Abstract
BackgroundRecently, we established that a C>T single nucleotide polymorphism (SNP) in the promoter of the VEGF receptor FLT1 gene generates a ½ site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required an estrogen receptor (ER) ½ site response element (ERE1) 225 nt upstream to the RE-T.Methodology/Principal FindingsHere we report the identification of a second ER ½ site (ERE2) located 145 bp downstream of the RE-T and establish that both EREs can impact p53-mediated transactivation of FLT1-T in a manner that is cell type and ER level dependent. Gene reporter assays and ChIP experiments conducted in the breast cancer-derived MCF7 cells revealed that the ERE2 site was sufficient for p53-mediated ERα recruitment and transactivation of the FLT1-T promoter/reporter construct. Surprisingly, unlike the case for other p53 target promoters, p53-mediated transactivation of FLT1-T constructs or expression of the endogenous FLT1 gene, as well as binding of p53 and ER at the promoter constructs, was inducible by doxorubicin but not by 5-fluorouracil. Furthermore, ER activity at FLT1-T was differentially affected by ER ligands, compared to a control TFF1/pS2 ER target promoter. The p53-related transcription factors (TFs) p73 and p63 had no effect on FLT1 transactivation.Conclusions/SignificanceWe establish a new dimension to the p53 master regulatory network where p53-mediated transcription from a ½ site RE can be determined by ER binding at one or more cis-acting EREs in manner that is dependent on level of ER protein, the type of ER ligand and the specific p53-inducing agent.
Highlights
The Vascular Endothelial Growth Factor Receptor-1 (VEGFR1), commonly known as FLT1, is a high affinity VEGF receptor belonging to the VEGFR transmembrane receptor tyrosin kinase family, expressed in a variety of cell types, including endothelial cells, hematopoietic stem cells, leucocytes, and osteoblasts [1]
To understand better the cooperativity between p53 and estrogen receptor (ER) in the regulation of FLT1 promoter and the response to genotoxic stress, we evaluated the role of ERs in the p53-dependent transactivation of the FLT1 using a clone of the p53 wild-type breast adenocarcinoma MCF7 cell line which is positive for ERa and has low ERb expression
While in HCT116 cells the disruption of the 2902 Estrogen Receptor response element (ERE) K site (i.e., ‘‘ere1’’) was sufficient to nearly abolish the transactivation from a 1 kb FLT1-T promoter construct [17], there was no impact of the disruption in MCF7 cells (Figure 1A)
Summary
The Vascular Endothelial Growth Factor Receptor-1 (VEGFR1), commonly known as FLT1, is a high affinity VEGF receptor belonging to the VEGFR transmembrane receptor tyrosin kinase family, expressed in a variety of cell types, including endothelial cells, hematopoietic stem cells, leucocytes, and osteoblasts [1]. The FLT1 protein, which possesses higher affinity for VEGFA but weak tyrosine kinase activity, or none at all in the case of the soluble form (sFLT1), can act as inhibitory or decoy [2] to the FLK1/VEGFR-2 receptor. The latter binds to VEGFA and represents a primary driver of angiogenesis in development and healthy conditions. We established that a C.T single nucleotide polymorphism (SNP) in the promoter of the VEGF receptor FLT1 gene generates a K site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required an estrogen receptor (ER) K site response element (ERE1) 225 nt upstream to the RE-T
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