INDUCTION OF THE MOUSE SERUM AMYLOID A3 GENE BY CYTOKINES REQUIRES BOTH C EBP FAMILY PROTEINS AND A NOVEL CONSTITUTIVE NUCLEAR FACTOR/

Serum amyloid A (SAA) is a major acute-phase protein synthesized and s ecreted mainly by the liver. In response to acute inflammation, its ex pression may be induced up to 1,000-fold, primarily as a result of a 2 00-fold increase in the rate of SAA gene transcription. We have previo usly demonstrated that a 350-bp promoter fragment from the mouse SAAS gene was necessary and sufficient to confer liver-specific and cytokin e-induced expression. Deletion studies identified a distal response el ement that is responsible for the cytokine response and has properties of an inducible transcriptional enhancer. In this study, we further a nalyzed the distal response element and showed that it consists of thr ee functionally distinct elements: the A element constitutes a weak bi nding site for C/EBP family proteins, the B element also interacts wit h C/EBP family proteins but with a much higher binding affinity, and t he 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/EBP alpha, C/EBP beta, and C/EBP delta were capable of interacting with elements A and B. Under n oninduced conditions, C/EBP alpha was the major binding factor; howeve r, upon cytokine stimulation C/EBP beta- and C/EBP delta-binding activ ities mere dramatically increased and became the predominant binding f actors. Consistent with these binding studies were the cotransfection experiments in which C/EBP beta and C/EBP delta were shown to be poten t 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 intera ct with C/EBP directly, it was nevertheless required for maximum trans activation by C/EBP delta. Our studies thus demonstrate that both C/EB P family proteins and SEF-1 are required to transactivate the SAA3 gen e.