NPM-RAR Enhances NFκB Transcriptional Activity

Abstract 5117

Acute Promyelocytic Leukemia (APL) is characterized by recurrent translocations of 17q21, which result in expression of fusion proteins of the retinoic acid receptor alpha (RARα). We have been studying the t(5;17)(q35;q21) variant of APL, which fuses the N-terminal 117 amino acids of nucleophosmin (NPM) to the C-terminus of RARα. NPM-RAR is capable of binding to retinoic acid response elements (RAREs), and we have previously shown that NPM-RAR can recruit both corepressor and coactivator complexes to retinoic acid target promoters, to modulate transcription in a ligand-dependent fashion, though with a different sensitivity to ligand than RARα. The N-terminal NPM-sequence of NPM-RAR contains a dimerization domain capable of mediating not only homodimerization of the fusion protein, but also heterodimerization with wild-type NPM, to potentially modulate NPM functionality. NPM serves as a molecular chaperone, and binds to a diverse set of proteins. Amongst these are the p50 and p65 subunits of NFκB. NPM has been shown to serve as a coactivator for the NFκB transcriptional complex. We hypothesized that NPM-RAR might interact with NPM to modulate NFκB dependent transcription. We focused on NFκB regulation of the Superoxide Dismutase 2 (MnSOD, SOD2) promoter, since NPM regulation of NFκB mediated transcription has been most extensively studied in this model system. We found that NPM-RAR enhances both basal and TNFα-induced transcription of an NFκB-reporter construct (containing a minimal MnSOD promoter/enhancer) in HepG2 and Hela cells. This effect was not dependent upon translocation of NFκB to the nucleus, indicating that the positive effect on NFκB was mediated downstream of the canonical activation pathway of NFκB. The MnSOD promoter does not contain a consensus RARE, and this effect was not seen in cells transfected with RARα, suggesting that this effect is not mediated through the DNA binding motifs of the NPM-RAR C-terminal RARα sequences. NPM-RAR does not directly interact with NFκB; preliminary experiments using siRNA to down-regulate NPM indicate that NPM-RAR enhancement of MnSOD transcription is dependent upon the presence of wild-type NPM. This leads us to propose a model whereby NPM-RAR binds to the NPM/NFκB transcriptional complex through interaction with NPM, to enhance NFκB mediated transcription.