Constitutive and NF-κB—like proteins in the regulation of the serum amyloid a gene by interleukin 1

Serum amyloid A (SAA) is a major acute-phase protein whose chronic production by the liver can lead to the fatal disorder of secondary amyloidosis. Control of SAA is mediated by several inflammatory cytokines, including interleukin 1 (IL-1). To study the cis-acting regulatory elements responsible for constitutive and IL-1—induced expression, DNA constructs containing varying lengths of the promoter region from the human SAA2β gene 5′ to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT), were generated and transfected into human hepatoma cells, HepG2. Both positive and negative regulatory elements were found in the 5′ flanking region of the human SAA2β gene. The more proximal region contains an IL-1 enhancer sequence GGGACTTTCC (SAAκB1; between −82 and −91), the binding site for the ubiquitous transcription factor NF-κB. IL-1 induction of the binding of nuclear factor to this sequence is maximal between 5 min and 30 min after incubation with IL-1 and negative in cells incubated for 60 min or longer. Mutation of the SAAκB1 sequence to a nonbinding form of NF-κB (CTCACTTTCC) abolishes the IL-1 effect. The SAA 5′ region also contained an upstream repressor element, shown by transfection experiments. Within this element, a second NF-κB binding site (SAAκB2; −626 to −635) was found, and mutation of SAAκB2 to a non—NF-κB-binding form results in an increase in both constitutive + IL-1 stimulated SAA transcription. Footprint analysis showed the presence of a constitutive nuclear factor(s) binding to sequences containing a C/EBP motif immediately 3′ to SAAκB2. This protein is displaced by a nuclear factor binding to SAAκB2 on stimulation with IL-1.