Adequate concentration of B cell leukemia/lymphoma 3 (Bcl3) is required for pluripotency and self-renewal of mouse embryonic stem cells via downregulation of Nanog transcription

Abstract

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the NF-κB family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity. [BMB Reports 2018; 51(2): 92-97].

Fig. 1

Bcl3 is highly expressed in mouse embryonic stem cells. (A) Western blotting analysis of Bcl3 in mouse embryonic fibroblasts (MEF) and E14tg2a cells (E14). GAPDH was used as an internal control. (B) Western blot analysis of Bcl3 in E14 under the leukemia inhibitor factor (LIF) depletion conditions. After seeding, the cells were cultured for 3 days, and then were transferred in LIF depletion media. The samples were extracted at the indicated times. β-actin was used as an internal control. (C) Quantitative RT-PCR assay of Bcl3 in ESC-derived embryoid bodies. Embryoid bodies were formed by hanging drop culture methods and extracted at the indicated times. Data are normalized to β-actin and shown relative to E14 cells. ***P < 0.005 vs E14 cells. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using one-way ANOVA. (D) Western blot analysis of Bcl3, Nanog, Sox2, and Oct4 in ESC-derived embryoid bodies. β-actin was used as an internal control.

Fig. 2

Knockdown of Bcl3 attenuated pluripotency of mouse embryonic stem cells. (A) Western blot analysis of Bcl3 in E14 cells transfected with mock vector (shMock) and Bcl3 shRNA (shBcl3). GAPDH was used as an internal control. (B) Cell proliferation assay of shMock and shBcl3. The cells were seeded on a 24-well culture plate at a density of 3 × 10³ cells/well and cultured for 3 days. ***P < 0.005 vs shMock. (C) Alkaline phosphatase staining of shMock and shBcl3. After AP staining, the colonies were scored, and the percentages of undifferentiated, mixed, and differentiated colonies were calculated. The bar graph shows the statistical evaluation. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. **P < 0.01 vs shMock. (D) Western blot analysis of pluripotent-related genes, Nanog, Oct4, and Sox2 in shMock and shBcl3. GAPDH was used as an internal control. (E) Quantitative RT-PCR of pluripotent related genes in shMock and shBcl3. The data are normalized to β-actin and expressed relative to shMock. The error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. **P < 0.01, ***P < 0.005 vs shMock.

Fig. 3

Overexpression of Bcl3 reduces pluripotency of mouse embryonic stem cells. (A) Western blot analysis of Bcl3 in E14 cells transfected with mock vector (ZsMock) and Bcl3 (ZsBcl3). β-Actin was used as an internal control. (B) The cell proliferation assay of ZsMock and ZsBcl3. **P < 0.01 vs ZsMock. (C) Alkaline phosphatase staining of ZsMock and ZsBcl3. The bar graph shows the statistical evaluation. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. *P < 0.05 vs ZsMock. (D) Western blot analysis and (E) quantitative RT-PCR of pluripotent related genes and differentiation related genes in ZsMock and ZsBcl3. The data are normalized to β-actin and expressed relative to ZsMock. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.005 vs ZsMock. (F) Luciferase reporter assay of Nanog in E14 cells. E14 was co-transfected with Nanog-5p and vectors as indicated. The transfected cells were cultured for 24 h and the luciferase activity was measured. Data were normalized to a Renilla luciferase control. The error bars indicate the mean ± SEM (n = 4). P values were calculated by using one-way ANOVA. ***P < 0.005 vs control, ^##P < 0.01 vs Nanog-5p-only transfected cells. (G) Single cells of ZsMock and ZsBcl3 were sorted into 96-well plates by FACS and cultured for 5 days, after which the wells were scored for the presence of colonies. *P < 0.05 vs. ZsMock. Error bars indicate the mean ± SEM (n = 3). (H) The morphology of E14_ZsMock and E14_ZsBcl3. The cells were grown for 5 days and sorted for GFP-positive cells by FACS. Representative fluorescence microscopy images at 50× (left) and 200× (right) magnification are shown.

Fig. 4

Regulation of Bcl3 expression is an essential for maintenance of mouse embryonic stem cell pluripotency. In the absence or overexpression of Bcl3, mouse embryonic stem cells lose pluripotency and differentiation is promoted. Reduction of Bcl3 in mESCs induces loss of self-renewal ability and induces differentiation. Overexpression of Bcl3 in mESCs also promotes differentiation through a reduction in Nanog promoter activity. After the suppression of Nanog promoter activity, the expression of pluripotency genes was decreased, and that of differentiation genes, was increased. Collectively, Bcl3 was required for pluripotency of mESCs and the regulation of Bcl3 expression is essential for the maintenance of mESC pluripotency.