title: Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia.
abstract: We have previously reported that the exposure of human HepG2 cells to hypoxic conditions results in the overexpression of human insulin-like growth factor II (IGF-II) mRNA whose size is 6.0 kb. This particular size of IGF-II mRNA is transcribed under the control of the IGF-II P3 promoter. In the present study, to delineate the molecular mechanism for the activation of the IGF-II gene, we examined the induction of P3 promoter activity in HepG2 cells by hypoxia in the transient expression system. In this system, hypoxia induced a linear increase within 24 h in the expression of luciferase that was driven by the IGF-II P3 promoter. To further delineate which factors mediate this response, the expression pattern of regulators of the P3 promoter, Egr-1, Sp1, and WT1, were analyzed by reverse transcription-PCR and Northern blot analysis. We found that hypoxia increased the expression of Egr-1 but not of Sp1. In contrast, the level of WT1, a repressor of IGF-II expression, was markedly decreased during hypoxia. The mRNA stability assay revealed that the induction of transcription is the mechanism of underlying Egr-1 mRNA elevation. We then investigated the effects of hypoxia on the DNA binding activity of Egr-1. Both electrophoretic mobility shift assay and supershift assay demonstrated that the DNA binding activity of the Egr-1 protein was increased by hypoxia. In addition, the level of Egr-1 protein was also increased under the hypoxia as determined by Western blot analysis. Cotransfection of HepG2 cells with an Egr-1 expression vector and an IGF-II P3 promoter-luciferase reporter plasmid showed that the transcription of IGF-II was activated by Egr-1 in a dose-dependent manner. Moreover, the elevation of IGF-II P3 promoter activity was induced synergistically by the cotreatment of hypoxia with Egr-1 overexpression. Deletion of sequences in the IGF-II P3 promoter containing Egr-1 binding sites did not respond to hypoxic stress. Taken together, these data strongly indicate that hypoxia-induced IGF-II expression in HepG2 cells is due to the enhanced activity of Egr-1 on the IGF-II P3 promoter and that the Egr-1 binding site in the IGF-II P3 promoter is essential for the transcriptional regulation of IGF-II under hypoxic conditions.
authors.affiliation: Department of Molecular Biology, Pusan National University, Korea.
authors.forename: S K
authors.initials: SK
authors.lastname: Bae
authors.forename: M H
authors.initials: MH
authors.lastname: Bae
authors.forename: M Y
authors.initials: MY
authors.lastname: Ahn
authors.forename: M J
authors.initials: MJ
authors.lastname: Son
authors.forename: Y M
authors.initials: YM
authors.lastname: Lee
authors.forename: M K
authors.initials: MK
authors.lastname: Bae
authors.forename: O H
authors.initials: OH
authors.lastname: Lee
authors.forename: B C
authors.initials: BC
authors.lastname: Park
authors.forename: K W
authors.initials: KW
authors.lastname: Kim
mesh_terms: D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008113:Liver Neoplasms; D008156:Luciferases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D007335:Insulin-Like Growth Factor II; D008156:Luciferases