Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response.
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.
An
M R
MR
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, 77555, USA.
Hsieh
C C
CC
Reisner
P D
PD
Rabek
J P
JP
Scott
S G
SG
Kuninger
D T
DT
Papaconstantinou
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects*; D007106:Immune Sera; D015151:Immunoblotting; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D009961:Orosomucoid / Q000096:biosynthesis / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007106:Immune Sera; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D009961:Orosomucoid; D010446:Peptide Fragments; D012333:RNA, Messenger; D014157:Transcription Factors