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Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5’-upstream region of GREB1exon 19 are necessary for GREB1 Is4transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENfloxmelanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

The decrease in mortality rate owing to asthma has slowed in recent years. A large proportion of patients with asthma remain uncontrolled in Japan. This study aimed to determine the prevalence of short-acting beta 2 agonists (SABA) overuse and its associated factors.

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5ʹ-untranslated region of the DKK1gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.

Molecular characteristics of carcinoma arising from mature cystic teratoma of the ovary (MCT) remain unclear due to its rarity. We analyzed RNA-sequencing data of 2322 pan-cancer [1378 squamous cell carcinomas (SCC), 6 adenosquamous carcinomas (ASC), and 938 adenocarcinomas (AC)] including six carcinomas arising from MCT (four SCCs, one ASC, and one AC). Hierarchical clustering and principal component analysis showed that gene expression profiles of carcinomas arising from MCT were different between each histological type and that gene expression profiles of SCCs arising MCT (MCT-SCCs) was apparently similar to those of lung SCCs. By epidermis-associated pathways activity based on gene set enrichment analysis, 1030 SCCs were divided into two groups: epidermis-signature high (head and neck, esophagus, and skin) and low (cervix, lung, and MCT). In addition to pan-SCC transcriptome analysis, cytokeratin profiling based on immunohistochemistry in the independent samples of 21 MCT-SCCs clarified that MCT-SCC dominantly expressed CK18, suggesting the origin of MCT-SCC was columnar epithelium. Subsequently, we investigated differentially expressed genes in MCT-SCCs compared with different SCCs and identified XCL1was specifically overexpressed in MCT-SCCs. Through immunohistochemistry analysis, we identified XCL1 expression on tumor cells in 13/24 (54%) of MCT-SCCs but not in MCTs. XCL1 expression was also significantly associated with the number of tumor-infiltrating CD8-positive T cells and PD-L1 expression on tumor cells. XCL1 produced by tumor cells may induce PD1/PD-L1 interaction and dysfunction of CD8-positive T cells in tumor microenvironment. XCL1 expression may be a novel biomarker for malignant transformation of MCT into SCC and a biomarker candidate for therapeutic response to an anti-PD1/PD-L1 therapy.

Aberrant expression of the secretory protein Dickkopf1 (DKK1) is associated with poor prognosis of esophageal squamous cell carcinoma (ESCC), but the underlying mechanism of how DKK1 is involved in aggressiveness of ESCC is not clear. In this study, we show that cytoskeleton-associated protein 4 (CKAP4) functions as a DKK1 receptor in ESCC cells. Immunohistochemical analyses of ESCC revealed that both DKK1 and CKAP4 are minimally expressed in associated normal esophageal squamous mucosa of non-tumor regions, but strongly expressed in tumor lesions. Forty-six of 119 cases (38.7%) were positive for both DKK1 and CKAP4. Those expressing both proteins showed poor prognosis and relapse-free survival. Multivariate analysis demonstrated that expression of both proteins was the poor prognostic factor. The Cancer Genome Atlas data set indicated that the mRNA levels of DKK1and CKAP4are significantly elevated in the tumor lesions compared to non-tumor regions. DKK1 bound to CKAP4 at endogenous levels. DKK1 induced the internalization of CKAP4 from and its recycling to the plasma membrane. AKT was activated in ESCC cells in which DKK1 was highly expressed and CKAP4 was localized to the plasma membrane. Knockdown of either DKK1 or CKAP4 inhibited AKT activity and cell proliferation in vitro and xenograft tumor formation. Wild-type CKAP4 or DKK1, but not a DKK1 mutant that was unable to bind to CKAP4, rescued phenotypes induced by CKAP4 or DKK1 knockdown, respectively. The anti-CKAP4 antibody also inhibited AKT activity and suppressed xenograft tumor formation. In contrast, in ESCC cells in which DKK1 was marginally expressed, knockdown of CKAP4 or anti-CKAP4 antibody affected neither AKT activity nor cell proliferation. These findings suggest that the DKK1-CKAP4 pathway promotes ESCC cell proliferation and that CKAP4 might represent a novel therapeutic target for ESCCs expressing both DKK1 and CKAP4.

Helicobacter pyloristrains that deliver the oncoprotein CagA into gastric epithelial cells are the major etiologic agents of upper gastric diseases including gastric cancer. CagA promotes gastric carcinogenesis through interactions with multiple host proteins. Here, we show that CagA also disrupts Wnt-dependent planar cell polarity (Wnt/PCP), which orients cells within the plane of an epithelium and coordinates collective cell behaviors such as convergent extension to enable epithelial elongation during development. Ectopic expression of CagA in Xenopus laevisembryos impaired gastrulation, neural tube formation, and axis elongation, processes driven by convergent extension movements that depend on the Wnt/PCP pathway. Mice specifically expressing CagA in the stomach epithelium had longer pyloric glands and mislocalization of the tetraspanin proteins VANGL1 and VANGL2 (VANGL1/2), which are critical components of Wnt/PCP signaling. The increased pyloric gland length was due to hyperproliferation of cells at the gland base, where Lgr5+stem and progenitor cells reside, and was associated with fewer differentiated enteroendocrine cells. In cultured human gastric epithelial cells, the N terminus of CagA interacted with the C-terminal cytoplasmic tails of VANGL1/2, which impaired Wnt/PCP signaling by inducing the mislocalization of VANGL1/2 from the plasma membrane to the cytoplasm. Thus, CagA may contribute to the development of gastric cancer by subverting a Wnt/PCP-dependent mechanism that restrains pyloric gland stem cell proliferation and promotes enteroendocrine differentiation.

The need for affordable onsite sensing device (AOSD) has been determined in the medical, environmental, and educational fields since mid 2000s. This article aims to explore the unique nature and definition of the AOSD, and conclusively culminated the discussion into a designed prototype trial device. The definition of AOSD was concluded as “a unique cost efficient device that can solve users’ actual small problems giving unique experimental capacity to improve their quality of life, quality of education, quality of environment, and quality of job under quite resource limited settings”. The best water hygiene indicator, E. coli, was selected as an example of analyte. A designed prototype trial E. coli AOSD showed very good performance in a community engagement program in LAO PDR.

Producing high quality essential oil from Eucalyptus globulusleaves, distillation is the primary method that is both economical and environmentally friendly. In order to improve the producing process, provide renewal information as reference for further practical applications, the effects of the storage conditions (air-drying, freezing, chilling, and oven-assisted drying) as well as the grinding (less than 4.75, 4.75–9.50, 9.50–22.40, and greater than 22.40 mm), ultrasound (ultrasonic cleaning bath, ultrasonic homogenizer), and agitation treatments (0 ~ 100 rpm) of the samples were studied. In addition, the yield differences by different methods (steam distillation and the water distillation) were compared. Considering both the yield and the operation time, the technique, which extracting small-sized (smaller than 4.70 mm) oven-assisted dried leaves with water distillation under an agitation rate of 25 rpm, should be ideal for the production of essential oil.

AbstractA separation for Cd2+and its interferences of common cations (Na+, NH+, K+, Mg2+, and Ca2+) was performed. Cation-exchange ion chromatography for Cd2+and common cations separation on a weakly acidic H+forming cation-exchange resin was investigated and applied to test the efficacy of activated carbon in the removal of Cd2+from simulated wastewater. The difference of retention volumes between Ca2+and Cd2+was 0.45 mL for oxalic acid with a concentration of 1.75 mM, and the retention volume drastically decreased when the oxalic acid concentration increased in the eluent. Considering the prevailing conditions for TOSOH TSK gel Super IC A/C at 40°C with a least pH limit of 2.0, 1.75 mM of oxalic acid was selected for rapid analysis with pH of 2.3. Applying 50 μL sample injection, the calibration curve was linear for tested standard samples ranging from 0.05 to 2.0 mg/L, the detection limit (S/N = 3) was 0.066 mg/L (0.583 μM), and the analysis was applicable with coexisting Ca2+up to 10.0 mg/L (250 μM) while other cations did not interfere. It was possible to analyze three samples within an hour. The application of this method on Cd2+adsorption indicated the effectiveness of the method for heavy metals’ analysis.

A separation for Cd2+and its interferences of common cations (Na+, NH+, K+, Mg2+, and Ca2+) was performed. Cation-exchange ion chromatography for Cd2+and common cations separation on a weakly acidic H+forming cation-exchange resin was investigated and applied to test the efficacy of activated carbon in the removal of Cd2+from simulated wastewater. The difference of retention volumes between Ca2+and Cd2+was 0.45 mL for oxalic acid with a concentration of 1.75 mM, and the retention volume drastically decreased when the oxalic acid concentration increased in the eluent. Considering the prevailing conditions for TOSOH TSK gel Super IC A/C at 40°C with a least pH limit of 2.0, 1.75mM of oxalic acid was selected for rapid analysis with pH of 2.3. Applying 50μl sample injection, the calibration curve was linear for tested standard samples ranging from 0.05 to 2.0 mg/L, the detection limit (S/N = 3) was 0.066 mg/L (0.583 μM), and the analysis was applicable with coexisting Ca2+up to 10.0 mg/L (250 μM) while other cations did not interfere. It was possible to analyze three samples within an hour. The application of this method on Cd2+adsorption indicated the effectiveness of the method for heavy metals’ analysis.

Graft-versus-host disease (GVHD) is one of the most important complications in allogeneic hematopoietic stem cell transplantation. Intensity of conditioning regimen is one of the risk factors, which is associated with acute GVHD, and some studies have shown that alteration of the administration order from busulfan to cyclophosphamide to cyclophosphamide to busulfan could decrease cytokine levels and organ toxicity.

Wnt signal plays important roles in polarization. Here intestinal epithelial cells are shown to form apicobasal polarization at a single-cell level in an extracellular matrix adhesion–dependent manner. Wnt5a signaling promotes single-cell polarization through balanced control between Rac1 and RhoA activities spatially.

Kasabach-Merritt phenomenon (KMP) is a rare condition and optimal treatments have not yet been established, especially for cases that are unresponsive to first-line therapy. We retrospectively reviewed 11 KMP cases treated over the past 13 years in our institute.

Background:Recent studies suggest an association between maternal periodontitis and preterm birth, although the association remains controversial. It was suggested that mechanisms such as a genetic predisposition for a hyperinflammatory response cause periodontitis and preterm births. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a nuclear hormone receptor and ligand‐dependent transcription factor. PPARγ inhibits the transcriptional activity of the genes that produce proinflammatory mediators and repress periodontitis. Recently, a common polymorphism, proline(PRO)‐to‐alanine(ALA) mutation at codon12 in exonB (Pro12Ala: rs 1801282) PPARγ, was reported to reduce the ability to transactivate responsive promoters. In this study, we tested whether the PPARγPro12Ala polymorphism was associated with maternal periodontitis and/or preterm birth. Methods:Genomic DNA was isolated from the venous blood of pregnant Japanese women (term birth: n = 72; preterm birth: n = 58). The PPARγPro12Ala genotype was determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism. Within 5 days after labor, clinical periodontal parameters were evaluated, and periodontopathic bacteria from the subgingival plaque were detected by species‐specific PCR. Results:The mean clinical attachment level (P= 0.012), mean probing depth (P= 0.031), mean gingival index (P= 0.037), and percentages of sites with bleeding on probing (P= 0.041) in women with the PPARγPro12Ala genotype were significantly higher than in women with the PPARγPro12Pro genotype. However, there was no association between preterm birth and periodontitis. Conclusion:We suggest that the PPARγPro12Ala polymorphism may represent a genetic susceptibility factor for the clinical measurements of periodontitis in a limited number of pregnant Japanese women, but it probably cannot influence the relationship between periodontitis and preterm birth.

Genetic diversity and genetic integrity were tested for wheat (Triticum aestivumL.) landraces conserved in two gene banks with considerably different germplasm management systems. We identified two sets of 17 wheat accessions derived from identical sources collected in Pakistan, which were later deposited at the National Institute of Agrobiological Sciences, Japan (NIAS) and the Plant Genetic Resources Program, Pakistan (PGRP). Regeneration of the conserved germplasm was infrequent at both gene banks. Amplified fragment length polymorphism (AFLP) profiles did not indicate prominent changes in the banding patterns of the wheat landraces conserved at the two gene banks. No significant differences were observed in allele frequency of the detected loci in comparison to the original collections (materials preserved without regeneration and multiplication), except in one accession at NIAS. We conclude that genetic diversity was conserved at these two gene banks with different management systems. However, among the 161 scored loci, 26 AFLP bands disappeared at the same loci in NIAS and PGRP accessions, four disappeared only in NIAS accessions, and 28 only in PGRP accessions. At one particular locus, the band disappeared in 12 PGRP accessions but not in NIAS accessions. The results indicate that unique selection might occur at PGRP and that regeneration strategies to reduce it will be needed in the future.

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11and NRAS/KRASmutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RASmutation or without any aberrations, patients with PTPN11mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11mutation than for those without (25 versus64%; p0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11mutations were also associated with poor prognosis for survival. Mutation in PTPN11was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p0.001). All patients who died after relapse had PTPN11mutation. These results suggest that JMML with PTPN11mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

Previous studies have shown that cannabinoid 2 (CB2)-receptor agonists might have analgesic effects on visceral hypersensitivity. To extend these results, we have determined the pharmacological characteristics of a newly designed CB2ligand, N-[(1S)-1-(aminocarbonyl)-2,2-dimethylpropyl]-3-(3-hydroxy-3-methylbutyl)-2-oxo-2,3-dihydro-1H-benzimidazole-1-carboxamide (PF-03550096), in vitro and in vivo. PF-03550096 showed high affinity to human (Ki= 7.9 ± 1.7 nM) and rat CB2receptors (Ki= 47 ± 5.6 nM). In a cell-based functional assay, PF-03550096 behaved as a full agonist and showed high selectivity for human CB2receptors. Orally administered PF-03550096 (3, 10 mg/kg) inhibited the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced decrease in colonic pain threshold with statistical significance. The inhibitory effect of PF-03550096 (10 mg/kg) was significantly reversed by a selective CB2antagonist, N-(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl-5-(4-chloro-3-methylphenyl)-1(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), while SR144528 itself did not modify colonic pain threshold. These results indicate that PF-03550096 is a potent CB2agonist and possesses efficacy in a rat model of visceral hypersensitivity.

Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RASmutations (8 with NRASmutations, 3 with KRAS2mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2- to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RASmutations at the time of hematologic improvement, whereas DNA obtained from the nails had the wild type. Additionally, numbers of circulating granulocyte-macrophage progenitors were significantly reduced during the clinical course. Thus, some patients with JMML with specific RASmutations may have spontaneously improving disease.

Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RAS mutations (8 with NRAS mutations, 3 with KRAS2 mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2- to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RAS mutations at the time of hematologic improvement, whereas DNA obtained from the nails had the wild type. Additionally, numbers of circulating granulocyte-macrophage progenitors were significantly reduced during the clinical course. Thus, some patients with JMML with specific RAS mutations may have spontaneously improving disease.

The Wnt signaling pathway is conserved in various species from worms to mammals, and plays important roles in cellular proliferation, differentiation, and migration. Wnt stabilizes cytoplasmic ß-catenin and then the accumulated ß-catenin is translocated into the nucleus, where it activates the transcriptional factor T-cell factor (Tcf)/lymphoid enhancer factor (Lef), and thereby stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Tight regulation of this response involves post-translational modifications of the components of the Wnt signaling pathway. Phosphorylation, ubiquitination, and sumoylation have been shown to affect the half-life of ß-catenin and the transcriptional activity of Tcf/Lef. The precise spatio-temporal patterns of these multiple modifications determine the driving force of various cellular responses.

The development of modeling systems for activity-based travel demand ushers in a new era in transportation demand forecasting and planning. A comprehensive multimodal activity-based system for forecasting travel demand was developed for implementation in Florida and resulted in the Florida Activity Mobility Simulator (FAMOS). Two main modules compose the FAMOS microsimulation model system for modeling activity-travel patterns of individuals: the Household Attributes Generation System and the Prism-Constrained Activity-Travel Simulator. FAMOS was developed and estimated with household activity and travel data collected in southeast Florida in 2000. Results of the model development effort are promising and demonstrate the applicability of activity-based model systems in travel demand forecasting. An overview of the model system, a description of its features and capabilities, and preliminary validation results are provided.

The development of modeling systems for activity-based travel demand ushers in a new era in transportation demand forecasting and planning. A comprehensive multimodal activity-based system for forecasting travel demand was developed for implementation in Florida and resulted in the Florida Activity Mobility Simulator (FAMOS). Two main modules compose the FAMOS microsimulation model system for modeling activity–travel patterns of individuals: the Household Attributes Generation System and the Prism-Constrained Activity–Travel Simulator. FAMOS was developed and estimated with household activity and travel data collected in southeast Florida in 2000. Results of the model development effort are promising and demonstrate the applicability of activity-based model systems in travel demand forecasting. An overview of the model system, a description of its features and capabilities, and preliminary validation results are provided.

Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of β-catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xIdax, a negative regulator of Wnt signaling. XIdax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpole brain. Ectopic expression of xIdax inhibits the target gene expression, suggesting that xIdax can inhibit canonical Wnt signaling. To examine the function of xIdax, a morpholino oligo for xIdax (xIdaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xIdaxMO-injected embryo, suggesting that xIdax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xIdax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development. Developmental Dynamics 230:79–90, 2004. © 2004 Wiley-Liss, Inc.

The Wnt signaling pathways are important in many developmental events. The canonical Wnt pathway is one of the three major Wnt-mediated intracellular signaling pathways and is thought to activate Dvl followed by the stabilization of β-catenin. In Xenopus, this pathway is involved in dorsal determination, anterior-posterior patterning during gastrulation, and neural induction. Here we describe a role for the Xenopus ELL ( Eleven-nineteen Lysine-rich Leukemia ) gene product in canonical Wnt signaling. Translocation of ELL has been associated with acute myeloid leukemia and the protein possesses three functional domains. We identified rELL-C from a rat brain cDNA library as a binding factor for Dishevelled (Dvl); it represents a partial sequence of rat ELL lacking the pol II elongation domain and has been shown to suppress canonical Wnt signaling. Next, we isolated two Xenopus homologs of ELL, xELL1 and xELL2. No obvious phenotypes were observed with microinjection of full-length xELL1 or xELL2 mRNA, however, microinjection with their occludin homology domain inhibited Wnt signaling at the level of Dvl and upstream of β-catenin. Intracellular localization of microinjected xELL1- and xELL2-GFP mRNAs showed localization of the full-length products in the nucleus and the occludin-homology domain products in cytoplasm. These results raise the possibility that ELL, which is thought to function as a transcription factor in nuclei, can serve other, novel roles to suppress canonical Wnt signaling in the cytoplasm. [ABSTRACT FROM AUTHOR]

The genetics of development and cancer have converged in the identification of intra‐ and extra‐cellular signaling pathways that are aberrantly regulated in cancer, and are also central to embryonic patterning. The Wnt signaling pathway has provided an outstanding example of this. The genes for β‐catenin, ARC, and Axin in the Wnt signaling pathway are often mutated in human cancers. In all such cases, the common denominator is the activation of gene transcription by β‐catenin. The resulting gene expression profile should provide a significant clue to the developmental mechanisms of cancers carrying defects in the Wnt signaling pathway. In this review, the functions of β‐catenin, APC and Axin, and the alterations of the three genes in human cancers are described. (Cancer Sci 2003; 94: 225–229)

The yeast Saccharomyces cerevisiaehas four genes,MCK1, MDS1(RIM11),MRK1, and YOL128c, that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). A gsk-3null mutant in which these four genes are disrupted showed growth defects on galactose medium. We isolated several multicopy suppressors of this growth defect. Two of them encoded Msn2p and phosphoglucomutase (PGM). Msn2p is a transcription factor that binds to the stress-response element (STRE). PGM is an enzyme that interconverts glucose-1 phosphate and glucose-6 phosphate and is regulated by Msn2p at the transcriptional level. Expression of the mRNAs ofPGM2and DDR2, whose promoter regions possess STRE sequences, on induction by heat shock or salt stress was reduced not only in an msn2 msn4(msn2homologue) double mutant but also in the gsk-3null mutant. STRE-dependent transcription was greatly inhibited in thegsk-3null mutant or mck1 mds1double mutant, and this phenotype was suppressed by the expression of Mck1p but not of a kinase-inactive form of Mck1p. Although Msn2p accumulated in the nucleus of the gsk-3null mutant as well as in the wild-type strain under various stress conditions, its STRE-binding activity was reduced in extracts prepared from the gsk-3null mutant or mck1 mds1double mutant. These results suggest that yeast GSK-3 promotes formation of a complex between Msn2p and DNA, which is required for the proper response to different forms of stress. Because neither Msn2p–GSK-3 complex formation nor GSK-3–dependent phosphorylation of Msn2p could be detected, the regulation of Msn2p by GSK-3 may be indirect.

POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl-terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl-terminal region including the SH3 domain of PAG2. Substitutions of Pro423and Pro426with Ala (POB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3β binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3β formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3β, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3β, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3β bound to AKAP220 decreased more markedly than the total GSK-3β activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3β bound to AKAP220 more strongly than the total GSK-3β activity. These results suggest that PKA and PP1 regulate the activity of GSK-3β efficiently by forming a complex with AKAP220.

ABSTRACTAxam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of β-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of β-catenin. An Axam fragment which contains both domains was able to decrease the level of β-catenin. On substitution of Ser for Cys547in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate β-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopusembryos, AxamC547Sshowed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate β-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.

Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of β-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of β-catenin. An Axam fragment which contains both domains was able to decrease the level of β-catenin. On substitution of Ser for Cys547in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate β-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopusembryos, AxamC547Sshowed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate β-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.

Using yeast two-hybrid screening, we have identified a mouse Postsynaptic density 95/Discs large/Zona occludens-1 (PDZ) protein that interacts with activin type II receptors (ActRIIs). We named the protein activin receptor-interacting protein 2 (ARIP2). ARIP2 was found to have one PDZ domain in the NH2-terminal region and interact specifically with ActRIIs among the receptors for the transforming growth factor β family by the PDZ domain. Interestingly, overexpression of ARIP2 enhances endocytosis of ActRIIs and reduces activin-induced transcription in Chinese hamster ovary K1 cells. In addition, immunofluorescence co-localization studies indicated the direct involvement of ARIP2 in the intracellular translocation of ActRIIs by PDZ domain-mediated interaction. Moreover, we have identified that the COOH-terminal region of ARIP2 interacts with Ral-binding protein 1 (RalBP1). RalBP1 is a potential effector protein of small GTP-binding protein Ral and regulates endocytosis of epidermal growth factor and insulin receptors. The studies using deletion mutants of RalBP1 and constitutively GTP and GDP binding forms of Ral indicate that ARIP2 regulates endocytosis of ActRIIs through the Ral/RalBP1-dependent pathway, and the GDP-GTP exchange of Ral is critical for this regulation.

Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4',6-Diamidino-2-phenylindole staining and 5-bromo-2'-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10(-4) M GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10(-4) M uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.

Duplin binds to β-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the β-catenin-mediated transactivation and Xenopusaxis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin α was isolated as a binding protein of Duplin. Importin α bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (DuplinΔ500–584) retained Duplin in the cytoplasm. DuplinΔ500–584bound to β-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopusembryos. The Duplin mutant without a β-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed β-catenin-dependent axis duplication and expression ofsiamois, a Wnt-regulated gene, but also inhibitedsiamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin α, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream β-catenin target genes.

Although casein kinase Iε (CKIε) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIε activates the Wnt signaling pathway in co-operation with Dvl. CKIε and Axin associated with different sites of Dvl, and CKIε and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIε alone did not accumulate β-catenin, but their co-expression accumulated greatly. Dvl and CKIε activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIε activated Tcf, it did not synergize with CKIε. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIε. Furthermore, Dvl and CKIε co-operatively induced axis duplication of Xenopusembryos. These results indicate that Dvl and CKIε synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIε to Axin is necessary for their synergistic action.

Axin acts as a negative regulator in Wnt signaling through interaction with various molecules involved in this pathway, including β-catenin, adenomatous polyposis coli, and glycogen synthase kinase 3β. We show here that Axin also regulates the effects of Smad3 on the transforming growth factor β (TGF-β) signaling pathway. In the absence of activated TGF-β receptors. Axin physically interacted with Smad3 through its C-terminal region located between the β-catenin binding site and Dishevelled-homologous domain. An Axin homologue, Axil (also called conductin), also interacted with Smad3. In the absence of ligand stimulation, Axin was colocalized with Smad3 in the cytoplasm in vivo. Upon receptor activation, Smad3 was strongly phosphorylated by TGF-β type I receptor (TβR-I) in the presence of Axin, and dissociated from TβR-I and Axin. Moreover, the transcriptional activity of TGF-β was enhanced by Axin and repressed by an Axin mutant which is able to bind to Smad3. Axin may thus function as an adapter of Smad3, facilitating its activation by TGF-β receptors for efficient TGF-β signaling.

ABSTRACTAxin acts as a negative regulator in Wnt signaling through interaction with various molecules involved in this pathway, including β-catenin, adenomatous polyposis coli, and glycogen synthase kinase 3β. We show here that Axin also regulates the effects of Smad3 on the transforming growth factor β (TGF-β) signaling pathway. In the absence of activated TGF-β receptors. Axin physically interacted with Smad3 through its C-terminal region located between the β-catenin binding site and Dishevelled-homologous domain. An Axin homologue, Axil (also called conductin), also interacted with Smad3. In the absence of ligand stimulation, Axin was colocalized with Smad3 in the cytoplasm in vivo. Upon receptor activation, Smad3 was strongly phosphorylated by TGF-β type I receptor (TβR-I) in the presence of Axin, and dissociated from TβR-I and Axin. Moreover, the transcriptional activity of TGF-β was enhanced by Axin and repressed by an Axin mutant which is able to bind to Smad3. Axin may thus function as an adapter of Smad3, facilitating its activation by TGF-β receptors for efficient TGF-β signaling.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3β (GSK-3β), β-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3β-dependent phosphorylation in the complex and the stability of β-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61β and -γ, interact with Axin. PR61β or -γ formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61β on Axin was different from those of GSK-3β, β-catenin, APC, and Dvl. Although PR61β did not affect the stability of β-catenin, it inhibited Dvl- and β-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed β-catenin-induced axis formation and expression of siamois, a Wnt target gene, inXenopusembryos, suggesting that PR61β acts either at the level of β-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of β-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of β-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopusembryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not β-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

ABSTRACTIn attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of ß-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax inXenopusembryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not ß-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3β (GSK-3β), β-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3β efficiently phosphorylates β-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axinassociating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3β-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of β-catenin in SW480 cells and inhibited Wnt-dependent axis duplication inXenopusembryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.