Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPβ and two uncharacterized protein complexes to the C/EBP–ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP–ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP–ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP–ATF composite site.

gaddl53 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gaddl53 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease ofgaddl53 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBPat appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gaddl53 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBPoa and gaddl53 mRNAs were elevated. However, C/EBPa induction occurred on day 3, while gaddl53 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gaddl53 expression without preventing C/EBPa expression or interfering with cellular differentiation. Thus, gaddl53 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell. gaddl53 is a mammalian gene whose expression is induced by a wide variety of stresses, including growth arrest and DNA damage (16,20,21). gaddlS3 encodes a 19-kDa protein related to the CCAAT/enhancer-binding protein (C/ EBP) family of transcription factors (28). The C/EBPs are bZip proteins characterized by a conserved leucine zipper domain, through which various C/EBP monomers dimerize, adjacent to a basic domain involved in DNA sequence recognition and binding. Although the gaddl53 protein forms stable heterodimers with other C/EBP proteins, it lacks a functional DNA-binding domain (28). Thus, the gaddlS3 protein-C/EBP heterodimer is unable to bind known C/EBP-binding sites, suggesting that gaddlS3 may function as a negative regulator of C/EBP transcription factors.It has been hypothesized that C/EBPa is a central regulator of genes encoding proteins involved in energy metabolism (22). This proposal is based on the following observations: levels of C/EBPa expression are high in tissues with high rates of metabolism, such as liver, adipose, and placenta; the developmental pattern of C/EBPot expression in the liver coincides with the onset of gluconeogenic activity; C/EBPa can trans activate the promoters of genes involved in energy metabolism; and livers of cl4CoS/clCS mice show reduced levels of C/EBPa expression. These mutant mice, containing radiation-induced deletions involving the albino locus on chromo...

Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site.