Microarray Analysis of the Overlap Between Genes Known to Be Induced by Hypoxia and/or Hypoxia-inducible Factor 1α (HIF-1α) and Genes Rapidly Induced by Estrogen in Rat Uterine Epithelial Cells.

Abstract

Estrogen is the master regulator of gene transcription in the uterine endometrium, acting primarily through its α receptor (ERα). Recent work in our laboratory identified hypoxia-inducible factor 1α (HIF-1α) as a transcription factor which, along with ERα, is necessary for 17β-estradiol (E2)-induced vascular endothelial growth factor (VEGF) expression in the uterus. VEGF causes increased microvascular permeability and edema in the endometrium, which is necessary for subsequent cell proliferation, angiogenesis, and implantation. HIF-1α is normally present in the uterus at low levels and is recruited to the VEGF promoter by E2 via activation of the PI3K/Akt pathway. HIF-1α is also known to regulate the transcription of over 50 other genes in various cell types, many of which are also induced by E2 in the uterus, including most of the glycolytic enzymes, Glut-1, erythropoietin, adrenomedulin, and leptin, in addition to VEGF. Therefore, we hypothesize that E2-induced expression of many genes will involve HIF-1α. To better understand the extent of the overlap between E2- and hypoxia/HIF-1-regulated genes, we used a microarray specific for genes known to be induced by hypoxia and/or to be regulated by HIF-1α (SuperArray Rat Hypoxia Signaling Pathway Oligo GEArray) to determine how many are also induced by E2 in the uterus. Immature female rats were treated with E2 for 0, 1, or 4 h, and the luminal epithelial (LE) cells isolated. Total RNA was extracted and amplified. A biotin tag was attached, and the samples were then hybridized to the array. Of the 114 genes represented, 11 were significantly up-regulated by E2 by 1 and/or 4 h. There was little or no hybridization with the majority of the genes, however, including VEGF, which RT-PCR analysis showed was strongly induced by E2 in the same samples. This indicated that the methodology may be too insensitive to detect many of the target genes, even after E2 stimulation. Nevertheless, increases in the expression of several genes known to be induced by E2 in the uterus were detected, including fos, the glycolytic enzymes enolase, aldolase, and lactate dehyrogenase A, and interleukin 6. In addition, several other genes not previously reported to be controlled by E2 in the uterus were significantly induced. Two of these the transcriptional regulators retinoic acid receptor α (RARα) and inhibitor of DNA binding 1 (Id-1) are of particular interest. RARα was the most strongly up-regulated gene (22-fold). This suggests that it may play an important role in the regulation of gene transcription by E2. Inhibitor of DNA binding 1 (Id-1), which was recently shown to play a role in angiogenesis, was up-regulated 5- and 7-fold by 1 and 4 h, respectively. RT-PCR analysis of both E2-treated rat LE cells and human endometrial cancer (ECC-1) cells was done to confirm these results. These findings support our hypothesis that many of the genes that play a role in cellular adaptations to hypoxia are also required for E2-induced endometrial growth. Future studies will determine exactly how ERα and HIF-1α interact to regulate the expression of this cohort of genes. This work was supported by the NIH/NHLBI-sponsored Institutional Training Grant in Cardiac and Vascular Cell Biology (HL72751) and the NICHD U54 Cooperative Centers Program (HD36207).