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 âÂÂ60 to âÂÂ37 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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/6010 |
| Date | 17 September 2007 |
| Creators | Higgins, Kelly Jean |
| Contributors | Safe, Stephen H. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 28162623 bytes, electronic, application/pdf, born digital |
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