Effect of Hedgehog Signaling Pathway on VDUP1 Expression in Human Embryonic Mesenchymal cells (HEPM)
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Authors
Bawazeer, Mona
Issue Date
2014-04-26
Type
poster
Language
en_US
Keywords
Alternative Title
Abstract
Vitamin D_3 Upregulated protein-1 (VDUP1) is a tumor suppressor gene. Its expression is reduced in tumor versus non-tumor cells although the mechanism for the reduction is not known. Studies on the developing Drosophila nervous system demonstrated that mutants lacking functional Hedgehog (Hh) signaling expressed a higher amount of VDUP1, suggesting that Hh signaling can regulate VDUP1 expression within the brain. The hypothesis is that Hh signaling within tumor cells results in reduced VDUP1 expression. HEPM cell line is known to exhibit activated Hh signaling. Transfection of HEPM cells with plasmid containing 8 copies of the consensus Gli binding site inserted upstream of luciferase (Gli-pluc) and ?-galactosidase (?-Gal) was used to assess Hh signaling. Transfection followed by treatment with recombinant Hh protein increased luciferase activity. Transfection with a plasmid containing the human VDUP1 promoter upstream of luciferase (VDUP1-pluc) and ?-Gal was used to assess VDUP1 promoter activity. Transfection followed by treatment with recombinant Hh protein resulted in decreased luciferase activity. HEPM cells treated with GANT61, an inhibitor of Gli, and stained with anti-VDUP1 and anti-HDAC showed increased VDUP1 immunofluorescence, primarily in the nucleus. These results confirmed active Hh signaling in HEPM cells with the ability to downregulate VDUP1 expression by inhibiting the pathway.