Diva/BclB regulates differentiation by inhibiting NDPKB/Nm23H2-mediated neuronal differentiation in PC-12 cells

Abstract

Background: Diva (death inducer binding to vBcl-2 and Apaf-1)/BclB is a Bcl-2 family member, which is known for its function in apoptosis. Diva/BclB has been shown to interact with NDPKB/Nm23H2, which is involved in cellular differentiation. Thus far, there has been no direct evidence of Diva/BclB having a role in differentiation. In the present study, we investigated the expression of Diva/BclB and NDPKB/Nm23H2 during differentiation in PC-12 cell line.

Results: Our results show that after differentiation, Diva/BclB expression was decreased and reciprocally, NDPKB/Nm23H2 expression was increased and it translocated into the nucleus. Overexpression of NDPKB/Nm23H2 promoted PC-12 neuronal differentiation by increasing neurite outgrowth and arresting cell cycle progression. There was a concurrent downregulation of Diva/Boo when NDPKB/Nm23H2 was overexpressed, which mirrors the effect of NGF on PC-12 cell differentiation. Overexpression of Diva/BclB did not change the expression level of NDPKB/Nm23H2, but inhibited its nuclear localization. Cells that overexpressed Diva/BclB presented a decreased percentage of differentiated cells and average neurite length was shortened. This was due to an increase in the formation of Diva/BclB and NDPKB/Nm23H2 complexes as well as Diva/BclB and β-tubulin complexes. Concomitantly, there was a decrease in formation of NDPKB/Nm23H2 and β-tubulin complexes. Overexpression of Diva/BclB also resulted in a higher percentage of S-phase cells.

Conclusion: Our results showed a novel role for Diva/BclB in neuronal differentiation. Its downregulation during neuronal differentiation may be necessary to allow NDPKB/Nm23H2 and β-tubulin interaction that promotes NDPKB/Nm23H2 mediated differentiation.

Figure 1

Reciprocal expression of Diva/BclB and NDPKB/Nm23H2 proteins in differentiated PC-12 cells. (A) Real time-PCR showed decreased expression of Diva/BclB mRNA and upregulation of NDPKB/Nm23H2 and β-tubulin expressions. The data was normalized using GAPDH expression. (B) Western blotting show protein levels follow the same trend after differentiation. (C) Immunocytochemistry staining with Diva/BclB (green) and NDPKB/Nm23H2 (red) antibodies show colocalization of both proteins in the cytoplasm. (D) Nuclear staining of NDPK B was observed in differentiated cells, and the percentage of cells with NDPKB/Nm23H2 in the nucleus was quantified after counting an average of 300 cells. Bar=40μm; *p<0.05, ***p<0.001.

Figure 2

Overexpression of NDPKB/Nm23H2 increases cell differentiation and neurite length. The PC-12 cells were harvested or fixed 24hrs post-transfection. (A) Immunoblotting showed that overexpression of NDPKB/Nm23H2 suppresses expression of Diva/BclB. (B) Immunocytochemistry staining with Diva/BclB (green) and NDPKB/Nm23H2 (red) antibodies show colocalization of both proteins in the cytoplasm. (C) The percentages of cells with NDPKB/Nm23H2 in the nucleus were greatly elevated after the protein was overexpressed. Both the percentage of differentiated cells (D) and average neurite length (E) of the differentiated cells were increased in the NDPKB/Nm23H2 overexpressing cells. An average of 150 cells per group was counted for graphical analyses, and the experiments were performed in triplicate (Bar=40μm, *p<0.05, **p<0.01, ***p<0.001).

Figure 3

Overexpression of Diva/BclB decreases cell differentiation and neurite length. After transfection with either the empty vector (control) or Diva/BclB overexpressing plasmid(Diva/BclB), immunoblotting (A) both showed an increase in Diva/BclB expression without any change to NDPKB/Nm23H2 expression. (B) Overexpression of Diva (green) resulted in stronger immunostaining in the cytoplasm. There was no change in NDPKB/Nm23H2 (red) expression. In the Diva/BclB cells, Diva/BclB expression remained strong even after 24 hrs of differentiation. There was strong colocalization of both proteins in the cytoplasm and at the neurite. (C) Overexpression of Diva/BclB prevents translocation of NDPKB/Nm23H2 into the nucleus after differentiation. It also decreases the percentage of differentiated cells (D) and their average neurite length (E). An average of 150 cells per group was counted for graphical analyses, and the experiments were performed in triplicate (Bar =40μm, *p<0.05, **p<0.01).

Figure 4

Diva/BclB prevents NDPKB/Nm23H2 and β-tubulin complex formation. After transfection, the cells were grown for a day under normal growth medium or differentiating medium for 24 hrs. The interaction between the proteins were analysed using the Duolink assay. Regardless of the differentiation status, there were more Diva/BclB and β-tubulin (A) and Diva/BclB and NDPKB/Nm23H2 (B) complexes, and fewer NDPKB/Nm23H2 and β tubulin (C) complexes formed in Diva-overexpressing cells. (Bar= 20; *p<0.05, **p<0.01).

Figure 5

Effect of Diva/BclB and NDPKB/Nm23H2 on the cell cycle and cellular proliferation of cells. (A) The number of cells in S phase are visualised by BrdU staining (green) in the nucleus. Diva/BclB had the highest proportion of stained nuclei, followed by control and NDPKB/Nm23H2 had the lowest proportion. (B) An average of 500 cells per group was counted, and quantitative analysis of the data showed that overexpression of NDPKB/Nm23H2 decreased the percentage of S-phase cells, while Diva/BclB increased cellular proliferation. (C) Growth curves of the control and transfected cells were plotted using MTS assay. Diva/BclB cells had a less inhibited growth curve, while NDPKB/Nm23H2 cells had a slower growth curve, when compared to control cells. (D-G) Flow cytometry data was collected after serum starvation for 24 hours (0hr), and at indicated times after serum addition. Graphical analysis using Summit software showed that overexpression of Diva/BclB resulted in a decrease in apoptotic cells and faster cell cycle progression. In contrast, overexpression of NDPKB/Nm23H2 resulted in slower cell cycle progression. (Bar=20μm, *p<0.05, **p<0.01).