Abstract
alpha 1-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/EBP alpha, C/EBP beta, and C/EBP delta, bind to the APRE. Furthermore, with liver nuclear protein from control animals, C/EPB alpha is the predominant form that binds to the APRE, whereas with nuclear proteins from acute phase-induced animals, C/EBP alpha is replaced by C/EBP beta. The mechanism of activation of the AGP gene during the acute phase response appears to involve an exchange of C/EBP alpha by C/EBP beta. C/EBP delta 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/EBP alpha 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/EBP alpha, C/EBP beta, and C/EBP delta are strong transcriptional trans-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.
Highlights
EBPa is the predominant formthat binds to thAe PRE, whereaswithnuclearproteinsfromacute phaseinduced animals, C/EBPa is replaced by C/EBPB
We describe functional analyses, which demonstrate that C/EBPa, CIEBPP, and C/ EBPG are strong transcriptional trans-activators of the Acid glycoprotein (AGP) gene, acute phase responsive element (APRE)
Theformation of the four complexes was sequencespecific sincebinding was totally abolished by a 100-fold molar excess of the wild-type APRE but not a 100-fold molar excess of themutatedAPRE(mt-I).Thesedata strongly suggest that multiple CCAAT/enhancerbinding protein (C/EBP)-like proteins can interact with a common DNA binding domain
Summary
One of the three mouse AGP genes, AGP-1, hasbeen cloned, andits5”flanking region was identifiedand sequenced (Cooper et al, 1987). Liver nuclear extracts from mals formed four complexes control and (Cl, C2, C3, LPS-stimulated aniand C4) with the AGP APRE (wt)but not with the AI’RE ( m t - 1 )in which the potential C/EBP binding site has been mutated (mutated sequence is shown under “Materials and Methods”)C. ompetition assays were performed. Theformation of the four complexes was sequencespecific sincebinding was totally abolished by a 100-fold molar excess of the wild-type APRE (wt) but not a 100-fold molar excess of themutatedAPRE(mt-I).Thesedata strongly suggest that multiple C/EBP-like proteins can interact with a common DNA binding domain. Purified C/EBPa bound avidly to the wild-type APRE, but binding was completely abolished when its cognate site was mutated (Fig. 4, lane 2). Titration experiments with hGR andC/EBPaalso suggested thatthese two transcription
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