Chondroitin sulfate, a long linear polysaccharide that is covalently bound to specific core proteins to form chondroitin sulfate proteoglycans, is distributed on cell surfaces and in the extracellular matrix. Chondroitin sulfate proteoglycans play important roles in physiological processes such as cell–cell interactions, cell proliferation, differentiation, and morphogenesis. Many physiological roles are attributed to chondroitin sulfate side chains. However, multiple glycosyltransferases involved in chondroitin sulfate biosynthesis in mammals are highly redundant, and functional analysis of chondroitin sulfate chains is difficult in mammals because of this redundancy1). Each of four single-gene knockouts, namely chondroitin synthase-1 (Chsy1), chondroitin polymerizing factor (Chpf), chondroitin GalNAc transferase-1 (Csgalnact1), and Csgalnact2, lead to viable, fertile mice, although each knockout genotype results in reduced chondroitin sulfate production and/or a sulfation imbalance in chondroitin sulfate chains (Figure 1). Thus, to clarify the functions of chondroitin sulfate chains in early mammalian embryogenesis, we focused on glucuronyltransferae-I (GlcAT-I). Because GlcAT-I is a single enzyme that transfers a glucuronic acid residue to trisaccharide-serine, Galβ1-3Galβ1-4Xylβ1-O-Ser, thus finalizing the formation of the common linkage region. GlcAT-I knockout would result in mutant mice completely lacking chondroitin sulfate and heparan sulfate chains (Figure 1)1). GlcAT-I knockout (B3gat3) results in embryonic lethality before the 8-cell stage because of cytokinesis failure. A complementary analysis in which the bacterial chondroitin sulfate-degrading enzyme chondroitinase ABC was used to remove chondroitin sulfate selectively from wild-type 2-cell embryos indicated that the failure of cytokinesis is causally linked to the loss of chondroitin sulfate2). These findings showed that chondroitin sulfate chains are indispensable for embryonic cell division.Chondroitin sulfate is required for proper cell division in early mammalian embryos based on our analyses of GlcAT-I-deficient and chondroitinase ABC-treated embryos; nevertheless, approximately 7% of the homozygous GlcAT-I-deficient embryos resulting from crosses between GlcAT-I+/- heterozygotes survive to the implantation stage2). Thus, these implantation-stage, GlcAT-I-deficient embryos could be used to produce embryonic stem cells that lack glycosaminoglycans. GlcAT-I-deficient mouse embryonic stem cells have been established and they completely lack chondroitin sulfate and heparan sulfate chains3). GlcAT-I-deficient mouse embryonic stem cells fail to exit the self-renewal program and they cannot initiate differentiation even in the absence of leukemia inhibitory factor3). In addition, GlcAT-I-deficient mouse embryonic stem cells cannot form embryoid bodies or differentiate into multiple lineages. Moreover, treatment of wild-type mouse embryonic stem cells with chondroitinase ABC affects embryoid body formation; in contrast, heparitinase-treated mouse embryonic stem cells develop normally into embryoid bodies3). Taken together, chondroitin sulfate is required to maintain the pluripotency of embryonic stem cells and chondroitin sulfate differs from heparan sulfate because it promotes the initial commitment of embryonic stem cells to differentiation. Notably, polysaccharides rich in A disaccharide units (CS-A) and those rich in E disaccharide units (CS-E) but not those rich in C disaccharide units (CS-C) bind to E-cadherin and enhance embryonic stem cell differentiation3). Furthermore, active RhoA and ERK1/2 phosphorylation levels are lower in chondroitinase ABC-treated embryonic stem cells than in controls3). In GlcAT-I-deficient mouse embryonic stem cells, active RhoA and ERK1/2 phosphorylation levels are reduced, whereas active RhoA and ERK1/2 phosphorylation levels are rescued when GlcAT-I-deficient mouse embryonic stem cells are cultured in the presence of CS-A or CS-E but not CS-C3). Therefore, CS-A and CS-E are selective ligands for a potential chondroitin sulfate receptor, E-cadherin, and these ligands lead to embryonic stem cell differentiation by activating the Rho signaling pathway. These results indicated that chondroitin sulfate controls the functional integrity of embryonic stem cells via binding to E-cadherin.The last step of cell division is cytokinesis in which two daughter cells physically separate. Cytokinesis failure leads to multinucleated cells. During cytokinesis, multiple proteins localize at the midbody, the central region of the intracellular bridge interconnecting daughter cells, and assemble abscission 58Regulation of cytokinesis and exosome formation by chondroitin sulfateHiroshi KitagawaCollaborators: Yuko Naito-Matsui, Ban Sato, and Satomi NadanakaKobe Pharmaceutical University
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