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PMID: 18052938 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Deprivation of protein or amino acid induces C/EBPbeta synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription.

The Biochemical journal ·Vol. 410 ·No. 3 ·2008-03-15 ·Pages 473-84

Thiaville MM, Dudenhausen EE, Zhong C, Pan YX, Kilberg MS

Abstract

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

MeSH Terms
Amino Acids/administration & dosage Animals Base Sequence Blotting, Western CCAAT-Enhancer-Binding Protein-beta/biosynthesis,metabolism Cell Nucleus/metabolism Cells, Cultured Chromatin Immunoprecipitation DNA Primers Dietary Proteins/administration & dosage Electrophoretic Mobility Shift Assay Immunohistochemistry Liver/metabolism RNA, Small Interfering/genetics Rats Rats, Sprague-Dawley Repressor Proteins/biosynthesis,metabolism Transcription, Genetic
Chemicals
Amino Acids CCAAT-Enhancer-Binding Protein-beta DNA Primers Dietary Proteins RNA, Small Interfering Repressor Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Thiaville Michelle M
Department of Biochemistry and Molecular Biology, Center for Nutritional Sciences, and Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Dudenhausen Elizabeth E
Zhong Can
Pan Yuan-Xiang
Kilberg Michael S
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
1470-8728
DOI
10.1042/BJ20071252
Published
2008-03-15
Pages
473-84
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC3589902
Subset
IM
Grants
NIDDK NIH HHS · R01 DK052064 · United States
NIDDK NIH HHS · R01 DK052064-13 · United States
NIDDK NIH HHS · R01 DK070647 · United States
NIDDK NIH HHS · DK-70647 · United States
External Links
PubMed source DOI: 10.1042/BJ20071252 PMC Full Text View journal details
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