西村 渉

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance.

The objective of this study was to investigate the levels of gene expression in the middle ear mucosa of 2 patients diagnosed with eosinophilic otitis media. One patient with severe hearing loss showed high expression levels of genes encoding IL-5 and IL-33 receptors.

The mechanisms of impaired glucose-induced insulin secretion from the pancreatic β-cells in obesity have not yet been completely elucidated. Here, we aimed to assess the effects of adipocyte-derived factors on the functioning of pancreatic β-cells. We prepared a conditioned medium using 3T3-L1 cell culture supernatant collected at day eight (D8CM) and then exposed the rat pancreatic β-cell line, INS-1D. We found that D8CM suppressed insulin secretion in INS-1D cells due to reduced intracellular calcium levels. This was mediated by the induction of a negative regulator of insulin secretion-NECAB1. LC-MS/MS analysis results revealed that D8CM possessed steroid hormones (cortisol, corticosterone, and cortisone). INS-1D cell exposure to cortisol or corticosterone increased Necab1 mRNA expression and significantly reduced insulin secretion. The increased expression of Necab1 and reduced insulin secretion effects from exposure to these hormones were completely abolished by inhibition of the glucocorticoid receptor (GR). NECAB1 expression was also increased in the pancreatic islets of db/db mice. We demonstrated that the upregulation of NECAB1 was dependent on GR activation, and that binding of the GR to the upstream regions of Necab1 was essential for this effect. NECAB1 may play a novel role in the adipoinsular axis and could be potentially involved in the pathophysiology of obesity-related diabetes mellitus.

BACKGROUND: Chronic rhinosinusitis (CRS) can be divided into endotypes by functional or pathophysiologic findings. OBJECTIVE: The aim of this study was to analyze the expression of cytokines, prostaglandin (PG) synthases, and their receptors related to the pathogenesis of CRS, especially those contributing to nasal polyp (NP) formation. METHODS: NPs and uncinate tissue (UT) samples were collected from 90 patients who underwent endoscopic sinus surgery. They included 75 patients with CRS (including 45 with eosinophilic CRS [eCRS] and 30 with non-eCRS) and 15 patients without CRS. A total of 30 genes were selected for our original DNA array plate to analyze the levels of expression of 10 cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, IL-25, IL-33, and TSLP), 4 prostaglandin synthases (prostaglandin D2 [PGD2] synthase, prostaglandin E2 synthase, COX-1, and COX-2), and their 16 receptors. Clustering analysis was performed according to the expression results, and clinical findings of patients from each cluster were investigated. RESULTS: The samples could be divided into 3 clusters. Cluster 1 showed elevated levels of expression of IL4, IL5, IL13, TSLP, IL1RL1 (ST2 [an IL-33 receptor]), HPGDS, and GPR44 (CRTH2, a PGD2 receptor); cluster 2 showed elevated levels of expression of IL17A and PTGES; and cluster 3 showed an elevated level of expression of IL25. Regarding clinical features, the main characteristics of each cluster were as follows: NPs from patients with eCRS for cluster 1, NPs and/or UT samples from patients with non-eCRS for cluster 2, and UTs from patients with non-CRS for cluster 3. CONCLUSION: The results suggest that there are associations between type 2 inflammation/PGD2 and eCRS and also between type 3 inflammation/prostaglandin E2 and non-eCRS.

Long-term and mild confinement or isolation in an enclosed environment can occur in situations such as disasters, specific political, economic or social events, nuclear shelters, seabed exploration, polar expeditions, and space travel. To investigate the effects of stress caused by long-term confinement in an enclosed environment in mammals, we divided 8-week-old C57BL/6J mice into four groups that were housed in a closed environment with a narrow metabolic cage (stress group), normal metabolic cage (control group), conventional cage (conventional group) or conventional cage with wire mesh floor (wire mesh group). The phenotypes of the mice were examined for four weeks, followed by behavioral tests. Weight gain suppression was observed in the stress group. Continuous analysis of these mice every two minutes for four weeks using an implanted measuring device showed a significantly decreased amount of spontaneous activity and subcutaneous temperature in the stress group. After housing in each environment for four weeks, the behavioral tests of mice in the stress group also revealed a shorter latency to fall off in the rotarod test and shorter stride length and interstep distance in the footprint test. Interestingly, the lower spontaneous activity of mice in the stress group was rescued by housing in conventional cages. These results suggest a temporary effect of long-term confinement in an enclosed environment as a chronic and mild stress on homeostasis in mammals.

The bicaudal D homolog 2 (BICD2) gene encodes a protein required for the stable complex of dynein and dynactin, which functions as a motor protein working along the microtubule cytoskeleton. Both inherited and de novo variants of BICD2 are reported with autosomal dominant spinal muscular atrophy with lower extremity predominance (SMALED2). Here, we report a male patient with a novel mutation in the BICD2 gene caused by a heterozygous substitution of arginine with cysteine at residue 162 (Arg162Cys); inherited from his asymptomatic mother. The patient showed typical clinical symptoms of SMALED2, which was genetically confirmed by sequencing. The Arg162Cys mutant clusters with four previously reported variants (c.361C>G, p.Leu121Val; c.581A>G, p.Gln194Arg; c.320C>T, p.Ser107Leu; c.565A>T, p.Ile189Phe) in a region that binds to the dynein-dynactin complex (DDC). The BICD2 domain structures were predicted and the Arg162Cys mutation was localized in the N-terminus coiled-coil segment 1 (CC1) domain. Protein modeling of BICD2's CC1 domain predicted that the Arg162Cys missense variant disrupted interactions with dynein cytoplasmic 1 heavy chain 1 within the DDC. The mutant did this by either changing the electrostatic surface potential or making a broader hydrophobic unit with the neighboring residues. This hereditary case supports the complex and broad genotype-phenotype correlation of BICD2 mutations, which could be explained by incomplete penetrance or variable expressivity in the next generation.

Dedifferentiation has been implicated in β cell dysfunction and loss in rodent diabetes. However, the pathophysiological significance in humans remains unclear. To elucidate this, we analyzed surgically resected pancreatic tissues of 26 Japanese subjects with diabetes and 11 nondiabetic subjects, who had been overweight during adulthood but had no family history of diabetes. The diabetic subjects were subclassified into 3 disease stage categories, early, advanced, and intermediate. Despite no numerical changes in endocrine cells immunoreactive for chromogranin A (ChgA), diabetic islets showed profound β cell loss, with an increase in α cells without an increase in insulin and glucagon double-positive cells. The proportion of dedifferentiated cells that retain ChgA immunoreactivity without 4 major islet hormones was strikingly increased in diabetic islets and rose substantially during disease progression. The increased dedifferentiated cell ratio was inversely correlated with declining C-peptide index. Moreover, a subset of islet cells converted into exocrine-like cells during disease progression. These results indicate that islet remodeling with dedifferentiation is the underlying cause of β cell failure during the course of diabetes progression in humans.

Glucose metabolism is regulated by insulin, which is produced from β-cells in the pancreas. Because insulin is secreted into vessels in response to blood glucose, vascular structures of the pancreas, especially the relationship between vessels and β-cells, are important for physiological and pathological glucose metabolism. Here, we developed a system to visualize vessels surrounding mature β-cells expressing transcription factor MafA in a three-dimensional manner. Optical clearing of the pancreas prevented light scattering of fluorescence driven by the bacterial artificial chromosome (BAC)-mafA promoter in β-cells. Reconstruction of confocal images demonstrated mature β-cells and the glomerular-like structures of β-cell vasculatures labeled with DyLight 488-conjugated lectin in normal mice as well as in low-dose streptozotocin-injected diabetes model mice with reduced β-cell mass. This technological innovation of organ imaging can be used to investigate morphological changes in vascular structures during transplantation, regeneration and diabetes development.

Pathological complete response (pCR) is considered to be a useful prognostic marker for neoadjuvant chemotherapy to improve the survival rate of patients with operable breast cancer. In the present study, we identified differentially expressed microRNAs (miRNAs) between pCR and non-pCR groups of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who received neoadjuvant chemotherapy with trastuzumab. Expression profiles were examined by miRNA microarrays using total RNA extracted from formalin-fixed, paraffin-embedded tissues from pretreatment biopsy specimens. Significant differences were observed in miRNAs associated with pCR between the luminal B-like (HER2-positive) and HER2posi-tive (nonluminal) subtypes, which were further classified according to their estrogen receptor (ER) status. Prediction models constructed with differentially expressed miRNAs performed well. In conclusion, the combination of miRNA profiles and ER status may improve the accuracy of pCR prediction in patients with HER2-positive breast cancer and enable the development of personalized treatment regimens.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3’ UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3’ UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

Early in pancreatic development, epithelial cells of pancreatic buds function as primary multipotent progenitor cells (1 degrees MPC) that specify all three pancreatic cell lineages, i.e., endocrine, acinar and duct. Bipotent "Trunk" progenitors derived from 1 degrees MPC are implicated in directly regulating the specification of endocrine progenitors. It is unclear if this specification process is initiated in the 1 degrees MPC where some 1 degrees MPC become competent for later specification of endocrine progenitors. Previously we reported that in Pdx1(tTA/+); tetO(MafA) (bigenic) mice inducing expression of transcription factor MafA in Pdx1-expressing (Pdx1(+)) cells throughout embryonic development inhibited the proliferation and differentiation of 1 degrees MPC cells, resulting in reduced pancreatic mass and endocrine cells by embryonic day (E) 17.5. Induction of the transgene only until E12.5 in Pdx1(+) 1 degrees MPC was sufficient for this inhibition of endocrine cells and pancreatic mass at E17.5. However, by birth (P0), as we now report, such bigenic pups had significantly increased pancreatic and endocrine volumes with endocrine clusters containing all pancreatic endocrine cell types. The increase in endocrine cells resulted from a higher proliferation of tubular epithelial cells expressing the progenitor marker Glut2 in E17.5 bigenic embryos and increased number of Neurog3-expressing cells at E19.5. A BrdU-labeling study demonstrated that inhibiting proliferation of 1 degrees MPC by forced MafA-expression did not lead to retention of those progenitors in E17.5 tubular epithelium. Our data suggest that the forced MafA expression in the 1 degrees MPC inhibits their competency to specify endocrine progenitors only until E17.5, and after that compensatory proliferation of tubular epithelium gives rise to a distinct pool of endocrine progenitors. Thus, these bigenic mice provide a novel way to characterize the competency of 1 degrees MPC for their ability to specify endocrine progenitors, a critical limitation in our understanding of endocrine differentiation.

Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease-associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non-coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP-containing locus in an allele-specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage-gated channel, KQT-like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non-risk alleles were immobilized onto the novel nanobeads and DNA-binding proteins were purified from the nuclear extracts of pancreatic cells using these DNA-immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF-Y) specifically bound the non-risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele-specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF-Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele-specific DNA-binding proteins, which may provide important clues for the functional impact of disease-associated non-coding SNPs.

The plasticity of adult somatic cells allows for their dedifferentiation or conversion to different cell types, although the relevance of this to disease remains elusive. Perturbation of beta cell identity leading to dedifferentiation may be implicated in the compromised functions of beta cells in diabetes, which is a current topic of islet research. This study aims to investigate whether or not v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker, is involved in maintaining mature beta cell phenotypes. The fate and gene expression of beta cells were analysed in Mafa knockout (KO) mice and mouse models of diabetes in which the expression of MafA was reduced in the majority of beta cells. Loss of MafA reduced the beta to alpha cell ratio in pancreatic islets without elevating blood glucose to diabetic levels. Lineage tracing analyses showed reduced/lost expression of insulin in most beta cells, with a minority of the former beta cells converted to glucagon-expressing cells in Mafa KO mice. The upregulation of genes that are normally repressed in mature beta cells or transcription factors that are transiently expressed in endocrine progenitors was identified in Mafa KO islets as a hallmark of dedifferentiation. The compromised beta cells in db/db and multiple low-dose streptozotocin mice underwent similar dedifferentiation with expression of Mafb, which is expressed in immature beta cells. The maturation factor MafA is critical for the homeostasis of mature beta cells and regulates cell plasticity. The loss of MafA in beta cells leads to a deeper loss of cell identity, which is implicated in diabetes pathology.

MafA and MafB are basic leucine zipper transcription factors expressed in mature pancreatic β- and α-cells, respectively. MafA is not only an insulin gene transcription factor but is also critical for the maturation and maintenance of β-cell function, whereas MafB is expressed in immature β-cells during development and in compromised β-cells in diabetes. In this study, we developed a mouse model to easily trace the promoter activity of MafA in β-cells as a tool for studying β-cell differentiation, maturation, regeneration and function using the expression of the fluorescent protein Kusabira Orange (KOr) driven by the BAC-mafA promoter. The expression of KOr was highly restricted to β-cells in the transgenic pancreas. By crossing MafA-KOr mice with MafBGFP/+ reporter mice, simultaneous monitoring of MafA and MafB expressions in the isolated islets was successfully performed. This system can be a useful tool for examining dynamic changes in the differentiation and function of pancreatic islets by visualizing the expressions of MafA and MafB.

Chemotherapy-induced nausea and vomiting is a serious adverse side-effect of anthracycline-based chemotherapy regimens, in patients with breast cancer. A combination of three drugs, 5-hydroxytryptamine (5-HT3) receptor antagonist, aprepitant and dexamethasone, is recommended for antiemetic therapy. Palonosetron (PALO), a novel 5-HT3 receptor antagonist has been identified to be effective against delayed nausea and vomiting. In this study, the results of PALO for patients who received anthracycline-based chemotherapy were compared with that of granisetron (GRA) using a crossover study design. This study evaluated the efficacy of antiemetics in the first cycle of chemotherapy, as well as the second and third cycles. A total of 21 patients and 19 patients were assigned to PALO and GRA treatment groups during the first cycle of chemotherapy, respectively. The patients switched to the other antiemetic drug for the second chemotherapy cycle (PALO followed by GRA or GRA followed by PALO). The patients could select PALO or GRA antiemetics for the third cycle, according to their preference. A total of 21 patients selected PALO and 18 patients selected GRA in the third cycle, and one patient was withdrawn from the study as their third cycle questionnaire was not obtained. No significant differences between PALO and GRA were identified in first and second cycles. However, during the third cycle, a significant difference was observed in acute-phase complete control of emetic events between the PALO and GRA groups, which was defined as no emetic episode, no additional antiemetic treatment and no more than mild nausea, between PALO and GRA. These results demonstrated that changing antiemetics may affect the efficacy of antiemetics. This study indicates that alteration of antiemetic regimens, including drug combination and order, may improve the efficacy of antiemetic treatment.

The luciferase reporter system is useful for the assessment of various biological processes in vivo. The transcription factor pancreatic and duodenal homeobox 1 (Pdx1) is critical for the formation and the function of pancreatic β-cells. A novel reporter system using secreted Gaussia princeps luciferase (GLuc) under the control of a Pdx1 promoter was generated and activated in rat and mouse β-cell lines. This Pdx1-GLuc construct was used as a transgene for the generation of reporter mice to monitor Pdx1 promoter activity in vivo via the measurement of secreted GLuc activity in a small aliquot of blood. Significantly increased plasma GLuc activity was observed in Pdx1-GLuc mice. Analysis of Pdx1-GLuc mice by bioluminescence imaging, GLuc reporter assays using homogenatesof various organs,andimmunohistochemistryrevealed thatGLucexpressionandactivity were exponentially higher in pancreatic β-cells than in pancreatic non-β-cells, the duodenum, and other organs. In addition, GLuc activity secreted into the culture medium from islets isolated from Pdx1-GLucmicecorrelatedwiththenumberofislets. ThetransplantationofPdx1-GLucisletsintosevere combined immunodeficiency mice elevated their plasma GLuc activity. Conversely, a partial pancreatectomy in Pdx1-GLuc mice reduced plasma GLuc activity. These results suggest that a secreted luciferase reporter system in vivo enables not only the monitoring of promoter activity but also a quantitative and minimally invasive assessment of physiological and pathological changes in small cell masses, such as pancreatic β-cells. Copyright © 2013 by The Endocrine Society.

Abstract. Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE-19 and RNA interference (RNAi). Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy.

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium -Pdx1, Tcf2 and Sox9 - suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of beta-cells, but with increasing maturation have increasing numbers of MafA(+) insulin(+) cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

The basic helix-loop-helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3(+) or Ki67(+) cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

The transcription factor MafA regulates glucose-responsive expression of insulin. MafA-deficient mice have a normal proportion of insulin(+) cells at birth but develop diabetes gradually with age, suggesting that MafA is required for maturation and not specification of pancreatic beta-cells. However, several studies show that ectopic expression of MafA may have a role in specification as it induces insulin(+) cells in chicken gut epithelium, reprograms adult murine acinar cells into insulin(+) cells in combination with Ngn3 and Pdx1, and triggers the lens differentiation. Hence, we examined whether MafA can induce specification of beta-cells during pancreatic development. When the MafA transgene is expressed in Pdx1(+) pancreatic progenitors, both pancreatic mass and proliferation of progenitors are reduced, at least partially due to induction of cyclin kinase inhibitors p27 and p57. Expression of MafA in Pdx1(+) cells until E12.5 was sufficient to cause these effects and to disproportionately inhibit the formation of endocrine cells in the remnant pancreas. Thus, in mice, MafA expression in Pdx1(+) pancreatic progenitors is not sufficient to specify insulin(+) cells but in fact deters pancreatic development and the differentiation of endocrine cells. These findings imply that MafA should be used to enhance maturation, rather than specification, of beta-cells from stem/progenitor cells. (C) 2009 Elsevier Inc. All rights reserved.

Mammalian MafA/RIPE3b1 is an important glucose-responsive transcription factor that regulates function, maturation, and survival of beta-cells. Increased expression of MafA results in improved glucose-stimulated insulin secretion and beta-cell function. Because MafA is a highly phosphorylated protein, we examined whether regulating activity of protein kinases can increase MafA expression by enhancing its stability. We demonstrate that MafA protein stability in MIN6 cells and isolated mouse islets is regulated by both p38 MAPK and glycogen synthase kinase 3. Inhibiting p38 MAPK enhanced MafA stability in cells grown under both low and high concentrations of glucose. We also show that the N-terminal domain of MafA plays a major role in p38 MAPK-mediated degradation; simultaneous mutation of both threonines 57 and 134 into alanines in MafA was sufficient to prevent this degradation. Under oxidative stress, a condition detrimental to beta-cell function, a decrease in MafA stability was associated with a concomitant increase in active p38 MAPK. Interestingly, inhibiting p38 MAPK but not glycogen synthase kinase 3 prevented oxidative stress-dependent degradation of MafA. These results suggest that the p38 MAPK pathway may represent a common mechanism for regulating MafA levels under oxidative stress and basal and stimulatory glucose concentrations. Therefore, preventing p38 MAPK-mediated degradation of MafA represents a novel approach to improve beta-cell function. (Molecular Endocrinology 23: 1281-1290, 2009)

OBJECTIVE-Diabetes results from a deficiency of functional beta-cells due to both an increase in beta-cell death and an inhibition of beta-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for beta-cells. RESEARCH DESIGN AND METHODS-We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin. RESULTS-Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in P-cell mass, confirming the specificity of the survivin effect in these cells. CONCLUSIONS-Our findings implicate survivin in the maintenance of beta-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in beta-cell regulation in diseased states, such as diabetes.

During pancreatic development insulin(+) cells co-express the transcription factors MafB and Pax6, and transition from a MafA(-) to MafA(+) state. To examine the role of Pax6 and MafB in the development of beta-cells, we analyzed embryonic pancreata from Pax6- and MqfB-deficient mice. Pax6 deficiency, as manifest in the Pax6(Sey - Neu) allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr(ENU)) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr(ENU)/kr(ENU)) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin+ cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for beta-cell maturation. (c) 2007 Elsevier Inc. All rights reserved.

Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both alpha- and beta-cells, and MafA and MafB showed selective expression in the beta- and alpha-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of beta-cells proceeds through a MafB(+) MafA(-) Ins(+) intermediate cell to MafB(-) MafA(+) Ins(+) cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1(high)) in MatB(+) Ins(+) cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both beta- and alpha-cells while MafA may regulate replication/survival and function of beta-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth. (c) 2006 Elsevier Inc. All rights reserved.

The transcription factor MafA/RIPE3b I is an important regulator of insulin gene expression. MafA binds to the insulin enhancer element RIPE3b (C1-A2), now designated as insulin MARE (Maf response element). The insulin MARE element shares an overlapping DNA-binding region with another insulin enhancer element A2.A2.2, a beta-cell-specific activator, like the MARE-binding factor MafA, binds to the overlapping A2 element. Our previous results demonstrated that two nucleotides in the overlapping region are required for the binding of both factors. Surprisingly, instead of interfering with each other's binding activity, the MafA and the A2-binding factors co-operatively activated insulin gene expression. To understand the molecular mechanisms responsible for this functional co-operation, we have determined the nucleotides essential for the binding of the A2.2 factor. Using this information, we have constructed non-overlapping DNA-binding elements and their derivatives, and subsequently analysed the effect of these modifications on insulin gene expression. Our results demonstrate that the overlapping binding site is essential for maximal insulin gene expression. Furthermore, the overlapping organization is critical for MafA-mediated transcriptional activation, but has a minor effect on the activity of A2-binding factors. Interestingly, the binding affinities of both Mafa and A2.2 to the overlapping or non-overlapping binding sites were not significantly different, implying that the overlapping binding organization may increase the activation potential of MafA by physical/functional interactions with A2-binding factors. Thus our results demonstrate a novel mechanism for the regulation of MafA activity, and in turn beta-cell function, by altering expression and/or binding of the A2.2 factor. Our results further suggest that the major downstream targets of MafA will in addition to the MARE element have a binding site for the A2.2 factor..

N-methyl-D-aspartate (NMDA) receptors have been suggested to be critical for the development of central sensitization, which may amplify postoperative pain. NMDA receptors are formed by GluRzeta (NR1) with any one of four GluRvarepsilon1-4 (NR2A-D) subunits. To clarify the involvement of NMDA receptors in postoperative pain, we examined the effect of the GluRepsilon2-selective antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenyl piperidino)-1-propanol (CP-101,606) on postoperative pain caused by plantar incision. We also applied the postoperative pain model to GluRepsilon1 and GluRepsilon4 knockout mice. CP-101,606 administered intrathecally 30 min prior to incision significantly increased mechanical withdrawal thresholds 2 h and 1-3 days after surgery and reduced postoperative pain dose-dependently. Neither GluRvarepsilon1 nor GluRepsilon4 knockout mice showed a difference in withdrawal thresholds as compared with wild-type mice. Pretreatment with CP-101,606 did not produce an additive analgesic effect in the mice. These results demonstrate that GluRepsilon2-containing NMDA receptors are involved in postoperative pain and that CP-101,606 may be effective in reducing it.

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-D-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.

MAGI-1 and CASK are membrane-associated guanylate kinases of epithelial junctions. MAGI-1 is localized at tight junctions in polarized epithelial cells, whereas CASK is localized along the lateral membranes. We obtained the KIAA0769 gene product through the yeast two-hybrid screening using MAGI-1 as a bait and named it Carom. Carom has a coiled-coil domain in the middle region, and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region. Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro. MAGI-1 and CASK bind to the distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding. The study using a stable transformant of Madine Darby canine kidney (MDCK) cells expressing GFP-Carom revealed that Carom was partially overlapped by MAGI-1 in MDCK cells, which have not yet established mature cell junctions, but became separated from MAGI-1 and colocalized with CASK in polarized cells. Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures. Carom is a binding partner of CASK, which interacts with CASK in polarized epithelial cells and may link it to the cytoskeleton.

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.

Background: SAPAP1 was originally identified as a protein interacting with the guanylate kinase domain of PSD-95. SAPAP1 also interacts with various proteins, including neurofilaments, synaptic scaffolding molecule (S-SCAM), nArgBP2, dynein light chain and Shank through different regions. Results: We expressed various regions of SAPAP1 in hippocampal neurones. The synaptic targeting of SAPAP1 was mediated by the N-terminal region and did not depend on the interaction with PSD-95 or S-SCAM. SAPAP1 was not involved in the synaptic localization of PSD-95 or S-SCAM, but affected that of Shank. The synaptic targeting of SAPAP1 was not suppressed by blocking NMDA or AMPA receptors. Fluorescent recovery after a photobleaching study revealed that SAPAP1 was immobile at synapses. Conclusion: SAPAP1 is a component of the static core of PSD, and its dynamics are different from those of the other PSD components, PSD-95, S-SCAM and BEGAIN.

Brain-enriched guanylate kinase-associated protein (BEGAIN) interacts with postsynaptic density (PSD)-95/synapse-associated protein (SAP) 90. In immunohistochemistry and immunocytochemistry, BEGAIN was detected in nuclei and at synapses in neurons. Nuclear localization was also confirmed through subcellular fractionation. BEGAIN was localized exclusively in nuclei when expressed in epithelial cells. These findings led us to analyze the mechanism to determine the subcellular localization of BEGAIN in neurons. Green fluorescent protein (GFP)-tagged BEGAIN appeared first in nuclei and subsequently accumulated at dendrites. Approximately 75 and 90% of GFP-BEGAIN clusters were colocalized with synaptophysin and PSD-95/SAP90, respectively. GFP-protein containing only the N-terminal region also formed foci in nuclei and clusters at dendrites. The N-terminal BEGAIN was not precisely targeted to synapses, although it was partially localized at synapses, possibly through dimer formation with endogenous BEGAIN. The truncated form of PSD-95/SAP90 containing the guanylate kinase domain blocked synaptic targeting of BEGAIN but did not affect cluster formation at dendrites. NMDA receptor antagonists blocked localization of GFP-BEGAIN at synapses but did not affect recruitment to dendrites. These results suggest that BEGAIN is recruited to dendrites by the N-terminal region independently of NMDA receptor activity and that synaptic targeting of BEGAIN depends on NMDA receptor activity and may be mediated by interaction with PSD-95/SAP90.

Membrane-associated guanylate kinase-interacting protein (MAGUIN) has been identified as a protein binding postsynaptic density (PSD)-95 and synaptic scaffolding molecule (S-SCAM). MAGUIN has one sterile alpha motif, one conserved region in connector enhancer of ksr (Cnk) (CRIC), one PSD-95/Dlg-A/ZO-1 (PDZ) and one pleckstrin homology (PH) domain. There are two isoforms, MAGUIN-1 and -2. MAGUIN-1 binds the PDZ domains of PSD-95 and S-SCAM by the C-terminus, whereas MAGUIN-2 does not bind to PSD-95 or S-SCAM. Here, we have determined that MAGUIN-2 is also localized at synapses and that the synaptic localization of MAGUIN depends on the pleckstrin homology domain. The overexpressed C-terminal PDZ-binding region inhibits the synaptic targeting of PSD-95. Furthermore, the synaptic targeting of MAGUIN does not require N-methyl-d-aspartate (NMDA) receptor activity. These findings suggest that MAGUIN-1 and -2 are recruited to synapses by the PH domain and that MAGUIN-1 subsequently interacts with PSD-95 at synapses.

Synaptic scaffolding molecule (S-SCAM) is a synaptic membrane-associated guanylate kinase with inverted domain organization (MAGI) that interacts with NMDA receptor subunits and neuroligin. In epithelial cells, the non-neuronal isoform of S-SCAM (MAGI-1) is localized at tight or adherens junctions. Recent studies have revealed that the polarized targeting of MAGI-1 to the lateral membrane is mediated by its C-terminal region and that MAGI-1 interacts with beta-catenin in epithelial cells. In this article, we report that S-SCAM interacts with beta-catenin in neurons. beta-Catenin is coimmunoprecipitated with S-SCAM from rat brain. Both S-SCAM and beta-catenin are localized at synapses and are partially colocalized. The C-terminal region of S-SCAM binds to the C-terminal region of beta-catenin. We have tested how the interaction between S-SCAM and beta-catenin plays a role in the synaptic targeting of S-SCAM and beta-catenin. S-SCAM is targeted to synapses via the C-terminal postsynaptic density-95/Dlg-A/ZO-1 (PDZ) domain. beta-Catenin is targeted to synapses with armadillo repeats. The over-expressed C-terminal region of beta-catenin blocks the synaptic targeting of S-SCAM. The overexpressed C-terminal region of S-SCAM is partially targeted to synapses and forms a small number of clusters. In the presence of overexpressed beta-catenin, the C-terminal region of S-SCAM forms more clusters at synapses. These data suggest that the synaptic targeting of S-SCAM is mediated by the interaction with beta-catenin.

Basaloid-squamous cell carcinoma of the esophagus (BSCC) is an extremely rare tumor. Histologically, this tumor should be differentiated from adenoid cystic carcinoma (ACC) and small cell undifferentiated carcinoma (SCUC). Biologically, this tumor is very aggressive, with a propensity for distant metastasis. We report a 64-year-old male with esophageal BSCC. The patient complained of dysphagia and was found to have a torous lesion in the esophagus on radiological examination. Upper gastrointestinal fiberscopy showed a localized ulcerative type tumor, which was diagnosed as squamous cell carcinoma (SCC) on biopsy. Surgery resulted in curative resection. A histological examination of the resected tumor showed features of BSCC. Immunohistochemical examination demonstrated AE3/1- and CAM 5.2-positive tumor cells, and laminin-positive cells in the periphery of the nests. These data were useful in differentiating this tumor from ACC and SCUC. Six months after surgery, the patient developed hepatic metastases, which were successfully treated by regional chemotherapy via the hepatic artery by using fluorouracil. The patient is currently being followed up at the outpatient clinic and shows no signs of any recurrence. (C) 2001 Blackwell Science Asia Pty Ltd.

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (CK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form light junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and CK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1. J. Cell. Physiol. 185:358-365, 2000. (C) 2000 Wiiey-Liss, Inc.

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/ delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/ Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.

Background/Aims: Hormone therapy by tamoxifen was performed on patients with adenocarcinoma of the pancreas and the effect of tamoxifen on their survival rate was evaluated inpatients with resected pancreatic adenocarcinoma (n=65). Material and Methods: Tamoxifen. lugs administered once daily (at 20mg) in addition, to immunochemotherapy (Tegaful, Mitomycin, Krestin or OK-432). Results: A remarkably beneficial effect of tamoxifen was found when compared with non-tamoxifen group (p<0.01). When the location of the pancreatic carcinoma was considered, the survival rate of the tamoxifen group with adenocarcinoma in the head and in, also the body/tail of the pancreas revealed the same statistically significant difference (p<0.01) individually. Regarding curability by surgery, the beneficial effect of tamoxifen was observed even in the survival rate of patients who had received noncurative surgery. Furthermore, the remarkable effect of tamoxifen was revealed in the male group, whose one-year survival rate for resected carcinoma in the head of the pancreas was 85.6% (n=16) as when. compared to 19.1% of non-tamoxifen group (n=15) and also in the female group whose one-year survival rate was 53.8% (n=1.3) compared with 21.4% of non-tamoxifen group (n=7) (p<0.01). Conclusion: With these data, we can see that the adjuvant therapy of tamoxifen in carcinoma of the pancreas confers a significant benefit to those patients who have received a surgical resection and reduction in the volume of the carcinoma in the pancreas.