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.

FIGURE 1.

Neural stem cells prepared in the form of neurospheres. a, neurospheres were prepared from the striata of mouse embryos (embryonic day 14.5) and cultured in Neurobasal-A medium supplemented with B27, basic FGF and EGF. b, cells forming neurospheres were stained with Rat 401 anti-nestin antibody or AK97 anti-SSEA-1 antibody. Most neurosphere-forming cells were positive for nestin (neural stem cell marker protein; green) and SSEA-1 (neural stem cell marker carbohydrate; green). coIgG and coIgM indicate subclass control IgG and control IgM (BD Biosciences), respectively. c, cells forming neurospheres were cultured in undifferentiation condition (undiffer) or differentiation condition (differ; 0 ng/ml basic FGF and EGF and 1% fetal bovine serum for 5 days) and then stained with antibodies to nestin, β-III tubulin, glial fibrillary acidic protein (GFAP), and galactocerebroside (GalC). β-III Tubulin⁺ neurons (green), GFAP⁺ astrocytes (green), and GalC⁺ oligodendrocytes (green) are found in the cells cultured in differentiation condition. Nuclei were stained with Hoechst 33258 (blue). Scale bars, 100 μm in a; 50 μm in b and c.

FIGURE 2.

Detection of HNK-1-carrier proteins in NSCs and differentiated cells. a, lysates prepared from primary (lane 1), secondary (lane 3), and tertiary (lane 5) neurospheres and cells differentiated from primary (lane 2), secondary (lane 4) and tertiary (lane 6) neurospheres were analyzed by Western blotting with NGR50 anti-HNK-1 antibody or anti-β-actin antibody (Blot). β-Actin was detected as a loading control. Arrow indicates a major band positive for HNK-1 with a molecular mass of 280 kDa. b, lysates of primary neurospheres and their differentiated cells (containing 20 μg of proteins) were incubated with PNGase F (0 or 50 units/20 μl) at 37 °C for 3 h to remove N-glycans and then analyzed by Western blotting with anti-HNK-1 and anti-β-actin antibodies. undiffer and differ indicate lysates from primary neurospheres and differentiated cells, respectively.

FIGURE 3.

HNK-1 epitopes were carried by TNC in NSCs. a, major HNK-1-carrier protein in secondary neurospheres (arrow) was immunoprecipitated with control mouse IgG or anti-HNK-1 antibody (IP) and then detected by silver staining. By LC-MS/MS analysis, the major HNK-1-carrier protein was identified as TNC. b, amino acid sequence of TNC is shown. The sequences of observed peptides are shown in green. The putative signal sequence is shown in blue. Twenty potential N-glycosylation sites are shown in red. The amino acid sequence of the alternatively spliced domain C is underlined. c, proteins from the secondary neurospheres were immunoprecipitated with control mouse IgG or H300 anti-TNC antibody (IP) and then subjected to Western blot analysis with anti-HNK-1 antibody. d, NSCs prepared from primary neurospheres were treated with PBS containing 3% fetal bovine serum and 0.1% Triton X-100 and then stained with anti-TNC and anti-HNK-1 antibodies as primary antibodies, and DyLight488-conjugated anti-rat IgG antibody (green) and Cy3-conjugated anti-mouse IgG antibody (red) as secondary antibodies. Nuclei were stained with 2 μg/ml Hoechst 33258 (blue).

FIGURE 4.

Expression of HNK-1-containing glycans on a larger TNC isoform. a, scheme shows the structural organization of TNC and the localization of the primer pairs used in this study to amplify the splicing domain of TNC. b, proteins of NSCs were analyzed by Western blotting with anti-HNK-1, anti-TNC, or anti-β-actin antibodies. Western blot analysis with anti-TNC antibody shows the existence of some alternatively spliced variants in NSCs (indicated by arrowheads). The largest TNC variant possesses the HNK-1 epitopes resulting from Western blotting with anti-HNK-1 antibody (indicated by an open arrowhead). β-Actin was detected as a loading control. An asterisk denotes nonspecific bands. c and d, isoform expression of TNC was analyzed by RT-PCR using a specific primer pair (shown in a) under a condition including primer sequences described previously (30). e, mRNA expression of TNC, Sox2, β-actin, HNK-1ST, GlcAT-S, GlcAT-P, Pax6, and Sam68, in undifferentiated (undiffer) NSCs and differentiated (differ) cells was analyzed by RT-PCR. Sox2 was detected as maker gene of undifferentiated NSCs. β-Actin was used as a control.

FIGURE 5.

Effect of HNK-1ST or TNC knockdown by siRNA. a, Western blot of NSCs treated with HNK-1ST, TNC, or negative control siRNAs. Samples were collected after 72 h of siRNA treatment and then subjected to Western blot analysis with anti-HNK-1, anti-TNC, and anti-β-actin antibodies. β-Actin was detected as a loading control. Expression levels of TNC (b) and HNK-1 (c) were measured by densitometric analysis using a scanning densitometer (n = 3). d, effects of knockdown of TNC or HNK-1ST by siRNA on proliferation of NSCs. NSCs transfected with HNK-1ST, TNC, or negative control siRNAs were cultured as neurospheres for 72 h (n = 4). The proliferation rates of neurosphere-forming cells were measured by WST-8 assay at 24, 48, 72 h after siRNA transfection. e, number of neurospheres formed by siRNA-transfected NSCs counted at 72 h after lipofection (n = 4). f, proliferation rates of neurosphere-forming cells in the presence of control IgG, anti-HNK-1 or anti-TNC antibodies (20 μg/ml) estimated by WST-8 assay at 72 h in vitro (n = 3). The anti-HNK-1 and anti-TNC antibodies inhibit the proliferation of NSCs.

FIGURE 6.

Cell death and cell survival in HNK-1 knockdown NSCs. a, cell death of NSCs transfected with HNK-1ST or TNC siRNA was evaluated by TUNEL assay. Tunicamycin was used as a control inducer of cell death (1 μg/ml for 10 h). There is no difference in the numbers of TUNEL⁺ cells between control siRNA, HNK-1ST siRNA, and TNC siRNA-transfected cells. Scale bar, 25 μm. b, activation of cell survival signaling and cell death signaling pathways (phosphoinositide 3-kinase-Akt pathway and caspase pathway, respectively) in NSCs transfected with HNK-1ST siRNA was evaluated by Western blotting with antibodies to Akt, phospho-Akt, caspase 3, and β-actin. The cell survival signaling and cell death signaling pathways in NSCs were not inhibited or activated by transfection with HNK-1ST siRNA.

FIGURE 7.

Down-regulation of EGF receptor and inhibition of the Ras-MAPK pathway in NSCs transfected with HNK-1ST or TNC knockdown by siRNA. a, Western blot of NSCs treated with HNK-1ST, TNC, or negative control siRNAs using anti-HNK-1, anti-EGF receptor (EGFR), anti-phospho-ERK1/2, anti-ERK, and anti-β-actin antibodies. The expression levels of EGF receptor (b) and phospho-ERK2 (c) were measured by densitometric analysis using a scanning densitometer (n = 3). d, importance of the Ras-MAPK pathway in EGF-induced proliferation of NSCs. NSCs were cultured in the presence or absence of EGF (10 ng/ml) and U0126, an inhibitor of MAPK kinase, for 72 h (n = 3). The proliferation rates of neurosphere-forming cells were measured by WST-8.