Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress.
GADD153, a ubiquitously expressed member of the CCAAT/enhancer-binding protein (C/EBP) family is induced by a wide variety of growth-arresting and DNA-damaging agents. Functionally, GADD153 has been postulated to act as a dominant-negative regulator of C/EBPs. Therefore we sought to gain evidence for interactions between GADD153 and other C/EBPs during cellular responses to stress. In this report we have demonstrated that treatment of rat pheochromocytoma PC12 cells with sodium arsenite leads to enhanced expression of C/EBP-beta and GADD153 (growth arrest and DNA damage inducible gene 153) but not other C/EBPs. Coimmunoprecipitation experiments provided evidence for the formation of endogenous GADD153-C/EBP-beta complexes in arsenite-treated cells. Additional experiments were performed to determine the role of such complexes in regulating GADD153 expression. Previous studies in our laboratory demonstrated that the GADD153 promoter contains a C/EBP binding site through which other C/EBPs interact to transactivate GADD153 expression in liver hepatoma cells. Here, we demonstrate that extracts prepared from arsenite-treated PC12 cells likewise show increased amounts of factors capable of binding to the GADD153-C/EBP site and that these complexes are comprised at least in part of C/EBP-beta. Forced expression of C/EBP-beta was found to be capable of transactivating the GADD153 promoter in PC12 cells cotransfected with plasmids expressing a GADD153 reporter gene and C/EBP-beta protein. However, overexpression of GADD153 inhibited the transactivation of the GADD153 promoter by C/EBP-beta. These findings provide evidence for an autoregulatory loop in which stress-induced GADD153 feeds back to attenuate GADD153 expression during the cellular response to stress.
Fawcett
T W
TW
Gene Expression and Aging Section, NIA, National Institutes of Health, Baltimore Maryland 21224, USA.
Eastman
H B
HB
Martindale
J L
JL
Holbrook
N J
NJ
D000310:Adrenal Gland Neoplasms; D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D016384:Consensus Sequence; D006224:Cricetinae; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D010673:Pheochromocytoma; D011401:Promoter Regions, Genetic; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013312:Stress, Physiological; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D018053:Arsenites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C497251:Ddit3 protein, rat; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051742:Transcription Factor CHOP; C015001:arsenite