An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene.
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.
Ron
D
D
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
Brasier
A R
AR
Wright
K A
KA
Tate
J E
JE
Habener
J F
JF
D000210:Acute-Phase Reaction; D000808:Angiotensinogen / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005796:Genes; D066298:In Vitro Techniques; D007249:Inflammation; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000502:physiology; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D014157:Transcription Factors; D000808:Angiotensinogen; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate