👤 Noriyuki Egusa

To explore the cellular behavior of stem cells derived from human exfoliated deciduous teeth (SHED) in response to inorganic pyrophosphate (PP SHED cells were isolated from the dental pulp tissues of human primary exfoliated teeth. Cell proliferation was examined using the MTT assay, colony-forming unit assay, and cell cycle analysis. Cell migration was evaluated using the scratch assay. Osteogenic differentiation was assessed by the expression of osteogenic marker genes and in vitro mineral deposition. Oil Red O staining was employed to determine intracellular lipid accumulation under adipogenic differentiation. For osteoclast differentiation, TRAP staining was used. The global gene expression profile was examined by RNA sequencing analysis. PP PP Show less

The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs. Show less

The formation of heparan sulfate (HS) chains is catalyzed by glycosyltransferases encoded by EXT (hereditary multiple exostosin gene) family members. Genetic screening for mutations affecting morphogen signaling pathways in Drosophila has identified three genes, tout-velu (ttv), sister of tout-velu (sotv), and brother of toutvelu (botv), which encode homologues of human EXT1, EXT2, and EXTL3, respectively. So far, in vitro glycosyltransferase activities have been demonstrated only for BOTV/DEXTL3, which harbors both N-acetylglucosaminyltransferase-I (GlcNAcT-I) and N-acetylglucosaminyltransferase-II (GlcNAcT-II) activities responsible for the chain initiation and elongation of HS, and no glucuronyltransferase-II (GlcAT-II) activity. Here we demonstrated that TTV/DEXT1 and SOTV/DEXT2 had GlcNAcT-II and GlcAT-II activities required for the biosynthesis of repeating disaccharide units of the HS backbone, and the coexpression of TTV with SOTV markedly augmented both glycosyltransferase activities when compared with the expression of TTV or SOTV alone. Moreover, the polymerization of HS was demonstrated on a linkage region analogue as an acceptor substrate by BOTV and an enzyme complex composed of TTV and SOTV (TTV-SOTV). In contrast to human, TTV-SOTV exhibited no GlcNAcT-I activity, indicating that BOTV/DEXT3, which is an EXT-Like gene and possesses GlcNAcT-I activity required for the initiation of HS, is indispensable for the biosynthesis of HS chains in Drosophila. Thus, all three EXT members in Drosophila, TTV, SOTV, and BOTV, are required for the biosynthesis of full-length HS in Drosophila. Show less

The proteins encoded by the EXT1, EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in the Caenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both alpha1,4-N-acetylglucosaminyltransferase and beta1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an alpha1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that reported for the mammalian system. Show less