Regulation of vascular endothelial growth factor receptor-2 in pancreatic and breast cancer cells by Sp proteins

摘要

Vascular endothelial growth factor receptor-2 (VEGFR2) is a keyangiogenic factor, and angiogenesis is an important physiological processassociated with neovascularization, growth, and metastasis of many differenttumors. The mechanism of VEGFR2 gene expression was investigated inMiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with aseries of VEGFR2 promoter deletion/mutated constructs, and the resultsindicated that the GC-rich ??????60 to ??????37 region of the promoter was essential forVEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Spproteins are expressed and bind to the proximal GC-rich region of the VEGFR2promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3,and Sp4 all contributed to VEGFR2 gene/protein expression in pancreaticcancer cells.VEGFR2 gene expression was also investigated in ZR-75 and MCF-7breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increasedVEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was inducedby E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSAand ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed inZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter.RNA interference was used to determine the relative contributions of Sp proteinson hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly,in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 andERa/Sp4 but not ERa/Sp1.In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels weredecreased. Analysis of the VEGFR2 promoter revealed that the same GC-richregion important for E2-mediated upregulation in ZR-75 cells was responsible forE2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells.EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins areexpressed in MCF-7 cells and bind to the proximal GC-rich region of theVEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, andERa/Sp protein-promoter interactions are accompanied by recruitment of thecorepressor SMRT using the ChIP assay.