Mechanism Research Of EGF-induced Hair Follicle-derived Mesenchymal Stem Cells Proliferation

The hair follicle is a very rich source of adult stem cells which not only actively take part in the hair development and regeneration but also are considered as ideal seed cells for tissue engineering. Clinical research showed that successful stem cell therapy requires 1×109 stem cells. So, maintenance of highly proliferative capacity and fully differentiation potentials is a necessary step in initiation of stem cell-based regenerative medicine.By binding to the EGF receptor(EGFR), EGF phosphorylates EGFR, activating downstream signaling pathways and subsequently affecting the cell's biological behavior. Although the major downstream signaling pathways of EGF-EGFR are known and include the ERK, PI3K-AKT, and JAK/STAT signaling pathways, these signaling pathways are cell specific and the pathway by which EGF affects HFMSCs has not been reported.Growth factor is a class of protein components that can stimulate cell growth, which is involved in cell proliferation, differentiation a, migration, and a series of biological processes. Fibroblast growth factor(FGF), epidermal growth factor(EGF), platelet-derived factor(PDGF) and vascular endothelial growth factor(VEGF) can promote cell proliferation. However, the application of growth factors for the expansion of human hair follicle mesenchymal stem cells in vitro has not been resolved. Appropriate growth factor types and doses are needed in promoting the proliferation of human hair follicle mesenchymal stem cells while maintaining the characteristics of stemness. In addition, signal pathways by which the growth factors promote the proliferation of human hair follicle mesenchymal stem cells is not clear.Our recent study showed epidermal growth factor(EGF) at as low as 1ng/ml significantly enhanced the proliferation of HF-MSCs. However, the underlying pathways of EGF in enhancing HF-MSCs' proliferation remains unclear. The goal of this study is to elucidate the downstream pathways of EGF in enhancing the proliferation of HF-MSCs.To this end HF-MSCs were isolated and cultured in the presence or absence of EGF. Cell proliferation and cell signaling pathway related to EGF receptor as well as cell cycle assay of HF-MSCs were performed by immunofluorescence staining, flow cytometry, cytochemistry and Western blot.Immune fluorescence staining and flow cytometry showed HF-MSCs exhibited surface markers of mesencymal stem cells and displayed trilineage differentiation potentials towards adipocytes, chondrocytes and osteoblasts. Cell proliferation assay showed EGF at 1-50 ng/ml significantly increased the proliferation of HF-MSCs. Western blot showed EGF significantly increased not only the expression of phosphorylated EGFR(P-EGFR) in time-response and dose-dependent manner in HF-MSCs but also the expression of phosphorylated ERK1/2(P-ERK1/2) and phosphorylated AKT(P-AKT) in HF-MSCs treated with 10ng/ml of EGF in time-dependent manner. However EGF at 10ng/ml did not show any effects in regulating the expression of phosphorylated STAT3(P-STAT3) in HF-MSCs. EGFR inhibitor(AG1478) at 0.2-5?M, PI3K-AKT inhibitor(LY294002) at 10-50?M, ERK inhibitor(U0126) at 2-50?M and STAT3 inhibitor(STA-21) at 20-50?M significantly blocked the proliferation of HF-MSCs induced by EGF. Moreover AG1478 at 2?M, LY294002 at 10?M, U0126 at 10?M significantly decreased the expressions of p-EGFR, p-ERK1/2 and p-AKT. Cell cycle assays revealed that EGF shifted HF-MSCs at G1 phase to S and G2 phase, which was accompanied by increased protein expression of cyclin D1, phosphorylated Rb, E2F1 and decreased p16 expression after treatment with 10ng/ml of EGF for 24 hours.In conclusion EGF induces proliferation of HF-MSCs through EGFR/ERK and AKT pathway, but not STAT-3 signaling pathway. G1/S transition was stimulated through EGF-EGFR signaling pathway in HF-MSCs by up-regulating of cyclin D1 expression and inhibiting p16 expression.