Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

Development of a potassium ion-selective fluorescent sensor based on 3-styrylated BODIPY

We have developed a red-emitting fluorescent K(+) probe, B3TAC, which also shows a wavelength shift upon binding to K(+). The probe was synthesized by conjugating a cryptand-based chelator, 2-triazacryptand [2,2,3]-1-(2-methoxyethoxy)benzene (TAC), to position 3 of the BODIPY fluorophore through a styryl linker. In water-acetonitrile mixed solvent, it responded to K(+) in the physiological concentration range with high selectivity over Na(+) and other metal ions. B3TAC is potentially useful for measuring cellular K(+) ion concentration, as well as for simple, naked-eye detection of K(+) in solution.     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Inorganic nitrogen source utilization byFagus crenata on different soil types

The nitrogen source utilization by Fagus crenata distributed on soils with different forms of inorganic nitrogen in a cool-temperate deciduous forest in central Japan was determined by measuring foliar ¹⁵N. Two soil habitat types along a slope were delineated based on nitrogen transformation patterns, i.e., soils with high net nitrification rates and with no or low net nitrification, respectively. Despite differences in soil types, the study species, F. crenata, was distributed along the entire slope. The foliar ¹⁵N value of F. crenata from the lower slope area was significantly lower than that from the upper slope. Given the finding of a previous study that the ¹⁵N of NO₃^– was lower than that of NH₄⁺, our results indicate that reliance on NO₃^– as a nitrogen source was greater in the lower slope area than in the upper slope area. Differences in the values of foliar ¹⁵N were about 1, which is far less than the 10 ¹⁵N value of soil inorganic N reported in the previous study. This discrepancy might suggest that the study species utilized NO₃^– even in the upper site where net nitrification had not been detected. Measurements of nitrate reductase activity, an index of NO₃^– uptake, also supported this interpretation. Nitrate reduction occurred in leaves and roots at both the lower and the upper sites. Thus, the study species may be able to use NO₃^– even in soils with no net nitrification, a factor that could allow the distribution of F. crenata along the entire length of the slope.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.     

Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

We previously reported that substantial amounts of IL-10, an immunomodulatory cytokine, are produced by cell suspensions of fresh human metastatic melanoma tissues. Production diminished with continuous culturing of cells, which suggests a pivotal interactive role between melanoma cells and the tumor microenvironment. In this study, we found that the culture media obtained from LPS-stimulated peripheral blood mononuclear cells induced IL-10 production by metastatic melanoma cells. Of the multiple cytokines present in the conditioned culture media, IL-6 was identified as the inducer of IL-10 production. A neutralizing antibody against IL-6 completely blocked the conditioned medium-induced IL-10 production. Metastatic melanoma cells that constitutively produce low amount of IL-10 increased IL-10 production in response to recombinant human IL-6 in a dose-dependent fashion. The response to exogenously added IL-6 was less significant in melanoma cells that produced high amounts of IL-6, probably due to pre-existing autocrine stimulation of IL-10 by endogenous IL-6. On the other hand, metastatic melanoma cells that do not constitutively produce IL-10 protein did not respond to exogenous IL-6. In IL-6-responsive melanoma cells, IL-6 increased STAT3 phosphorylation and inhibition of STAT3 signaling using siRNA or inhibitors for JAKs diminished IL-6-induced IL-10 production. In addition, inhibition of MEK and PI3K, but not mTOR, interfered with IL-10 production. Taken together, the data suggest that blocking of these signals leading to IL-10 production is a potential strategy to enhance an anti-melanoma immune response in metastatic melanoma.