NFkappa B Interacts with Serum Amyloid A3 Enhancer Factor to Synergistically Activate Mouse Serum Amyloid A3 Gene Transcription

We had previously identified a distal regulatory element (DRE) in the mouse serum amyloid A3 (SAA3) promoter that functions as a cytokine-inducible transcription enhancer. Within this DRE, three functional elements interact with CCAAT/enhancer-binding protein (C/EBP) and SAA3 enhancer factor (SEF) transcription factors. In this study, we show that cotransfection of the SEF expression plasmid with an SAA3/luciferase reporter resulted in 3-5-fold activation of the SAA3 promoter. When SEF-transfected cells were further stimulated with conditioned medium or interleukin-1, SAA3 promoter activity was dramatically increased, suggesting that SEF may cooperate functionally with other interleukin-1-inducible transcription factors to synergistically up-regulate SAA3 gene transcription. Indeed, cotransfection of SEF and NFkappaBp65 expression DNAs resulted in synergistic activation of the SAA3 promoter. Intriguingly, no consensus NFkappaB-binding site was found in the SAA3 promoter region; rather a putative NFkappaB-binding sequence with 3-base pair mismatches was identified in the DRE. When this sequence was used in an electrophoretic mobility shift assay, it interacted with NFkappaBp50, albeit with binding affinities that were several hundredfold lower than that with the consensus NFkappaB probe. Functional cooperation between SEF and NFkappaB was further strengthened by the finding that SEF and NFkappaB formed stable cytokine-inducible protein-protein complexes. Finally, despite its weak binding, mutation of this NFkappaB-binding site nevertheless dramatically reduced both NFkappaBp65- and cytokine-mediated induction of SAA3 promoter. Therefore, the molecular basis for the functional synergy between SEF and NFkappaB may, in part, be the ability of SEF to recruit NFkappaB through physical interactions that lead to enhancement or stabilization of NFkappaB binding to the SAA3 promoter element.