Foxm1 transcription factor is required for maintenance of pluripotency of P19 embryonal carcinoma cells

Abstract

Transcription factor Foxm1 plays a critical role during embryonic development and its expression is repressed during retinoic acid (RA)-induced differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, correlated with downregulation of expression of pluripotency markers. To study whether Foxm1 participates in the maintenance of pluripotency of stem cells, we knock down Foxm1 expression in P19 cells and identify that Oct4 are regulated directly by Foxm1. Knockdown of Foxm1 also results in spontaneous differentiation of P19 cells to mesodermal derivatives, such as muscle and adipose tissues. Maintaining Foxm1 expression prevents the downregulation of pluripotency-related transcription factors such as Oct4 and Nanog during P19 cell differentiation. Furthermore, overexpression of FOXM1 alone in RA-differentiated P19 cells (4 days) or human newborn fibroblasts restarts the expression of pluripotent genes Oct4, Nanog and Sox2. Together, our results suggest a critical involvement of Foxm1 in maintenance of stem cell pluripotency.

Figure 1.

The expression of Foxm1 was repressed during RA-induced P19 cell differentiation and knockdown of Foxm1 decreased alkaline phosphatase activity and the expression of pluripotency genes. (A and B) Gene expression analysis of Foxm1, Oct4, Nanog and Sox2 during RA-induced differentiation of P19 cells by RT-PCR or quantitative real-time PCR (qPCR) (A) or western blotting (B). Cyclophilin or β-actin was used as the loading control. (C) P19 cells, P19 cells infected with AdGFP control virus or AdFoxm1siRNA virus (10 pfu/cell, 3 days) were stained for alkaline phosphatase. (D and E) Knockdown of Foxm1 decreased the expression of pluripotency markers in P19 cells. P19 cells were infected with AdGFP or AdFoxm1siRNA (10 pfu/cell) and gene expression analysis of Foxm1, Oct4, Nanog and Sox2 was performed by RT-PCR or qPCR (D) or western blotting (E).

Figure 2.

Knockdown of Foxm1 resulted in spontaneous mesodermal differentiation of P19 cells. (A) Knockdown of Foxm1 increased the expression of differentiation marker Gata4 in P19 cells. P19 cells were infected with AdGFP or AdFoxm1siRNA (10 pfu/cell) and gene expression analysis of Foxm1 and Gata4 was performed by RT-PCR. (B) Knockdown of Foxm1 resulted in significant decrease of SSEA-1 in P19 cells. Flow cytometry was used to measure cell populations of SSEA-1 in P19 cells and AdFoxm1siRNA-infected P19 cells (3d). MEFs were used as negative control cells and mouse ES cells were used as positive control cells. (C) Teratoma formation was abrogated in P19 cells with decreased levels of Foxm1. Teratomas collected from the nude mice injected with Adcontrol or AdFoxm1siRNA-infected P19 cells are shown. (D) Teratomas formed with AdFoxm1siRNA-infected P19 cells expressed mesodermal marker Brachyury and cardiac muscle-specific Actc1. The total RNAs isolated from teratomas of P19 cells or AdFoxm1siRNA-infected P19 cells were analyzed for selected genes by RT-PCR. (E) Knockdown of Foxm1 in P19 cells resulted in differentiation of mesodermal derivatives in teratoma formation. Hematoxylin and eosin-stained sections from teratomas of AdFoxm1siRNA-infected P19 cells after 3 weeks were shown.

Figure 3.

Foxm1 binds to and stimulates Oct4 promoter. (A) The predicted positions of putative Foxm1 binding sites in –5 kb mouse Oct4 promoter by gene sequence analysis and the positions of primers designed for ChIP assays. (B) FoxA1 bound to endogenous Oct4 promoter. ChIP assays were used to show direct binding of Foxm1 to endogenous Oct4 promoter regions. The chromatin of P19 cells or RA-induced (3d) P19 cells was cross-linked, sonicated, and immunoprecipitated (IP) with either Foxm1 antiserum or rabbit serum (control) and the amount of promoter DNA associated with the IP chromatin was quantitated by RT–PCR with primers specific to different Oct4 promoter regions. The predicted size of the PCR product was 170 bp (ChIP1) or 187 bp (ChIP2). (C) The –3-kb region of Oct4 promoter mediated the transcription activity of Foxm1. The Oct4 promoters with different length were constructed into luciferase reporter plasmid. The different reporter plasmid (1.5 µg) and loading control pRL-CMV luciferase reporter plasmid (20 ng) were transfected into P19 cells with the CMV-FOXM1 expression vector (200 ng) or a CMV empty expression vector (200 ng). Protein lysates were prepared at 24 h following transfection, and used to measure dual Luciferase enzyme activity. (D) The activities of Oct4 promoters with different length were inhibited at different degree during RA-induced P19 cell differentiation. The different reporter plasmid (1.5 µg) was transfected into P19 cells, followed by RA-induced differentiation. Three days later, the luciferase activities were measured. (E) Only the Oct4 promoter that binds Foxm1 was inhibited by Foxm1 depletion. The different reporter plasmid (1.5 µg) was transfected into P19 cells, followed by AdFoxm1siRNA infection. Two days later, the luciferase activities were measured. (F) Point mutations on Foxm1 binding sites of the –3 kb Oct4 promoter inhibited Foxm1-mediated transcription activity. Intact or mutated –3 kb Oct4 promoter-luciferase reporter plasmids (1.5 µg) were transfected into P19 cells with the CMV-FOXM1 expression vector (200 ng). The luciferase activities were measured at 24 h following transfection. The asterisks indicate statistically significant changes: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4.

Maintained expression of FOXM1 prevented the downregulation of pluripotency marker expression during P19 cell differentiation. (A) Overexpression of FOXM1 in P19 cells by AdFOXM1 infection. P19 cells were infected with AdGFP or AdFOXM1 (10 pfu/cell) and mRNA levels of exogenous FOXM1, endogenous Foxm1, and loading control Cyclophilin were measured by RT-PCR. (B and C) P19 cells infected with AdGFP or AdFOXM1 were induced to differentiation according to the standard protocol one day after viral infection. Gene expression analysis of selected pluripotency genes was performed by RT–PCR (B) or western blotting (C). (D and E) The overexpression of FOXM1 activated expression of Foxm1, Oct4, Nanog and Sox2 in the differentiated P19 cells. P19 cells were differentiated by RA treatment and 4 days later the cells were infected with AdGFP or AdFOXM1. The expression analysis of selected genes was performed by RT-PCR (D) or western blotting (E). (F) The overexpression of FOXM1 activated expression of of SSEA-1 in differentiated P19 cells. Flow cytometry was used to measure cell populations of SSEA-1 in P19 cells (RA 4d), P19 cells (RA 4d)/AdGFP (3d), P19 cells (RA 4d)/AdFOXM1 (3d) and P19 cells. P19 cells (RA 4d) were used as negative control cells and P19 cells were used as positive control cells. (G) P19 cells (RA 4d)/AdGFP (3d) and P19 cells (RA 4d)/AdFOXM1 (3d) were stained for alkaline phosphatase.

Figure 5.

The overexpression of FOXM1 activated the expression of pluripotency-related genes in human newborn fibroblasts. (A) The morphology of FOXM1-overexpressed human newborn fibroblasts. Human newborn fibroblasts were infected three times by AdFOXM1 (100 pfu/cell) at 4 days interval between each infection. Pictures of human newborn fibroblasts or the cells post AdFOXM1 3X infections were taken at 200× magnification using a TE2000 microscope (Nikon). (B) FOXM1-overexpressed human newborn fibroblasts obtained high levels of alkaline phosphatase activity. The human newborn fibroblasts infected 3X with AdGFP or AdFOXM1 were stained for alkaline phosphatase. (C) The overexpression of FOXM1 activated expression of OCT4, NANOG and SOX2 in human newborn fibroblasts. The human newborn fibroblasts infected with AdFOXM1 were analyzed for selected genes by RT-PCR (C). The human EC cell line NT2/D1 was used as a positive control of human pluripotent stem cells.