Interaction of ganglioside GD3 with an EGF receptor sustains the self-renewal ability of mouse neural stem cells in vitro

Abstract

Mounting evidence supports the notion that gangliosides serve regulatory roles in neurogenesis; little is known, however, about how these glycosphingolipids function in neural stem cell (NSC) fate determination. We previously demonstrated that ganglioside GD3 is a major species in embryonic mouse brain: more than 80% of the NSCs obtained by the neurosphere method express GD3. To investigate the functional role of GD3 in neurogenesis, we compared the properties of NSCs from GD3-synthase knockout (GD3S-KO) mice with those from their wild-type littermates. NSCs from GD3S-KO mice showed decreased self-renewal ability compared with those from the wild-type animals, and that decreased ability was accompanied by reduced expression of EGF receptor (EGFR) and an increased degradation rate of EGFR and EGF-induced ERK signaling. We also showed that EGFR switched from the low-density lipid raft fractions in wild-type NSCs to the high-density layers in the GD3S-KO NSCs. Immunochemical staining revealed colocalization of EGFR and GD3, and EGFR could be immunoprecipitated from the NSC lysate with an anti-GD3 antibody from the wild-type, but not from the GD3S-KO, mice. Tracking the localization of endocytosed EGFR with endocytosis pathway markers indicated that more EGFR in GD3S-KO NSCs translocated through the endosomal-lysosomal degradative pathway, rather than through the recycling pathway. Those findings support the idea that GD3 interacts with EGFR in the NSCs and that the interaction is responsible for sustaining the expression of EGFR and its downstream signaling to maintain the self-renewal capability of NSCs.

Fig. 1.

Accelerated loss of self-renewal ability in GD3-KO NSCs is accompanied by a decreased EGFR expression level. (A) Immunofluorescence of GD3 and nestin stain in neurospheres from GD3S^+/+ and GD3S^−/− mice. (B) Quantification of sphere number formed from every 10,000 cells demonstrates a neurosphere-forming defect in GD3S^−/− NSCs. (C) Lysates (50 µg protein) of neurospheres from embryonic day (E) 14 embryos and P10 postnatal mice at passage 2 were immunoblotted with the indicated antibodies. (D) Lysates (50 µg protein) of monolayer NSCs growing in different growth factor conditions (20 ng/mL EGF and/or 20 ng/mL bFGF) for 48 h were immunoblotted with the indicated antibodies. (E) Lysates (50 µg protein) of neurospheres from E14 embryos with different passages were immunoblotted with EGFR. Data represent three independent experiments with three replicates of neurosphere pool for neurosphere number quantification. All values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. (Scale bars, 50 µm.)

Fig. 2.

Accelerated EGFR degradation in GD3S^−/− NSCs. NSCs were grown as monolayers in the presence of 5 ng/mL EGF and 20 ng/mL bFGF for 16 h. EGF degradation was induced by addition of 50 ng/mL EGF and 25 µg/mL cycloheximide at 37 °C for the indicated time. (A) Lysates (40 µg protein) were immunoblotted with antibodies against EGFR, p-EGFR, and β1-integrin. Densitometric analysis indicated that EGFR degradation was faster in GD3S^−/− NSCs than in GD3S^+/+ cells, whereas β1-integrin showed a slight increase with EGFR degradation. (B) Lysates (40 µg protein) were immunoblotted with antibodies against p-Akt, t-Akt, p-ERK1/2, or t-ERK1/2. Densitometric analysis revealed that EGF-induced p-ERK1/2 signaling is reduced in GD3S^−/− NSCs and that p-Akt signaling is not influenced in the same way. (C) Flow cytometry was used to analyze cell membrane surface expression of EGFR with 50 ng/mL EGF treatment at 0.5 h. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 3.

Rescue effect of GD3 expression on EGF level by exogenous transfection of ST-II-GFP. (A) NSCs transfected with GFP-vector or ST-II-GFP were grown as a monolayer culture and immunostained with an anti-GD3 antibody, mAb R24, at 48 h after transfection. In GD3S^−/− NSCs, only cells with efficiently transfected ST-II-GFP had GD3 expression. (B) EGFR expression level was detected in monolayer cultures of NSCs at 48 h after GFP-vector or ST-II-GFP transfection. 75 kD GFP represents the ST-II-GFP. (C) Densitometric analysis revealed an increase of EGFR in GD3S^−/− NSCs with ST-II-GFP transfection compared with cells with GFP-vector transfection. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. (Scale bar, 10 µm.)

Fig. 4.

GD3 interacts with EGFR and plays a role in EGFR localization in the lipid raft fractions. (A) Colocalization of EGFR with GD3 in GD3⁺ NSCs under different EGF concentrations, as indicated. Box-enclosed areas are magnified on right. (B) Immunoprecipitation of neurosphere lysates with mAb R24 and blotted with EGFR or β1-integrin. (C) Distribution of indicated proteins from E14 passage 2 GD3S^+/+ and GD3S^−/− neurosphere lysates in the lipid raft fractions prepared by a detergent-free method. (D) Densitometric analysis of EGFR and β1-integrin distribution in each fraction showed fraction shifts of EGFR, but not of β1-integrin in GD3S^−/− NSCs, compared with the GD3S^+/+ NSCs. (Scale bar, 5 µm.)

Fig. 5.

GD3 regulates EGFR endocytosis. (A) Colocalization of EGFR with EEA1, LAMP 1, and Rab11 in monolayer NSCs under normal culture conditions (20 ng/mL EGF and 20 ng/mL bFGF). The colocalization coefficiency was measured by Zeiss LSM-enhanced colocalization software. At least 20 cells were measured for each condition in each experiment. (B) NSCs were grown as monolayers in the presence of 5 ng/mL EGF and 20 ng/mL bFGF for 16 h, and EGFR internalization was induced by addition of 50 ng/mL EGF. Colocalization coefficiency was measured by fixing cell coverslips and coimmunostaining EGFR with EEA1, LAMP 1, or Rab11 at the indicated time. Mander’s overlap coefficient, which indicates the actual overlap of two signals, was used to estimate the extent of colocalization of two channels. Values are expressed as mean ± SEM; n = 3. *P < 0.05; **P < 0.01. (Scale bar, 5 µm.)