Exp Mol Med.  2009 Dec;41(12):935-945. 10.3858/emm.2009.41.12.099.

The roles of glycosphingolipids in the proliferation and neural differentiation of mouse embryonic stem cells

• Jung JU
• Ko K
• Lee DH
• Ko K
• Chang KT
• Choo YK

• KMID: 1099869
• DOI: http://doi.org/10.3858/emm.2009.41.12.099

Abstract

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.

Keyword

cell differentiation; ceramide glucosyltransferase; extracellular signal-regulated MAP kinases; glycosphingolipids; embryonic stem cells; neurons

MeSH Terms

Animals
*Cell Proliferation
Cells, Cultured
Down-Regulation
Embryonic Stem Cells/*cytology/metabolism
Glucosyltransferases/genetics/metabolism
Glycosphingolipids/genetics/*metabolism
Mice
Mitogen-Activated Protein Kinases/metabolism
*Neurogenesis
Neurons/*cytology/metabolism
RNA, Messenger/genetics