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. 1990 Mar;10(3):1023–1032. doi: 10.1128/mcb.10.3.1023

An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene.

D Ron 1, A R Brasier 1, K A Wright 1, J E Tate 1, J F Habener 1
PMCID: PMC360957  PMID: 2106065

Abstract

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.

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