Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

Autoimmune Gastro-Pancreatitis with Anti-Protein Disulfide Isomerase-Associated 2 Autoantibody in Aire-Deficient BALB/cAnN Mice

Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.     

Retinoic acid induces discrete Wnt-signaling-dependent differentiation in F9 cells

Retinoic acid (RA) induces F9 cells, the mouse teratocarcinoma cells, to differentiate into primitive endoderm and further into visceral and parietal endoderm depending on the culture conditions. To elucidate the instructive mechanisms involved in the differentiation steps we investigated the effects of Wnt-signaling members, Wnt3a and β-catenin, on the differentiation of F9 cells and β-catenin-deficient F9 cells (βT cells). RA up-regulated the expression of differentiation markers for primitive, visceral and parietal endoderm in F9 cells but not for visceral endoderm in βT cells. Wnt3a or leukemia inhibitory factor (LIF) inhibited the RA-induced differentiation in F9 cells. LIF but not Wnt3a could inhibit differentiation in βT cells. RA evoked ZO-1α+ signals at cell-to-cell contacts in F9 cells in a Wnt3a sensitive manner. The results suggest that Wnt3a inhibits differentiation into endoderm through a pathway involving β-catenin, and β-catenin might be necessary in the process leading from primitive to visceral endoderm in F9 cells.     

Cytoplasmic Regulation of the Movement of E-Cadherin on the Free Cell Surface as Studied by Optical Tweezers and Single Particle Tracking: Corralling and Tethering by the Membrane Skeleton

The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH₂-terminal half. These cadherins were labeled with 40-nm colloidal gold or 210-nm latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (D_micro) of 1.2 × 10⁻¹⁰ and 2.1 × 10⁻¹⁰ cm²/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm² for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average D_micro for Fusion measured by SPT was small (0.2 × 10⁻¹⁰ cm²/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.     

The Loss of the Expression of α Catenin, the 102 kD Cadherin Associated Protein, in Central Nervous Tissues during Development

A monoclonal antibody specific for α catenin, the 102kD cadherin-associated protein, has been characterized and used to describe the expression and distribution pattern of α catenin in adult mice and mouse embryos. This monoclonal antibody recognized an epitope in the middle part of the α catenin molecule of various vertebrate species, and bound to neither vinculin nor αN catenin, which are cytoskeletal proteins with sequence similarity to α catenin. At the early mouse embryo stage (neurulae stage) α catenin was expressed and concentrated at cell-to-cell contact sites together with various types of cadherins in all tissues. In embryos at 12.5 days of gestation, the α catenin expression was gradually diminished selectively in central nervous tissues such as brain and spinal cord, and in most of the adult central nervous tissues the α catenin expression was hardly detected. In adult non-nervous tissues most of the cells examined expressed α catenin. Especially in well-polarized tissues such as epithelial cells, α catenin appeared to be highly concentrated at cell-to-cell adherens junctions where cadherins act as adhesion molecules.
This loss of α catenin expression in central nervous tissues was observed not only in mice but also in other vertebrate species such as fish and newt, suggesting that this phenomenon has important implications from the view point of nervous tissue development.     

Mechanism of antibody-mediated protection against herpes simplex virus infection in athymic nude mice: requirement of Fc portion of antibody

Experiments were performed to investigate the resistance of the host due to antibody-mediated mechanisms to herpes simplex virus (HSV) infection. Transfer of hyperimmune anti-HSV mouse serum inhibited the development of skin lesions and prolonged the survival of lethally HSV-infected nude mice. Relatively high concentrations of antibody were required to achieve this protection. Antisera prepared in heterologous animals were also effective, while administration of anti-cowpox virus serum or interferon provided no protection. This type of protection is therefore due to specific antibody and cannot be attributed to interferon. In order to delineate the requirement for antibody in antibody-mediated protection, human gamma globulin preparations were transferred to lethally HSV-infected nude mice. Transfer of intact human gamma globulin (GG) was effective in controlling infection. S-sulfonation of GG did not diminish the protective ability. However, purified F(ab')2 did not have any protective action even when it was administered frequently to maintain serum neutralizing antibody titer. GG was effective in C5-deficient mice lethally infected with HSV. These results indicate that in vivo antibody-mediated protection to HSV infection requires the Fc region of the intact IgG molecule and suggest that antibody-dependent cell-mediated cytotoxicity may be operative in vivo.     

Defining the roles of beta-catenin and plakoglobin in cell-cell adhesion: isolation of beta-catenin/plakoglobin-deficient F9 cells

F9 teratocarcinoma cells in which beta-catenin and/or plakoglobin genes are knocked-out were generated and investigated in an effort to define the role of beta-catenin and plakoglobin in cell adhesion. Loss of beta-catenin expression only did not affect cadherin-mediated cell adhesion activity. Loss of both beta-catenin and plakoglobin expression, however, severely affected the strong cell adhesion activity of cadherin. In beta-catenin-deficient cells, the amount of plakoglobin associated with E-cadherin dramatically increased. In beta-catenin/plakoglobin-deficient cells, the level of E-cadherin and alpha-catenin markedly decreased. In these cells, E-cadherin formed large aggregates in cytoplasm and membrane localization of alpha-catenin was barely detected. These data confirmed that beta-catenin or plakoglobin is required for alpha-catenin to form complex with E-cadherin. It was also demonstrated that plakoglobin can compensate for the absence of beta-catenin. Moreover it was suggested that beta-catenin or plakoglobin is required not only for the cell adhesion activity but also for the stable expression and cell surface localization of E-cadherin.     

Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan

We examined the genetic background of nonalcoholic fatty liver disease (NAFLD) in the Japanese population, by performing a genome-wide association study (GWAS). For GWAS, 392 Japanese NAFLD subjects and 934 control individuals were analyzed. For replication studies, 172 NAFLD and 1,012 control subjects were monitored. After quality control, 261,540 single-nucleotide polymorphisms (SNPs) in autosomal chromosomes were analyzed using a trend test. Association analysis was also performed using multiple logistic regression analysis using genotypes, age, gender and body mass index (BMI) as independent variables. Multiple linear regression analyses were performed to evaluate allelic effect of significant SNPs on biochemical traits and histological parameters adjusted by age, gender, and BMI. Rs738409 in the PNPLA3 gene was most strongly associated with NAFLD after adjustment (P = 6.8 × 10^?14, OR = 2.05). Rs2896019, and rs381062 in the PNPLA3 gene, rs738491, rs3761472, and rs2143571 in the SAMM50 gene, rs6006473, rs5764455, and rs6006611 in the PARVB gene had also significant P values (<2.0 × 10^?10) and high odds ratios (1.84–2.02). These SNPs were found to be in the same linkage disequilibrium block and were associated with decreased serum triglycerides and increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in NAFLD patients. These SNPs were associated with steatosis grade and NAFLD activity score (NAS). Rs738409, rs2896019, rs738491, rs6006473, rs5764455, and rs6006611 were associated with fibrosis. Polymorphisms in the SAMM50 and PARVB genes in addition to those in the PNPLA3 gene were observed to be associated with the development and progression of NAFLD.     

Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4⁺ T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein–Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3′UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3′UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3′UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3′UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.