CCAAT/enhancer-binding protein alpha antagonizes transcriptional activity of hypoxia-inducible factor 1 alpha with direct protein-protein interaction.
Hypoxia-inducible factor 1 (HIF-1), a master heterodimeric transcriptional regulator consisting of HIF-1alpha and HIF-1beta subunits for cellular response to hypoxia, plays an important role in carcinogenesis, while CCAAT/enhancer-binding protein alpha (C/EBPalpha) is proposed to act as a tumor suppressor in C/EBPalpha-expressing tissues. Previously, we reported that ectopically expressed HIF-1alpha protein interacts with and enhances transcriptional activity of C/EBPalpha, 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/EBPalpha and the basic helix-loop-helix domain of HIF-1alpha were essential for such an interaction. More intriguingly, we provided the first demonstration that C/EBPalpha competed with HIF-1beta for direct binding to HIF-1alpha protein. Correspondingly, C/EBPalpha 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/EBPalpha 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/EBPalpha protein.
Yang
L
L
Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences-Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Jiang
Y
Y
Wu
S F
SF
Zhou
M Y
MY
Wu
Y L
YL
Chen
G Q
GQ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism / Q000502:physiology*; D019556:COS Cells; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012313:RNA / Q000378:metabolism; D015533:Transcriptional Activation; D020298:U937 Cells
D000970:Antineoplastic Agents; D022763:CCAAT-Enhancer-Binding Protein-alpha; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012313:RNA; D004247:DNA