Role of Prion protein-EGFR multimolecular complex during neuronal differentiation of human dental pulp-derived stem cells

Abstract

Cellular prion protein (PrP^C) is expressed in a wide variety of stem cells in which regulates their self-renewal as well as differentiation potential. In this study we investigated the presence of PrP^C in human dental pulp-derived stem cells (hDPSCs) and its role in neuronal differentiation process. We show that hDPSCs expresses early PrP^C at low concentration and its expression increases after two weeks of treatment with EGF/bFGF. Then, we analyzed the association of PrP^C with gangliosides and EGF receptor (EGF-R) during neuronal differentiation process. PrP^C associates constitutively with GM2 in control hDPSCs and with GD3 only after neuronal differentiation. Otherwise, EGF-R associates weakly in control hDPSCs and more markedly after neuronal differentiation. To analyze the functional role of PrP^C in the signal pathway mediated by EGF/EGF-R, a siRNA PrP was applied to ablate PrP^C and its function. The treatment with siRNA PrP significantly prevented Akt and ERK1/2 phosphorylation induced by EGF. Moreover, siRNA PrP treatment significantly prevented neuronal-specific antigens expression induced by EGF/bFGF, indicating that cellular prion protein is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that PrP^C interact with EGF-R within lipid rafts, playing a role in the multimolecular signaling complexes involved in hDPSCs neuronal differentiation.

Keywords: EGF Receptor; dental pulp-derived stem cells; mesenchymal stem cells; prion protein; prions; rafts.

Figure 1.

Differentiation of hDPSCs. (A) Morphology of hDPSCs from dental pulp untreated and treated with EGF/bFGF for 14 days. Scale bars, 50 μm. (B) Immunofluorescence analysis of neuronal marker β3-tubulin mAb expression. Scale bars, 50 μm.

Figure 2.

PrP^C expression in hDSPCs. (A) Flow cytometry analysis of PrP^C expression at 21 and 28 days from dental pulp separation and after additional 7 and 14 days with EGF/bFGF. Histograms represent log fluorescence vs cell number, gated on cell population of a side scatter/forward scatter (SS/FS) histogram. Cell number is indicated on the y-axis and fluorescence intensity is represented on the x-axis. Each panel was compared with the corresponding IgG negative isotype control. A representative experiment among 3 is shown. (B) Double staining flow cytometry analysis of PrP/CD44 in control hDPSCs (28 days from pulp separation) and PrP/ β3-tubulin after stimulation with EGF/bFGF for additional 14 days. Histograms represent log fluorescence PE vs log fluorescence CY5, gated on cell population of a side scatter/forward scatter (SS/FS) histogram. CD44-PE fluorescence is indicated on the y-axis and PrP-CY5 fluorescence intensity is represented on the x-axis. Each panel was compared with the corresponding IgG negative isotype control. A representative experiment among 3 is shown. (C) Western blot analysis of PrP^C expression at 21 and 28 days from dental pulp separation and after additional 7 and 14 days with EGF/bFGF, using anti-PrP SAF32. Loading control was evaluated using anti-β-actin. Densitometric analysis of bands from the representative western blot is reported in panel on the right as Mean ± SD. (D) Immunofluorescence analysis of hDPSC untreated o treated with EGF/bFGF, using anti-PrP SAF32.

Figure 3.

Analysis of PrP^C association with gangliosides and EGF-R by coimmunoprecipitation. hDPSCs, untreated or treated with EGF/bFGF for 14 days, were lysed in lysis buffer, followed by immunoprecipitation with anti-PrP SAF32. A mouse IgG isotypic control was employed. The immunoprecipitates were spotted onto nitrocellulose, and incubated with anti-GM2 (A), anti-GD3 (B) and anti-EGF-R (C), as described in Materials and methods. A representative experiment among 3 is shown. Bar graph in the right panel shows densitometric analysis. Results represent the Mean ± SD from 3 independent experiments, *p < 0.01. The immunoprecipitates were checked using the anti-PrP 6H4 (D).

Figure 4.

Effects of PrP^C silencing on ERK and Akt phosphorylation induced by EGF. hDPSCs, untreated or treated with 20 ng/ml EGF, in the presence or in the absence of pre-treatment with siRNA PrP or scrambled siRNA, were analyzed by Western blot, using anti-pERK1/2, anti-total ERK1/2 (A), anti-pAkt and anti-total Akt (B), Densitometric analysis is shown in the right. Results represent the Mean ± SD from 3 independent experiments, *p <0.01 siRNA PrP treated cells vs EGF treated cells. As control, scrambled siRNA was employed in each experiment.

Figure 5.

Plasma membrane PrP^C is required for signal transduction. hDPSCs cells, treated with siRNA PrP or scrambled for 72 hours, were stimulated with anti PrP SAF61 mAb for 10 min and analyzed by Western blot, using anti-pERK1/2, anti-total ERK1/2 (A), anti-pAkt, and anti-total Akt (B). Densitometric analysis is shown in the right panel. Results represent the Mean±SD from 3 independent experiments, *p <0.01 siRNA PrP treated cells vs SAF61 treated cells.

Figure 6.

Role of PrP^C during neuronal differentiation of hDPSCs induced by EGF/bFGF. (A) Flow cytometry analysis of PrP^C expression in hDPSCs, untreated or treated with siRNA PrP for 72 hours. Histograms represent log fluorescence vs cell number, gated on cell population of a side scatter/forward scatter (SS/FS) histogram. Cell number is indicated on the y-axis and fluorescence intensity is represented on the x-axis. Each panel was compared with the corresponding IgG negative isotype control. A representative experiment among 3 is shown. (B-C) Western blot analysis of β3-tubulin, and NFH expression in hDPSCs, untreated or treated with 20 ng/ml EGF and 40 ng/ml bFGF for 14 days, in the presence or in the absence of pre-treatment with siRNA PrP or scrambled siRNA for 72 hours. Densitometric analysis is shown in the right panel. Results represent the Mean±SD from 3 independent experiments. *p <0.01 siRNAPrP treated cells vs EGF/bFGF treated cells.