JOURNAL ARTICLE

Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia

S K Bae, M H Bae, M Y Ahn, M J Son, Y M Lee, M K Bae, O H Lee, B C Park, K W Kim
Cancer Research 1999 December 1, 59 (23): 5989-94
10606246
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.

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