Antioxidant-induced Nuclear Translocation of CCAAT/Enhancer-binding Protein β

Abstract

Antioxidants have been associated with a diminished risk of cancer at various anatomical sites, including the colon (1, 2). The primary mechanism of chemoprevention by antioxidants is through the reduction of DNA-damaging free radicals (3). We have reported that two antioxidants, pyrrolidinedithiocarbamate (PDTC) 1 and vitamin E, induce G 1 cell cycle arrest and apoptosis in various human cancer lines including breast, colon, and lung (4). These cell cycle perturbations were mediated by induction of p21, a powerful inhibitor of the cell cycle, through a mechanism involving activation and binding of C/ EBP␤ to the p21 promoter.C/EBP␤ is a member of a diverse group of nuclear transcription factors that contain a leucine zipper motif required for dimer formation and a basic DNA-binding domain that facilitates interactions between these factors and the regulatory domains of promoters and/or enhancers of target genes (5-11).C/EBP␤ activates several acute phase protein genes through the NF-IL6 responsive elements (8,12) and also has been implicated in adipocyte differentiation and inflammatory and immune responses (13). Thus, C/EBP␤ is a pleiotropic transactivator involved in a myriad of signal transduction and cell differentiation events.Control of C/EBP␤ expression and activity is complex and poorly understood. It is known that C/EBP␤ gene is transcriptionally activated by IL-1 and lipopolysaccharide (5), whereas in other instances its binding to cognate DNA sequences is enhanced by cytokines (5, 11). Additionally, C/EBP␤ can be a target for post-translational modification. Various kinases, including cAMP-dependent protein kinase (PKA) (14), protein kinase C (PKC) (15), a Ras-dependent MAP kinase (16), and a calcium calmodulin-dependent kinase (17) have been shown to phosphorylate C/EBP␤ in vitro. These phosphorylation events modulate DNA binding and transcriptional activity of C/EBP␤. However, it is unknown if the phosphorylation status of C/ EBP␤ influences its subcellular compartmentalization. Elevation of cAMP levels in PC-12 cells or activation of tumor necrosis factor receptors in hepatocytes leads to a redistribution of C/EBP␤ from the cytosolic to nuclear compartment (9, 18). As a first step toward understanding the antioxidant-mediated increase in C/EBP␤ DNA binding activities, we evaluated the effect of the antioxidant PDTC on the post-translational modification and subcellular localization of C/EBP␤ in DKO-1 cells, a human colorectal cancer cell line. Our results indicate that PDTC induces a rapid and sustained translocation of C/EBP␤ protein from the cytoplasm to the nucleus, resulting in induction of p21. Moreover, we demonstrate that these antioxidantmediated events are regulated by PKA-mediated phosphorylation of Ser 299 in C/EBP␤. EXPERIMENTAL PROCEDURESMaterials-DKO-1 cells were obtained from Dr. Takehiko Sasazuki (Department of Genetics, Kyushu University, Fukuoka, Japan). FLAG antibodies were purchased from Amersham, and antibodies raised against p21 and the C/EBP proteins ␣ (14AA), ␤ (C-19), and ...

Regulated Nuclear-Cytoplasmic Localization of CCAAT/ Enhancer-binding Protein δ in Osteoblasts

“…The regulated nuclear import of C/EBP␦ defined here appears to resemble the pathway of nuclear translocation of the related transcription factor, C/EBP␤. As shown by several investigators, C/EBP␤ resided in the cytoplasm in unstimulated cells and accumulated in the nucleus after treatment with agents that activated PKA with kinetics similar to those observed here for C/EBP␦ (28,36). However, C/EBP␤ is a substrate for PKA, and its phosphorylation is required for its nuclear translocation (28).…”

“…C/EBP␦ Is Not a Direct Substrate for PKA in Vitro-The next series of experiments was designed to determine whether C/EBP␦ was phosphorylated by PKA. Other studies have shown that the related transcription factor, C/EBP␤, is a substrate for PKA (27,28), and inspection of the protein sequence of C/EBP␦ revealed several potential PKA phosphorylation sites. To test the hypothesis that C/EBP␦ is a substrate for PKA, recombinant C/EBP␦ was generated in E. coli, purified, and used in in vitro kinase assays with the purified, recombinant catalytic subunit of PKA.…”

Growth Hormone Regulates Phosphorylation and Function of CCAAT/Enhancer-binding Protein β by Modulating Akt and Glycogen Synthase Kinase-3

“…The increased binding associated with dephosphorylation corresponded with the increased LAP binding induced by GH treatment, suggesting that GH-promoted dephosphorylation enhances LAP binding. C/EBP␤ contains multiple phosphorylation sites, including sites for Ras-MAPK (Thr-235), calcium/calmodulin-dependent protein kinase (Ser-276), protein kinase C (Ser-105), and protein kinase A (Ser-105, Ser-173, Ser-233, Ser-299) (21,24,(55)(56)(57). However, several reports indicate that phosphorylation of C/EBP␤ by PKA and/or PKC attenuates site-selective DNA binding (57), whereas others suggest that phosphorylation may increase binding (58).…”

Roles of CCAAT/Enhancer-binding Proteins in Regulation of Liver Regenerative Growth

“…Hence, glucagon and epinephrine, which increase adenylyl cyclase activity and promote the accumulation of hepatic cAMP in the early pre-replicative period, are likely to play a role in the induction of these C/EBP isoforms after PH. The cAMP-dependent kinase, protein kinase A, has also been shown to influence the function of C/EBP␤ protein by phosphorylating Ser 299 (21). Norepinephrine and other ␣-adrenergic agents that promote increases in intracellular calcium and activate calmodulin-sensitive kinases in the regenerating liver may also regulate C/EBP␤ because there is evidence that Cam kinase II phosphorylation of C/EBP␤ alters its DNA binding activity (20).…”