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 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the alpha(2)-macroglobulin and Aalpha-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes.
Highlights
The defense processes initiated in most vertebrates after infection or tissue injury are termed the acute-phase response [1]
We demonstrated that distal response element (DRE) consists of three functionally distinct elements: the A element, a weak binding site for C/EBP family proteins; the B element, which interacts with C/EBP family proteins but with a much higher affinity; and the C element that interacts with a constitutive nuclear factor, which was named SAA3 enhancer factor (SEF)
We previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene was necessary and sufficient to confer cytokine-induced expression in hepatoma cells [19]
Summary
Vol 274, No 35, Issue of August 27, pp. 24649 –24656, 1999 Printed in U.S.A. (Received for publication, February 25, 1999, and in revised form, May 14, 1999). In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines.
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