Egr-1 is not upregulated in response to hypoxic and oxygenation conditions in human glioblastoma in vitro

Abstract

The early growth response factor 1 (Egr-1) gene (also known as krox24, NGFI-A, TIS8 or zif268) belongs to a family of immediate early response genes. This family of proteins contains a conserved zinc finger DNA-binding domain and can bind to a GC-rich sequence in the promoter region of target genes. Egr-1 expression is rapidly and transiently activated in many different cell types during development. In adult tissues, a variety of signals, including serum, growth factors, cytokines and hormones, stimulate Egr-1 expression. In several studies, it was demonstrated that the transcription factor Egr-1 is regulated by hypoxia, and it is hypothesized that Egr-1 is responsible for the hypoxia-induced regulation of the N-Myc downregulated gene 1 (NDRG1) in human tumor cells. In the present study, Egr-1 regulation was examined in the human glioblastoma cell lines U373, U251, GaMG and U87-MG under extreme hypoxic aeration conditions (0.1% O2) for 1, 6 and 24 h, 24-h extreme hypoxia with reoxygenation for 24 and 48 h, respectively, as well as oxygenated conditions (21% O2 and 5% CO2) in vitro. Protein and mRNA levels were detected in the lysates by Western blotting and RT-PCR, respectively. Egr-1 expression under hypoxic conditions was compared with the well-known and characterized hypoxia-induced gene regulator hypoxia-inducible factor-1α (HIF-1α) in parallel experimental sets. Cells incubated for 24 h with 100 µM desferroxamine served as a positive control for hypoxia, and β-tubulin and β-actin were used as loading controls. The experimental data indicate that Egr-1 was not upregulated under extreme hypoxic conditions (0.1% O2) or by reoxygenation after hypoxia in different glioblastoma cells in vitro. In conclusion, the regulation of Egr-1 in reaction to hypoxic development, at both the protein and mRNA levels, is not a general phenomenon. In contrast to previously published data, no Egr-1 regulatory events were observed in glioblastoma under hypoxic conditions in vitro. We suggest that Egr-1 regulation in human tumors in reaction to hypoxia could be a cell-specific post-translational event. Therefore, at least in glioblastoma, HIF-1α can be considered a major regulator of NDRG1 under hypoxic conditions. Further extensive analysis of tumor cells from different origins under similar physiological conditions is necessary to increase our knowledge of the conditions and functional role of Egr-1 in the regulation of hypoxia-induced gene expression.