Thus far, reports on Diva/BclB have only covered its role in apoptosis and oocyte maturation [5, 15, 16]. The results in this study propose a novel function for Diva/BclB, by demonstrating that the protein is involved in the negative regulation of neuronal differentiation, and that it achieves this by inhibiting NDPKB/Nm23H2 function.
In this study, we showed that with NGF-induced differentiation, Diva/BclB expression decreases and NDPKB expression increases. The regulation of these 2 proteins is likely through the sustained ERK signalling by NGF, which results in phosphorylation as well as binding of CREB and AP-1 family members so as to facilitate gene transcription [17, 18].
Overexpression of NDPKB/Nm23H2 encouraged PC12 neuronal differentiation by promoting neurite outgrowth and retarding cell proliferation. Interestingly, there was Diva/BclB downregulation in the NDPKB/Nm23H2 overexpressing cells, which is a salient point since decreased expression of Diva/BclB was also observed during NGF-induced differentiation. Thus, this supported that Diva/BclB was a negative modulator of neuronal differentiation that had to be downregulated and suggested that the induced downregulation of Diva/BclB by NDPKB/Nm23H2 could be a positive feedback mechanism for NGF-induced neuronal differentiation.
Overexpression of Diva suppressed NDPKB/Nm23H2 function by retaining NDPKB/Nm23H2 in the cytoplasm, and attenuated the number of NDPKB/Nm23H2 and β-tubulin complexes. In turn, there was an increase in S-phase cells and shorter neurites were observed after NGF-induced neuronal differentiation.
The regulatory molecules that control the rate of neurite growth and the signals that determine when and where the axons and dendrites have to grow are still largely unknown. Overexpression of negative regulators of neuronal differentiation have been shown to block neurite outgrowth and branching by inhibiting the Erk/MAPK and Rac1 but not Akt signaling pathway in response to NGF [3, 19]. In our study, Diva/BclB inhibited neurite outgrowth as more Diva/BclB and NDPKB/Nm23H2 as well as Diva/BclB and β-tubulin complexes were formed and the amount of NDPKB/Nm23H2 and β-tubulin complexes decreased significantly. Previous reports have reported the co-immunoprecipitation of NDPK/Nm23 with β-tubulin, and the number of complexes increases during the cellular differentiation process [12, 20]. Tubulin phosphorylation by NDPKB/Nm23H2 would allow the microtubules to attach more efficiently to the MAPs and increase microtubule stability, thereby facilitating neurite outgrowth [21–24]. Therefore, this study suggests that the physiological role of Diva/BclB in undifferentiated cells might be to inhibit the association between NDPKB/Nm23H2 and β-tubulin. Expression of Diva/BclB is downregulated during differentiation to allow more NDPKB/Nm23H2 and β-tubulin complexes to form, thereby encouraging neurite outgrowth. This is interesting since Diva/BclB has been shown to interact with Hungtington-interacting protein 1-related (H1P1R) protein and microtubule binding protein, translationally controlled tumor-associated protein (TCTP) and thereby postulated to regulate the cytoskeleton [14, 25]. Our study has plugged the gap to show regulation of the cytoskeleton by Diva/BclB and its consequential effect on neurite outgrowth.
In differentiated cells, the overexpression of Diva/BclB in PC-12 cells resulted in intense immunostaining in the cytoplasm, indicating the colocalisation of the 2 proteins, and significantly decreased the number of cells with NDPKB/Nm23H2 in their nucleus. This suggests that overexpression of Diva/BclB was able to sequester NDPKB/Nm23H2 in the cytoplasm and prevent its translocation into the cytoplasm after receiving the signal for differentiation. This is interesting because overexpression of NDPKB/Nm23H2 results in nuclear expression and decreased cellular proliferation. In contrast, the effect of overexpressing Diva/BclB is a higher proliferation rate and decreased nuclear expression of NDPKB/Nm23H2. Hence, this suggests that Diva/BclB is able to regulate proliferation by controlling the nuclear translocation of NDPKB/Nm23H2. NDPKs have been reported to translocate to the nucleus, although there is controversy whether NDPKA/Nm23H1 and NDPKB/Nm23H2 visit the nucleus together or separately [22, 26]. Also, reasons for NDPKB/Nm23H2 movement into the nucleus are also unclear, though the translocation has been observed to occur at certain cell phase cycles, thereby suggesting NDPK/Nm23 are required in the nucleus for gene regulation or to supply dNTPs for replication . NDPKB/Nm23H2 has been identified as human transcription factor PuF, a sequence-specific DNA-binding protein with affinity for a nuclease-hypersensitive element (NHE) of the c-MYC gene promoter [28, 29]. NDPKB/Nm23H2 has also been demonstrated to be capable of DNA binding, DNA cleavage, as well as transcriptional activity that is independent of phosphoryl transfer and NDPK activity . Thus, we report a novel observation of the translocation of NDPKB/Nm23H2 into the nucleus after differentiation, which causes inhibition of proliferation and retarded cell cycle progression, although the exact mechanisms are yet to be further elucidated.