Expression of rat serum amyloid A1 gene involves both C/EBP-like and NF kappa B-like transcription factors

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. During inflammation, its expression is increased by 1000-fold as the result of greatly increased gene transcription. In this study, we analyzed the cis-acting regulatory elements and trans-acting factors important for the expression of the rat SAA1 gene. A DNA fragment containing 304 base pairs (bp) of 5'-flanking sequences of the SAA1 gene was fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and the resulting construct, pSAA1/CAT (-304), was used to assess the function of the 5'-flanking sequences by transient transfection assay. pSAA1/CAT (-304) was not expressed or expressed at very low levels in both the liver- and nonliver-derived cells. However, when stimulated with conditioned medium prepared from mixed lymphocyte cultures, recombinant interleukin 1, or 12-O-tetradecanoylphorbol-13-acetate, expression of the pSAA1/CAT (-304) hybrid gene was induced 15-20-fold, but only in liver-derived cells. Further functional analysis demonstrated that a 66-bp DNA fragment conferred cytokine responsiveness onto a heterologous thymidine kinase promoter both in liver and nonliver cells. Footprint analysis with the Hep3B nuclear proteins revealed four protected regions in the 5'-flanking region of the SAA1 gene. The pattern of protection was identical with nuclear extracts prepared from either unstimulated or conditioned medium-treated Hep3B cells. Two of these footprint regions were identified as binding sites for C/EBP or C/EBP-related proteins, with the distal region having about 10-fold higher binding affinity than the proximal region. One additional cis-element formed a specific protein-DNA complex only with the nuclear proteins from TPA- or conditioned medium-treated Hep3B cells. This cis-element shares sequence identity with nuclear factor NF kappa B binding sites. The finding of a NF kappa B binding site within the 66-bp cytokine-responsive fragment further suggests its functional importance in the regulation of SAA1 gene expression. Our results suggest that C/EBP- and NF kappa B-related proteins may be important regulatory factors that contribute both to tissue specificity and to the high rate of SAA transcription in response to inflammatory mediators.