Transcriptional activity analysis of p65(-1), a new p65 isoform of NF-kB complex.

Nuclear Factor-?B (NF-?B) are ubiquitous transcription factors (TF) that exercise a variety of crucial physiological functions by binding as homodimers or heterodimers to specific DNA target sites. In basal conditions, NF-?B is localized in the cytoplasm complexed with its inhibitor IkB. Upon receipt of a specific signal, NF-kB is released from IkB and translocates to the nucleus. The NF-kB/Rel family members include p65, p50/p105, p52/p100, c-Rel and RelB proteins. These proteins are characterized by the Rel Homology Domain (RHD), an N-terminal region in which lie the dimerization, nuclear-localization and DNA-binding domains. In mouse and human a new alternative splicing form of p65, named p65(-1), has been reported. This new isoform contains a previously unknown exon, (named exon -1) located upstream to the first known exon of p65 (exon 0), and is detected mainly in the brain. Transcription of the exon -1 leads to an alternative splicing between exon -1 and exon 1, thus skipping exon 0. p65(-1) binds DNA through canonical NF-kB responsive elements, but in contrast to p65 activates transcription at very low levels. NF-kB plays an important role in modulating biological pathways of other transcription factors as AP1, GR and CREB. All these TFs are involved in fundamental cellular mechanisms as stress response, cell proliferation, inflammation, differentiation, apoptosis and oncogenesis. Previous studies demonstrated that p65(-1) enhances transcriptional activity of glucocorticoids receptors (GR) upon treatment with the selective GR agonist dexamethasone. In order to study the mechanism of action of p65(-1) on TFs as AP1 and CREB, we performed transfection experiments on HeLa cells. For this purpose, HeLa cells were cotransfected with a luciferase reporter gene driven by a specific consensus sequence for the TF of interest and with a plasmid constitutively expressing either p65(-1) or p65. Our results show that p65(-1), compared to p65, has different biochemical properties. In fact, p65(-1) can regulate the transcriptional level of the reporter driven by specific consensus sequences. These results highlight the important role of p65(-1) in regulating different pathways. It could furthermore explain the complex and sometime opposite functions attributed to NF-kB in the brain.