THE ROLE OF DIVA IN CELL DIFFERENTIATION

Abstract

Diva (death inducer binding to vBcl-2 and Apaf-1) is a Bcl-2 family member, and has been reported to play roles in apoptosis and oocyte maturation. Diva has also been shown to interact with Nm23-H2/NDPK B, which is involved in cellular differentiation. The main aim of this study was to elucidate the function and possible mechanisms for Diva in cellular differentiation in the brain.

In the present study, in PC-12 cells, Diva expression was decreased after differentiation and reciprocally, NDPK B expression was increased and it translocated into the nucleus. Endogenous Diva was also shown to interact with both ß-tubulin and NDPK B.

Overexpression of Diva in PC-12 cells did not change the expression level of NDPK B, but inhibited its nuclear localization. Diva-overexpressing cells had a decreased percentage of differentiated cells and average neurite length was shortened. This was due to the formation of more Diva/NDPK B and Diva/ß-tubulin complexes, at the expense of NDPK B/ß-tubulin complexes. Diva overexpressing cells also had a significantly elevated proliferation rate, with increased expression of cyclin D1, pRB and E2F-1 and concomitant decrease of p27 and p15 levels.

Overexpression of NDPK B in PC-12 cells resulted in decreased Diva expression and increased ß-tubulin expression. There was an increase in percentage of differentiated cells and average neurite length due to more NDPK B/ß-tubulin complexes being formed. NDPK B overexpression also inhibited cellular proliferation, by decreasing cyclin D1 and D3 levels as well as increasing p27 and p15 levels. In contrast, transfection of Diva and NDPK B plasmids individually into primary mesenchymal stem cells (MSCs) resulted in the appearance of a small percentage of oligodendrocyte-like cells, and there was a large amount of Diva/NDPK B complexes only in these cells.

Hence, the present results suggest a novel role for Diva in the negative regulation of neuronal differentiation. Its downregulation during neuronal differentiation is necessary to allow increased amounts of NDPK B/ß-tubulin complexes that promote neurite outgrowth. Decreased Diva expression is also required for translocation of NDPK B into the nucleus to inhibit cell proliferation during differentiation. An elevated level of NDPK B in the nucleus leads to increased expressions of cyclins, pRB and E2F-1 as well as decreased expressions of p27 and p15. Overexpression of Diva and NDPK B in MSCs resulted in the appearance of oligodendrocyte-like cells, which suggest a possible role for Diva in cell fate determination as well.