Vitamin D derivatives (VDDs) exhibit anticancer effects both in-vitro and in-vivo. However, the ability of VDDs to treat cancer in clinic still remains uncertain due to hypercalcemia at pharmacologic doses and incomplete understanding of the underlying molecular mechanisms. While ERK1/2 has been widely investigated and known to transmit signals for cell survival, growth, and differentiation, the involvement of the ERK5 MAPK pathway in these processes is still unclear. Here, using acute myeloid leukemia (AML) cell lines (HL60 and U937) and leukemic blasts isolated from patients with AML, we determined if ERK5 has a role in 1,25-dihydroxyvitamin D3 (1,25D)-induced differentiation distinct from that of ERK1/2. We found that in cells treated with a low concentration of 1,25D (10-9 M) inhibition of ERK5 by BIX02189 or XMD8-92 results in higher expression of a general myeloid marker CD11b and superoxide-generating activity but lower expression of the monocytic marker CD14. Furthermore, morphologic analysis of HL60 and U937 cells demonstrated that combinations of 1,25D and ERK5 inhibitors induce marked changes in cell phenotype compatible with macrophage-like differentiation. In contrast, inhibition of the ERK1/2 pathway by PD98059 reduced the expression of all differentiation markers tested. This shows that differentiation-related ERK5 and ERK1/2 pathways are not redundant. The results obtained with pharmacological inhibitors were confirmed using ERK5 siRNA. Down-regulation of ERK5 was accompanied by reduced phosphorylation of the known downstream ERK5 targets (the transcription factors cJun and MEF2C). We also observed that ERK5 positively regulated the expression of C/EBPb, but not C/EBPa. The above findings indicate that activated ERK5 is important for monocytic differentiation but may suppress the development of terminal macrophage-like phenotype. Our results suggest that ERK5 inhibitors can be evaluated as enhancers of the therapeutic effects of VDDs in clinical trials of AML. This study was supported by the Israel Science Foundation grant 635/11 (to M.D.) and NIH-NCI grant RO1CA 44722-22 (to G.P.S.).
