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Vascular endothelial growth factor receptor-2 (VEGFR2) is a key angiogenic factor, and angiogenesis is an important physiological process associated with neovascularization, growth, and metastasis of many different tumors. The mechanism of VEGFR2 gene expression was investigated in MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a series of VEGFR2 promoter deletion/mutated constructs, and the results indicated that the GC-rich –60 to –37 region of the promoter was essential for VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp proteins are expressed and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3, and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic cancer cells. VEGFR2 gene expression was also investigated in ZR-75 and MCF-7 breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced by 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. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter. RNA interference was used to determine the relative contributions of Sp proteins on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly, in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and ERa/Sp4 but not ERa/Sp1. In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich region important for E2-mediated upregulation in ZR-75 cells was responsible for E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in MCF-7 cells and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4 are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and ERa/Sp protein-promoter interactions are accompanied by recruitment of the corepressor SMRT using the ChIP assay.

Pax4 is a paired-domain-containing transcription factor that plays a crucial role in the development of pancreatic β- and δ-cells. In the absence of Pax4, no β- or δ-cells develop, but an increase in the number of α-cells is observed. To gain insight into Pax4 function, a rat insulinoma cDNA library was screened and two Pax4-related clones were isolated. One clone encoded a 349 amino acid protein with a molecular weight of 38K that corresponded to the full-length sequence of Pax4. The second cDNA, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH-terminus. Intracellular localisation studies indicated that Pax4 was sequestered specifically in the cytoplasm of β-cells. To determine the effect of Pax4 on islet cell gene expression, Pax4 was co-transfected with a series of human insulin and islet amyloid polypeptide (IAPP) promoter constructs into the β-cell line MIN6, and transcriptional activity was measured by reporter gene assay. Pax4 was found to have an inhibitory effect on the human insulin gene promoter, which was mapped, to the region -229 to -258. Electrophoretic mobility shift assay was used to show that Pax4 could bind to the C2 element located at -253 to -244 within this region. Pax4 was also found to have an inhibitory effect on the IAPP promoter, which was mapped to a sequence downstream of -138. Using a GAL4 reporter system, the repressive properties of Pax4 was further mapped to separate regions of the promoter between amino acids 2-230 and 231-349.

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