The Bysl gene product, bystin, is essential for survival of mouse embryos

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin apparently disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.     

Cell-cell interaction-dependent regulation of N-acetylglucosaminyltransferase III and the bisected N-glycans in GE11 epithelial cells. Involvement of E-cadherin-mediated cell adhesion

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and alpha1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.     

Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling

alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an alpha1,6-linkage to form core fucosylation in mammals. We recently found that disruption of the Fut8 gene induces severe growth retardation and early postnatal death. To investigate the molecular mechanism involved, we have established embryonic fibroblasts of Fut8+/+ and Fut8-/-, derived from wild-type and Fut8-null mice, respectively. Interestingly, the epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in Fut8-/- cells, compared with Fut8+/+ cells, while there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between two cells. The inhibition of EGFR phosphorylation was completely restored by re-introduction of the Fut8 gene to Fut8-/- cells. Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8-/- cells. Finally, we found that the core fucosylation of N-glycans is required for the binding of the EGF to its receptor, whereas no effect was observed for the expression levels of EGFR on the cell surface. Collectively, these results strongly suggest that core fucosylation is essential for EGF receptor-mediated biological functions.     

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.     

N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function

The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.     

Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer

The goal of this study was to identify glycosyltransferases that are specifically expressed in pancreatic cancer. To investigate the gene expression of glycosyltransferases between pancreatic cancer and normal pancreatic tissues, we performed DNA-microarray (involving about 1000 oligosaccharide-related genes) using RNA mixtures of pancreatic cancer cells and normal pancreatic tissues. Eighty-six genes were up-regulated and thirty-two were down-regulated in pancreatic cancer, compared to normal pancreatic tissue. Among these changes, it is noteworthy that the expression of GnT-IVa was decreased and the expression of GnT-IVb was increased in pancreatic cancer, compared to normal pancreatic tissues. Although GnT-IVa and -IVb are involved in the same reaction as a glycosyltransferase, their chromosomal localization is different. When 5 cases of pancreatic cancer tissues were examined using the real-time RT-PCR method, the expression of GnT-IVb was dominant in tumor tissues and the expression of GnT-IVa was dominant in the surrounding normal tissues. The expression of GnT-IVa was increased in all 3 cell lines that had been treated with 5-aza-C and butyrate. These results suggest that the down-regulation of GnT-IVa in pancreatic cancer cells is due to an epigenetic abnormality in the gene.     

Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.     

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.