1,25-Dihydroxyvitamin D3 regulation of phorbol ester receptors in HL-60 leukemia cells

In this study the relationship between cell binding of phorbol 12,13-dibutyrate (PDBu) and induction of differentiation by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was examined. Binding of 3H]PDBu increased within 12 h of 1,25-(OH)2D3 treatment, and a 60-130% increase in 3H]PDBu receptor levels was observed within 24 h. By 48 h, however, 3H]PDBu binding was not different from control. Scatchard analysis of 3H]PDBu binding showed no statistical differences in Kd value (Kd approximately equal to 30 nM) between 1,25-(OH)2D3-treated and control cells 22 h post-treatment; however, a 2-fold increase in Bmax was observed in treated (338 +/- 24 pmol/10(9) cells) compared to control cultures (170 +/- 14 pmol/10(9) cells). Stimulation of 3H]PDBu binding was dependent on 1,25-(OH)2D3 concentrations over a range of 1-100 nM. Homogenates from 1,25-(OH)2D3-treated HL-60 cells also demonstrated an increase (70%) in 3H]PDBu binding to the Ca2+/phospholipid-dependent enzyme protein kinase C as assessed by incubation of cell homogenates with 3H]PDBu in the presence of saturating phosphatidylserine and calcium concentrations. This suggests that the increase in 3H]PDBu binding cannot be entirely explained by modulation of the latter two agents. Cycloheximide (5 microM), an inhibitor of protein synthesis, ablated the 1,25-(OH)2D3-stimulated increase in 3H]PDBu binding to intact HL-60 cells. These data demonstrate that an increase in 3H]PDBu binding occurs early in the course of 1,25-(OH)2D3-induced differentiation, results from an increased number of 3H]PDBu-binding site, and is dependent on protein synthesis.