Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

Abstract

Background: Skin tissue homeostasis is maintained by a balance between the proliferation and differentiation of epidermal stem cells (EpSCs). EpSC proliferation and differentiation are complex processes regulated by many factors and signaling pathways. This study aimed to explore the connection between the Nanog and the Wnt/β-catenin pathway in the proliferation and differentiation of EpSCs.

Results: Our results demonstrated that during the study period, EpSC underwent differentiation when incubated in the presence neuropeptide substance P (SP), there was an opposing expression trend of Nanog and β-catenin after SP treatment, which could be antagonized by the Wnt antagonist, Dkk-1. The transduced EpSCs had a greater proliferative ability than the SP treatment group and they did not undergo differentiation upon SP treatment. More important, β-catenin expression was down-regulated but phosphorylated β-catenin expression and phosphorylated GSK-3β expression was up-regulated upon Nanog overexpression.

Conclusions: These results strongly suggest that Nanog plays an important role in maintaining the proliferation and differentiation homeostasis of EpSCs by promoting β-catenin phosphorylation via GSK-3β to inhibit the activity of the Wnt/β-catenin signaling pathway. This is important for precise regulation of proliferation and differentiation of EpSC in the application of tissue engineering.

Keywords: Differentiation; Epidermal stem cells; Nanog; Proliferation; Wnt/β-catenin.

Figure 1

Characterization of the isolated EpSCs. The morphology of the isolated cells are bird nest-like or slabstone-like (A). The isolated cells positively express CD34 (B) and β1 integrin (C) as detected by immunofluorescence. (D) EpSCs treated with 10^-7 M for 12 days showed polygonal or long spindle morphology. (E) CK18 expression in these SP-induced EpSCs indicated these cells were differentiated, as demonstrated by immunocytochemistry. (F) Wnt agonist triggers EpSC differentiation, as demonstrated by CK18 immunocytochemistry. (G) CD34 positive expression in the SP with Dkk-1 treated EpSCs demonstrated that these cells were still stem cells, as detected by immunofluorescence. (H) Fluorescence microscopy image of the transduced EpSCs. (I) The transduced EpSCs did not undergo differentiation upon SP treatment. Scale bar =20 μm.

Figure 2

Proliferative ability of EpSCs in vitro , as measured by the CCK-8 assay.

Figure 3

Expression of β-catenin and Nanog in EpSCs treated with SP. β-catenin and Nanog were measured at different time points (day 0-day 12) at the mRNA (q-PCR) and protein (western blot) level (A and B). The bands of each protein were quantitatively analyzed (C and D). Both q-PCR and western blot analysis showed that β-catenin expression was significantly higher on or after day 3 than at day 0. Nanog and β-catenin expression had an inverse relationship (*P < 0.05).

Figure 4

Expression of β-catenin and Nanog in EpSCs treated with Wnt antagonist, Dkk-1. Real-time PCR analysis of β-catenin mRNA (A) and Nanog mRNA (B) were obtained at different time points (day 1- day 12) in the control, SP treated group and SP with Dkk-1 treated group. The result showed that there were significant differences between the SP group and SP with Dkk-1 group on or after day 3 (*P < 0.05). At day 12, protein of Nanog and β-catenin were also detected (C). The bands of both proteins in each group were analyzed quantitatively (D and E). Werstern blot results were similar to q-PCR results (Con: control; SP: SP treated group; SP + Dkk-1: SP with Dkk-1 treated group).

Figure 5

Expression of β-catenin and Nanog in EpSCs infected with lentivirus. Real-time PCR analysis of Nanog mRNA (A) and β-catenin mRNA (B) were obtained at different time points (day 1- day 12) in the control, control lentivirus vector infected group and transduced group. The results showed that there were no significant differences between the control and control lentivirus vector infected group. However, significant differences existed in the transduced group with the control on or after day 3(*P < 0.05). At day 12, protein of Nanog and β-catenin were also detected (C). The bands of both proteins in each group were analyzed quantitatively (D and E). Western blot results were similar to q-PCR results (Con: control; LV: control lentivirus vector infected group; LV-Nanog: transduced group).

Figure 6

Overexpression of Nanog inhibits the Wnt signaling pathway. Real-time PCR analysis of gene expression ((A) Nanog, (B) β-catenin, and (C) c-myc) of the EpSCs in the control, SP-treated group, transduced group, and the combination treatment group. At the protein level, Nanog, c-myc, and total β-catenin were also detected (D). The bands of Nanog and c-myc protein in each group were analyzed quantitatively (E and F). (*P < 0.05) (Con: control; SP: substance P treated group; LV-Nanog: transduced group).

Figure 7

Overexpression of Nanog promotes β-catenin phosphorylation. The protein of total and phosphorylated GSK-3β,total and phosphorylated β-catenin were detected (A). The ratio of phosphorylated GSK-3β to total GSK-3β was an indicator to inactivation of β-catenin (B). The bands of β-catenin protein in each group were analyzed quantitatively (C and D). The ratio of phosphorylated β-catenin to total β-catenin was regarded as the inactive β-catenin (E). (*P < 0.05) (Con: control; SP: substance P treated group; LV-Nanog: transduced group).