice lacking Rho GDP-dissociation inhibitor-alpha (Arhgdia(-/-) mice), renal abnormalities, including heavy albuminuria and podocyte damage, were associated with increased Rac1 (but not RhoA) and mineralocorticoid receptor signaling in the kidney, without alteration in systemic aldosterone status. Pharmacological intervention with a Rac-specific small-molecule inhibitor diminished mineralocorticoid receptor overactivity and renal damage in this model. Furthermore, albuminuria and histological changes in Arhgdia(-/-) mice were suppressed by mineralocorticoid receptor blockade, confirming the pathological role of Rac1-mineralocorticoid receptor interaction. Our results provide evidence that signaling cross-talk between Rac1 and mineralocorticoid receptor modulates mineralocorticoid receptor activity and identify Rac1 as a therapeutic target for chronic kidney disease.

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.

Actin dynamics play multiple roles in promoting cell movement, changing cell shapes, and establishing intercellular adhesion. Cell contact events are involved in tissue morphogenesis, immune responses, and cancer cell invasion. In epithelial cells, cell–cell contacts mature to form apical junctions with which the actin cytoskeleton physically associates. Living cell imaging shows, however, that the apical junctional complex is less dynamically regulated than the actin cytoskeleton, indicating that their interaction does not remain stable. Given that several cell adhesion modules are clustered at apical junctions, the sum of weak or transient interactions may create linkages that can be strong yet easily remodeled. Here we describe how subcellular protein interactions are coordinated to induce changes in actin organization and dynamics, in response to the status of apical junctions.

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α6 and integrin β1. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1 −/− mice. We observed that basal endothelial permeability in lungs of RhoGDI-1 −/− mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1 −/− lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

SummaryEither herpesvirus entry mediator (HVEM, TNFRSF14) or nectin-1 (PVRL1) is sufficient for herpes simplex virus (HSV) infection of cultured cells. The contribution of individual receptors to infection in vivo and to disease is less clear. To assess this, Tnfrsf14−/− and/or Pvrl1−/− mice were challenged intravaginally with HSV-2. Infection of the vaginal epithelium occurred in the absence of either HVEM or nectin-1 but was virtually undetectable when both receptors were absent, indicating that either HVEM or nectin-1 was necessary. Absence of nectin-1 (but not HVEM) reduced efficiency of infection of the vaginal epithelium and viral spread to the nervous system, attenuating neurological disease and preventing external lesion development. While nectin-1 proved not to be essential for infection of the nervous system, it is required for the full manifestations of disease. This study illustrates the value of mutant mice for understanding receptor contributions to disease caused by a human virus.

The tumor suppressor p53 is a key transcriptional factor regulating the induction of cellular senescence by oncogenic signals. The activity of p53 is regulated by recruitment into promyelocytic leukemia (PML)-nuclear bodies (NBs) as well as by stabilization through posttranslational modifications such as phosphorylation and acetylation. Here we found that MORC3 (microrchidia3)-ATPase activated p53 and induced cellular senescence in normal human and mouse fibroblasts but not p53−/− fibroblasts. Conversely, genotoxic stress–induced phosphorylation and stabilization of p53 but barely increased its transcriptional activity in Morc3−/− fibroblasts. MORC3 localized on PML-NBs in presence of PML and mediated recruitment of p53 and CREB-binding protein (CBP) into PML-NBs. In contrast, expression of ATPase activity-deficient mutant MORC3-E35A or siRNA repression of MORC3 impaired the localization of p53 and Sp100 but not CBP on PML-NBs. These results suggest that MORC3 regulates p53 activity and localization into PML-NBs. We identified a new molecular mechanism that regulates the activity of nuclear proteins by localization to a nuclear subdomain.

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1 −/− or IFNAR −/− mice, whereas a normal NK antitumor response was induced in PKR −/− , MyD88 −/− , IFN-β −/− , and wild-type mice. IFNAR was a prerequisite for the induction of IFN-α/β and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR −/− mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1 −/− mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

Nectins and nectin-like molecules (Necls) are structurally related transmembrane proteins primarily involved in cell adhesion. Nectins and afadin, the adaptor or anchoring protein, stabilize the epithelium and endothelium and establish apical-basal polarity of epithelial cells, independently or in cooperation with other cell adhesion molecules. Necls facilitate cell–cell communication implicated in cell movement and proliferation, immune responses, and cancer cell phenotypes. Necls interact with nectins and specific ligands at cell–cell contacts, whereas Necls associate with integrin αvβ3 and growth factor receptors on the same cell surface. Besides their roles in cell adhesion, nectins regulate the activities of Rho family small G proteins which play critical roles in maintaining the apical junctions of epithelial cells through reorganization of the actin cytoskeleton. Since mice lacking the Rho GDP-dissociation inhibitor (GDI)α show massive proteinuria and degeneration of renal epithelial cells, nectins and other cell adhesion molecules may play roles in the structural and functional aspects of renal diseases. Here we summarize our knowledge of nectins and Necls and discuss cell adhesion biology in the kidney.

Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) α and Rho GDIβ (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDIα and Rho GDIβ genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDIα and Rho GDIβ thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.

Rab3A small G protein is a member of the Rab family and is most abundant in the brain, where it is localized on synaptic vesicles. Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and this change in activity is associated with the trafficking cycle of synaptic vesicles at nerve terminals. Rab3 GTPase-activating protein (GAP) stimulates the GTPase activity of Rab3A and is expected to determine the timing of the dissociation of Rab3A from synaptic vesicles, which may be coupled with synaptic vesicle exocytosis. Rab3 GAP consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150. Recently, mutations in p130 were found to cause Warburg Micro syndrome with severe mental retardation. Here, we generated p130-deficient mice and found that the GTP-bound form of Rab3A accumulated in the brain. Loss of p130 in mice resulted in inhibition of Ca 2+ -dependent glutamate release from cerebrocortical synaptosomes and altered short-term plasticity in the hippocampal CA1 region. Thus, Rab3 GAP regulates synaptic transmission and plasticity by limiting the amount of the GTP-bound form of Rab3A.

Neurites recognize their specific partners during the formation of interneuronal connections. In hippocampal pyramidal neurons, axons attach to dendrites for their synaptogenesis, but the dendrites do not form stable contacts with each other, suggesting the presence of a mechanism to allow their selective associations. Nectin-1 (N1), an immunoglobulin domain adhesive protein, is preferentially localized in axons, and its heterophilic partner, N3, is present in both axons and dendrites; we tested their potential roles in interneurite recognition. The overexpression of N1, causing its mislocalization to dendrites, induced atypical dendrodendritic as well as excessive axodendritic associations. On the contrary, the genetic deletion of N1 loosened the contacts between axons and dendritic spines. Those actions of nectins required cadherin–catenin activities, but the overexpression of cadherin itself could not accelerate neurite attachment. These results suggest that the axon-biased localization of N1 and its trans-interaction with N3 in cooperation with the cadherin machinery is critical for the ordered association of axons and dendrites.

Synapses are specialized intercellular junctions whose specificity and plasticity are mediated by synaptic cell adhesion molecules. In hippocampus, the mossy fibers form synapses on the apical dendrites of the CA3 pyramidal cells where synaptic and puncta adherentia junctions (PAJs) are highly developed. Synaptic junctions are the sites of neurotransmission, while PAJs are regarded as mechanical adhesion sites. Cell–cell adhesion molecules nectin-1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides of PAJs, respectively. To reveal the definitive role of nectins, we analyzed nectin-1−/− and nectin-3−/− mice. In both the mutant mice, the number of PAJs at the synapses between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells was reduced. In addition, the abnormal mossy fiber trajectory was observed. These results indicate that nectins are involved in the formation of PAJs, which maintain the proper mossy fiber trajectory.

We have previously shown that transforming growth factor-beta1 (TGF-beta1) markedly stimulates the invasive capacity of rat ascites hepatoma AH130 W1 cells in vitro and in vivo. A differential hybridization procedure was used to isolate genes that were specifically up-regulated in TGF-beta1 treated W1 cells. Among ten independent cDNA clones, we focused on LMO7 and a variant isoform, LMO7S, that was generated by alternative splicing. LMO7 had PDZ and LIM domains, while LMO7S had only PDZ domain. TGF-beta1 up-regulated expression levels of LMO7 and LMO7S. LMO7 expression was up-regulated in the highly metastatic clone MM1.

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.