Nls-Rar Alpha Is A Novel Transcriptional Factor

Acute promyelocytic leukemia (APL) is characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RAR-alpha) fusion protein. PML-RAR alpha can be cleaved by neutrophil elastase (NE) in several positions in cells in the promyelocytic stage, nuclear location signal (NLS) -negative PML and NLS-RAR alpha may be the products of PML-RAR alpha by NE. The function of NLS-RAR alpha may be affected by the addition of NLS, which would alter its localization in cells, as the role of NLS is to identify proteins for transport to the nucleus. Preliminary experiments demonstrated that the overexpression of NLS-RAR alpha in HL-60 cells could promote cellular proliferation and inhibit cellular differentiation. Following treatment with all-trans retinoic acid (ATRA), the degree of cellular differentiation was enhanced. In the present study, the localization of NLS-RAR alpha was identified and its activity as a novel transcriptional factor was assessed, which may be critical in the development of APL. The location of NLS-RAR alpha was detected in the nucleus and cytoplasm by indirect immunofluorescence and western blot analysis, with expression in the nucleus revealed to be increased compared with that in the cytoplasm. Next, native-PAGE was performed and NLS-RAR alpha and RXR alpha were revealed to form heterodimers in the nucleus. In addition, co-immunoprecipitation revealed an interaction between NLS-RAR alpha and retinoid X receptor-alpha (RXR alpha). An electrophoresis mobility shift assay (EMSA) indicated that NLS-RAR alpha could bind retinoic acid response elements (RAREs) in the presence of ATRA. Indeed, NLS-RAR alpha could bind RAREs just as WTRAR alpha could, including the RAREs direct repeat-2 (DR-2) and DR-5. In addition, results from a luciferase reporter gene assay demonstrated that NLS-RAR alpha could mediate the activity of RAREs that it bound. Together, these results indicated that NLS-RAR alpha may be a novel transcription factor that contributes to leukemogenesis by competitively binding RAREs as heterodimers with RXR alpha, just as PML-RAR alpha does, thus repressing the gene transcription essential for myeloid differentiation. These findings indicate the potential role of NLS-RAR alpha targeted therapy in APL.