Glycobiology Advance Access originally published online on March 23, 2005
Glycobiology 2005 15(7):687-699; doi:10.1093/glycob/cwi055
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Role for up-regulated ganglioside biosynthesis and association of Src family kinases with microdomains in retinoic acid-induced differentiation of F9 embryonal carcinoma cells
3 Department of Biomembrane and Biofunctional Chemistry, Hokkaido University, Kita 21-Nishi 10, Kita-ku, Sapporo 001-0021, Japan; 4 Division of Project Research, Creative Research Initiative "Sousei" Hokkaido University, Kita 21-Nishi 10, Kita-ku, Sapporo 001-0021, Hokkaido, Japan; and 5 Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan; 6 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, Kita 21-Nishi 10, Kita-ku, Sapporo 001-0021, Japan
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed; e-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp
Received on December 15, 2005; revised on March 15, 2005; accepted on March 16, 2005
Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, because 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 (SL) 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 (GSLs) 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 this resulted in delayed expression of Disabled-2 (Dab-2), suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterol-enriched membrane microdomains by methyl-ß-cyclodextrin (MßCD) also delayed differentiation. Both MßCD and D-PDMP blocked the accumulation of Src family kinases (SFKs) to microdomains. However, D-PDMP did not block flotillin accumulation, yet MßCD 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.
Key words: differentiation / F9 embryonal carcinoma cell / gangliosides / microdomains / Src family kinsases