trans-activation of the α₁-acid glycoprotein gene acute phase responsive element by multiple isoforms of C/EBP and glucocorticoid receptor

α₁-Acid glycoprotein (AGP) is a major acute phase protein synthesized primarily by the liver. The AGP gene is transcriptionally activated in hepatocytes during the acute phase response to bacterial lipopolysaccharide. In this study, we analyzed an acute phase responsive element (APRE) located between nucleotide residues -127 to -104 relative to the transcription initiation site of the mouse AGP gene. Binding studies show that several trans-acting factors interact with the APRE. Using monospecific antibodies we demonstrate that three isoforms of the CCAAT/enhancer-binding protein (C/EBP) family, namely C/EBPα, C/ EBPβ, and C/EBPδ, bind to the APRE. Furthermore, with liver nuclear protein from control animals, C/ EBPα is the predominant form that binds to the APRE, whereas with nuclear proteins from acute phase-induced animals, C/EBPα is replaced by C/EBPβ. The mechanism of activation of the AGP gene during the acute phase response appears to involve an exchange of C/EBPα by C/EBPβ. C/EBPδ does not play a role in this reaction. Interestingly, the C/EBP binding site of the APRE partially overlaps a functional glucocorticoid responsive element. We present evidence that both purified C/EBPα and glucocorticoid receptor bind strongly to the APRE. By site-specific mutation, we have identified the C/EBP and glucocorticoid receptor binding sites in the APRE. These mutants were used in expression vectors to demonstrate that both C/EBP and glucocorticoid receptor are essential for maximal response to interleukin-6 and dexamethasone. These results demonstrate that the APRE is a composite binding site for multiple factors that are responsible for the transcriptional control of the mouse AGP. Finally, functional analyses indicate that C/EBPα, C/EBPβ, and C/EBPδ are strong transcriptional irons-activators of the AGP APRE in hepatoma cells. These data suggest that the regulatory activity of the C/EBP with the APRE in the liver may require interactions with adjacent proteins.