HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Abstract

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development. Neural stem cells (NSCs)4 are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells, such as neurons, astrocytes, and oligodendrocytes (1-3). Environmental factors of NSCs, such as various growth factors, the extracellular matrix (ECM), and cell adhesion molecules, are known to play important roles in the maintenance of the stem cell population throughout specific cell lineage pathways (4 -7).Glycoconjugates, including glycoproteins, proteoglycans, and glycolipids, are expressed mainly on the cell surface as ECM, and they are known to regulate cell-to-cell communications. Certain glycoconjugates also serve as excellent biomarkers at various stages of the cellular differentiation of NSCs and play important functional roles in determining cell fate (8 -12). For example, stage-specific embryonic antigen-1 (SSEA-1), which is well known as a specific maker of undifferentiated cells including mouse embryonic stem cells, is expressed on NSCs and associated with cell migration (8, 10, 13). Recently, we have also demonstrated that cells positive for GD3 ganglioside (NeuAc␣2-8NeuAc␣2-3Gal␤1-4Glc␤1-1ЈCer) isolated from mouse brains of various ages possess characteristics of neural stem cells (11).The human natural killer-1 (HNK-1) carbohydrate epitope (CD57) was originally reported as a specific antigenic determinant for human natural killer cells (14) but is now widely known ...

Lewis X-carrying N-Glycans Regulate the Proliferation of Mouse Embryonic Neural Stem Cells via the Notch Signaling Pathway

“…NSC Culture-NSCs were prepared in the form of neurospheres according to methods described previously (22,24). In brief, single cell suspensions prepared from the striata of ICR mouse embryos (embryonic day 14.5) were cultured in Neurobasal-A medium (Invitrogen) containing B27 serum-free supplement (Invitrogen), L-glutamine (Invitrogen), 20 ng/ml basic FGF (PeproTech, Rocky Hill, NJ), and 20 ng/ml of epidermal growth factor (EGF; PeproTech).…”

“…It has been shown that the cells forming neurospheres have self-renewal ability, express neural stem cell markers, and are capable of differentiating into neurons, astrocytes, and oligodendrocytes (22,24).…”

“…End-labeling (TUNEL) Assay-Apoptotic NSCs were detected with the TUNEL assay as described previously (22). The siRNA-transfected NSCs were plated onto chamber slides and cultured for 3 days.…”

“…Quantitative Reverse Transcription (RT)-PCR AnalysisQuantitative RT-PCR was performed as described previously (22,34). In brief, total RNAs were isolated from NSCs using TRIzol reagent (Invitrogen).…”

“…Recently, we showed that mouse NSCs specifically express the extracellular matrix protein tenascin-C (TNC), which is modified with human natural killer (HNK)-1 carbohydrate, and this glycoconjugate regulates cellular proliferation via modulation of the Ras-MAPK pathway (22). This study suggested that glycoprotein glycans might be active regulators that maintain the stemness of NSCs through specific signaling pathways, which prompted us to undertake comprehensive, quantitative glycosylation profiling of NSCs before and after differentiation.…”