Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response

Abstract

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.