Carcinogenesis Advance Access originally published online on November 16, 2007
Carcinogenesis 2008 29(2):291-298; doi:10.1093/carcin/bgm262
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CCAAT/enhancer-binding protein 
antagonizes transcriptional activity of hypoxia-inducible factor 1 with direct protein–protein interaction
1 Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences–Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
* To whom correspondence should be addressed. Tel: +86 21 63846590 ext. 776573; Fax: +86 21 64154900; Email: chengq{at}shsmu.edu.cn
Hypoxia-inducible factor 1 (HIF-1), a master heterodimeric transcriptional regulator consisting of HIF-1
and HIF-1β subunits for cellular response to hypoxia, plays an important role in carcinogenesis, while CCAAT/enhancer-binding protein (C/EBP) is proposed to act as a tumor suppressor in C/EBP-expressing tissues. Previously, we reported that ectopically expressed HIF-1 protein interacts with and enhances transcriptional activity of C/EBP, which favors leukemic cell differentiation. Here we further showed that such an interaction also occurred in their endogenously expressing state of leukemic U937 cells. Glutathione S-transferase pull-down assay proposed that the protein–protein interaction was direct, and transactivation domains of C/EBP and the basic helix-loop-helix domain of HIF-1 were essential for such an interaction. More intriguingly, we provided the first demonstration that C/EBP competed with HIF-1β for direct binding to HIF-1 protein. Correspondingly, C/EBP overexpression significantly inhibited the DNA-binding ability of HIF-1 and expressions of hypoxia-responsive element-driven luciferase and HIF-1-targeted genes vascular endothelial growth factor, glucose transporter-1 and phosphoglycerate kinase 1. In parallel, suppression of C/EBP expression by specific small hairpin RNA increased DNA-binding ability of HIF-1 and expression of these HIF-1-targeted genes in leukemic U937 cells. These results would provide new insights for antitumor potential of C/EBP protein.
Abbreviations: bHLH, basic helix-loop-helix; ChIP, chromatin immunoprecipitation; Glut-1, glucose transporter-1; GST, glutathione S-transferase; HIF-1, hypoxia-inducible factor 1; PAS, Per-Arnt-Sim; PGK1, phosphoglycerate kinase 1; PCR, polymerase chain reaction; shRNA, short hairpin RNA; TAD, transactivation domain
Received August 2, 2007; revised November 5, 2007; accepted November 10, 2007.