[NLS-RARα transports into the nucleus by binding to importin α1/β2(KPNA2/KPNB1) and inhibits the differentiation of U937 cells].

Objective To investigate the inhibitory effect of abnormal nuclear localization of the nuclear localization signal-retinoic acid receptor α (NLS-RARα) on cell differentiation and its mechanism of nuclear transport. Methods Over-expression of HA-NLS-RARα and empty vector in HEK293T cells and U937 cells were achieved through a lentivirus vector and were assigned as NLS-RARα over-expression (NR) group and negative control (NC) group. Extracted nucleoproteins and cytosolic proteins of NC and NR groups of HEK293T cells and U937 cells were detected by Western blot analysis in order to demonstrate the localization of NLS-RARα. Meanwhile, immunofluorescence assay was performed to explore the localization of NLS-RARα. The real-time quantitative PCR and Western blot analysis were used to detect difference in the mRNA and protein expression of CD11b and CEBPβ in the NR cells treated with 1, 25-dihydroxyvitamin D3 (1, 25D3) compared with NC cells treated with 1, 25D3. Mass spectrometric analysis and co-immunoprecipitation were conducted to screen the transport proteins which were associated with NLS-RARα, which was followed by the verification of nuclear accumulation of NLS-RARα by the transfection of transport protein small interfering RNA. Results Western blot assay and immunofluorescence showed that NLS-RARα was mainly located in the nucleus. And the qRT-PCR analysis and western blot assay showed a significant decrease in the mRNA and protein expression of CD11b and CEBPβ in the NR group compared with the NC group. It demonstrated that NLS-RARα inhibited cell differentiation. Mass spectrometric analysis and COIP demonstrated that KPNA2 (importin α1) and KPNB1 (importin β1) interacted with NLS-RARα, and the knockdown of KPNA2/KPNB1 inhibited the nuclear accumulation of NLS-RARα. Conclusion Abnormal localization of NLS-RARα inhibits cell differentiation via binding to KPNA2 and KPNB1 into the nucleus.