Page 118 - 2019_01-Haematologica-web

Y. Lu et al.
AB
CD
E
F
Figure 5. 2BP directly binds with RARα. (A-B) Cellular thermal shift assay (CETSA) was performed on NB4 cells as described in the Methods section. The effects of 2BP on RARα, vinculin and PKM2 at different temperatures (A) and different doses (B) were evaluated by Western blot analysis. (C) SPR analysis of the binding between 2BP and RARα. The recombinant RARα protein was immobilized on an activated CM5 chip. 2BP was then flowed across the chip at increasing concentra- tions. (D) The recombinant RARα protein was incubated with biotin-2BP for the times indicated, and the mixtures were blotted with anti-biotin antibody. The mem- brane stained with Ponceau S is shown on the bottom. (E-F) NB4 cells were treated with PA, 2BP, 16BP or 12BP (chemical structure shown on E) for 3 days and CD11b-positive cells were counted by flow cytometry and RARα expression was detected by western blot are shown. ***P<0.001 against vehicle-treated group. All experiments were repeated three times with the same results.
RARα–disrupted PML nuclear body, which was decreased by the addition of 2BP (Online Supplementary Figure S6A). Western blot analysis using an antibody against PML- RARα fusion protein showed that 2BP could increase the amount of PML-RARα in NB4 cells (Online Supplementary Figure S6B), suggesting that 2BP may also stabilize PML- RARα fusion protein.
To further confirm the role of the stabilized RARα pro- tein in the synergistic differentiation-inducing effect of 2BP in combination with ATRA, AM580, a selective RARα agonist,41 was applied to NB4 cells. The results showed that, similar to that seen under ATRA treatment, 2BP could enhance AM580-induced differentiation of NB4 cells (Online Supplementary Figure S7A-B). Moreover, administration of AM580 induced a loss of RARα pro- teins, which could also be accumulated by addition of 2BP (Online Supplementary Figure S7C). On the other hand, Ro 41-5253, a highly specific RARα antagonist,42 could effec- tively antagonize the synergistic differentiation-inducing effect of the 2BP and ATRA combination in NB4 cells (Online Supplementary Figure S7A-B). These data indicated that maintenance of RARα protein contributed to the enhancing effect of 2BP on ATRA-induced differentiation.
Next, we asked whether the transcriptional activity of
RARα protein was also enhanced by the ATRA/2BP com-
bination following the accumulation of protein level.
Consistent with previous reports, ATRA, but not 2BP,
increased the expression of RARα target genes including
RARβ, CCAAT/enhancer-binding protein α(C/EBPα),
retinoic acid inducible gene-E (RIG-E), RIG-I, RIG-G, inter-
feron regulatory factor 1 (IRF-1), transglutaminase
2(TGM2), and ubiquitin-like modifier activating enzyme
2BP directly targets RARα
2BP has been reported to be active in covalently binding
7(UBE1L) gene and decreased that of myeloperoxidase 35-40
(MPO) in NB4 cells (Figure 4H). More intriguingly, the modulation of these expressions by ATRA was signifi- cantly enhanced by the cotreatment of 2BP with ATRA in NB4 cells (Figure 4H). Taken together, these data indicated that 2BP accumulates RARα protein and enhances ATRA- dependent transcriptional activity of RARα.
target proteins because it is from the α-halo-carbonyl group.10 To further elucidate the mechanism of 2BP on ATRA-induced APL differentiation, we detected the possi- bility of RARα protein as a cellular target of 2BP. To this
108
haematologica | 2019; 104(1)