PrPC and EGFR: a multimolecular complex involved during neuronal differentiation of human dental pulp-derived stem cells

Aims. The expression of PrPC makes mesenchymal stem cells good candidates to develop in vitro system for the study of prion infectivity and multiplication. Previous works suggest that lipid rafts and their components, as gangliosides, are essential for neuronal differentiation of different type of stem cells. Since PrPC is constitutively expressed in lipid rafts and in a wide variety of stem cells, the purpose of this study is to investigate the possible role of PrPC during neuronal differentiation of human dental pulp-derived stem cells (hDPSCs). Methods. hDPSCs were isolated from third molar of healthy adult subjects (13 to 19 years old) and they were maintained in Dulbecco’s Modified Eagle’s Medium low glucose, containing 100 units/ml penicillin, 10 mg/ml streptomycin, plus 0,1% amphotericin plus foetal bovin serum (FBS) qualified 10%, at 37°C in humified CO2 atmosphere. All samples were collected with informed consent of the patients according to ethics considerations and after subscribed suitable set of forms and with the approval of the ethics committee. Results. hDPSCs express multipotent mesenchymal stromal specific surface antigens, such as CD44, CD90, CD105 and STRO1, but they are negative for hematopoietic markers, such as CD14 and CD19. Moreover, after stimulation with EGF/bFGF, hDPSCs reduce their growth and, after two weeks, it is possible to observe neurites outgrowth and the expression of specific neuronal markers, such as B3-tubulin and NFH. Also, we showed that hDPSCs express precociously PrPC at low concentration and its expression increases after two weeks of treatment with EGF/bFGF. Then, we analyzed the association of PrPC with gangliosides and EGF receptor (EGF-R) during neuronal differentiation process. In untreated hDPSCs, PrPC associate constitutively with GM2 while for GD3 only after neuronal differentiation. Otherwise, EGF-R associate weakly in untreated hDPSCs while it is more markedly expressed after neuronal differentiation. To analyze the functional role of PrPC in the signal pathway mediated by EGF/EGF-R, a siRNA PrP was applied to ablate PrPC 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. Conclusions. In this study we have analyzed the role of PrPC in hDPSCs isolated from third molars of health patient. Taken together, our results suggest that PrPC interacts with EGF-R within lipid rafts, playing a role in the multimolecular signaling complexes involved in hDPSCs neuronal differentiation.