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Glycobiology Advance Access published online on March 23, 2005
Glycobiology, doi:10.1093/glycob/cwi055
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received December 15, 2004
Revised March 15, 2005
Accepted March 16, 2005

Article

Role for upregulated ganglioside biosynthesis and association of Src family kinases with microdomains in retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Takashige Sato 1, Asif Mohammad Zakaria 1, Satoshi Uemura 1, Atsushi Ishii 2, Yoshiko Ohno-Iwashita 3, Yasuyuki Igarashi 1, and Jin-ichi Inokuchi 4*

1 Department of Biomembrane and Biofunctional Chemistry, Hokkaido University, Kita 21-Nishi 10, Kita-ku, Sapporo 001-0021, Japan
2 Division of Project Research, Creative Research Initiative "Sousei" Hokkaido University, Kita 21-Nishi 10, Kita-ku, Sapporo 001-0021, Japan
3 Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
4 Department of Biomembrane and Biofunctional Chemistry, Hokkaido University; Core Research for Evaluational Science and Technology program (CREST), Japan Science and Technology Corporation (JST), Graduate School of Pharmaceutical Sciences, Frontier Research Center for Post-Genomic Science and Technology

* To whom correspondence should be addressed.
Jin-ichi Inokuchi, E-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp


  Abstract

Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, since they are similar to the inner cell mass of early mouse embryos and can differentiate into primitive endoderm (PrE) following retinoic acid (RA) treatment. During F9 cell differentiation, the carbohydrate chains of glycoproteins and their corresponding glycosyltransferases are known to undergo rapid changes. However, there have been no corresponding reports on the expression of gangliosides. We have developed a custom cDNA array that is highly sensitive for the genes responsible for sphingolipid biosynthesis and metabolism. Using this, we found that, of the 28 selected genes, 26 exhibited increased expression during F9 differentiation into PrE. Although neutral glycosphingolipids were expressed, at similar levels before and after differentiation, a greater than 20-fold increase in total ganglioside content was evident in PrE. Glucosylceramide synthase inhibitors, (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its analog) depleted gangliosides and resulted in delayed expression of Disabled-2 suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterolenriched membrane microdomains by methyl-{beta}-cyclodextrin (M{beta}CD) also delayed differentiation. Both M{beta}CD and D-PDMP blocked the accumulation of src family kinases (SFKs) to microdomains. However, D-PDMP did not block flotillin accumulation, yet M{beta}CD did. Additionally, confocal laser microscopy revealed the formation of distinct functional microdomains integrating SFKs with gangliosides and cholesterol during PrE differentiation. Thus, we demonstrate the outstanding up-regulation of ganglioside biosynthesis and its importance in the formation of distinct microdomains incorporating SFKs with gangliosides during RA-induced differentiation of F9 cells.

Keywords: Differentiation, Gangliosides, Microdomains, Src family kinsases, Retinoic acid, F9 embryonal carcinoma cell.
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