NLS-RAR? Inhibits The Effects Of All-trans Retinoic Acid On NB4 Cells By Interacting With P38? MAPK

Objective: Retinoic acid receptor alpha(NLS-RAR?)with nuclear localization signal,which forms from the cleavage of PML-RAR? by neutrophil elastase(NE),possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL).However,the potential mechanism underlying the effects of NLS-RAR? on APL is still not entirely clear.Here,we investigated the effects of NLS-RAR? on APL NB4 cells and its mechanism.Methods: The expressions of myeloid differentiation markers,C/EBP?and CD11 b,p38? MAPK and the proliferation of NB4 cells were detected by Western blot and CCK-8 assay respectively after treating cells with ATRA.The transcriptional activity of p38? MAPK was detected by dual luciferase reporter assay after the eukaryotic expression vector of p38?MAPK was constructed by molecular cloning technique.NLS-RAR? was overexpressed mediating by lentivirus and its efficiency of NLS-RAR?overexpression,the expressions of myeloid differentiation markers,C/EBP? and CD11 b,p38? MAPK in NB4 cells resulted from NLS-RAR?were detected by Western blot.In the meantime,the proliferation of NB4 cells was determined by CCK-8 assay.The localization of NLS-RAR? and its interaction with p38? MAPK was analysed by indirect immunofluorescence assay and co-immunoprecipitation assay,respectively.To investigate whether ATRA directly activates p38? MAPK and then recruits NLS-RAR? or recruitment of NLS-RAR? eventually causes activation of p38? MAPK,the inhibitor of p38? MAPK was applied.Results: We found that the amount of differentiation makers,C/EBP?and CD11 b,and phosphorylated-mitogen-activated protein kinase(p-p38?MAPK)in NB4 cells were elevated in ATRA treated group(p<0.05).Meanwhile,the cell proliferation level was decreased(p<0.05),and the expression level of p38? MAPK had no difference(p>0.05).We also had successfully established eukaryotic expression plasmid of p38? MAPK and found that luciferase activity was increased(p<0.05)in p38? MAPK eukaryotic expression plasmid transfection group.Next,we had successfully established NB4 cells with NLS-RAR? overexpression and found that the expression levels of differentiation makers,C/EBP? and CD11 b,was decreased(p<0.05)in NLS-RAR? overexpression group when cells were not treated with ATRA.Meanwhile,the amount of p38? MAPKand p-p38? MAPK had no change(p>0.05).However,the amount of differentiation makers,C/EBP? and CD11 b,and p-p38? MAPK were decreased(p<0.05)in NLS-RAR? overexpression group when cells were treated with ATRA.Meanwhile,the cell proliferation level was elevated(p<0.05),and the expression level of p38? MAPK had no change(p>0.05).Further more,immunofluorescence and co-immunoprecipitation assays confirmed NLS-RAR? interacted with p38? MAPK directly.Finally,the application of an inhibiter of p38?MAPK,PD169316,found that the amount of differentiation makers,C/EBP? and CD11 b,and p-p38? MAPK in LV-NLS-RAR?-NB4 cells were decreased(p<0.05)in ATRA+ PD169316 treated group when compared to the ATRA treated group.Meanwhile,the cell proliferation level was elevated(p<0.05),and the expression levels of NLS-RAR? and p38?MAPK had no change(p>0.05).What's more,the application of PD169316 in 293T cells found that NLS-RAR? can interact with p38? MAPK each other in both PD169316 treated group and PD169316 untreated group.Conclusion: our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38? MAPK after recruiting p38? MAPK-combinded NLS-RAR?,while NLS-RAR? could inhibit the effects of ATRA on NB4 cells by down regulating p-p38? MAPK in the process.