Induction of the Mouse Serum Amyloid A3 Gene by Cytokines Requires both C/EBP Family Proteins and a Novel Constitutive Nuclear Factor

Abstract

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1,000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We have previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene was necessary and sufficient to confer liver-specific and cytokine-induced expression. Deletion studies identified a distal response element that is responsible for the cytokine response and has properties of an inducible transcriptional enhancer. In this study, we further analyzed the distal response element and showed that it consists of three functionally distinct elements: the A element constitutes a weak binding site for C/EBP family proteins, the B element also interacts with C/EBP family proteins but with a much higher binding affinity, and the C element interacts with a novel constitutive nuclear factor, SEF-1. Site-specific mutation studies revealed that all three elements were required for maximum promoter activity. C/EBPα, C/EBPβ, and C/EBPδ were capable of interacting with elements A and B. Under noninduced conditions, C/EBPα was the major binding factor; however, upon cytokine stimulation C/EBPβ- and C/EBPδ-binding activities were dramatically increased and became the predominant binding factors. Consistent with these binding studies were the cotransfection experiments in which C/EBPβ and C/EBPδ were shown to be potent transactivators for the SAA3 promoter. Moreover, the transactivation required an intact B element despite the presence of other functional C/EBP-binding sites. Interestingly, although element C did not interact with C/EBP directly, it was nevertheless required for maximum transactivation by C/EBPδ. Our studies thus demonstrate that both C/EBP family proteins and SEF-1 are required to transactivate the SAA3 gene.