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Robert S. Cargill, Trevor J. McFarland, Michael H. Davies, Deborah C. Otteson, M R. Powers, J T. Stout, Binoy Appukuttan; TEAD4 Splice Variants from Mouse Neural Retina Increase VEGF Promoter Activity in Muller Glia and a HIF1-negative Cell Line. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5305.
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TEAD4 has been found to influence VEGF promoter activity in 293T and endothelial cells (EC), and is upregulated in the neural retina of a mouse model of oxygen-induced retinopathy (OIR). In EC, TEAD4 is upstream of HIF1-α signaling, enhancing HIF1-α transcription through binding MCAT-like sequences in the HIF1-α promoter. Here, we examine the effect of four murine isoforms of TEAD4 on VEGF promoter activity in Muller glia and other mammalian cells under normoxic and hypoxic conditions. We also investigate whether HIF1 influences this activity.
Four splice variants of TEAD4 (427, 384, 202, and 159) were previously cloned from OIR neural retina. MIO-M1 human and C57M10 mouse Muller glia were cotransfected with each isoform and a reporter plasmid encoding a secretable alkaline phosphatase (SEAP) driven by 1050bp of the mouse VEGF promoter. Cells were incubated in normoxic or hypoxic conditions, and media was assayed for SEAP. TEAD4 isoforms were also transfected individually, and secreted VEGF165 levels were measured by ELISA. The reporter expression assay was repeated in HIF1-negative C4 mouse hepatoma cells and compared to 1C1C7 controls.
All four isoforms enhanced VEGF promoter activity in MIO-M1 and 293T cells, but surprisingly not within C57M10 cells. Promoter activity was increased from 3- to 6-fold under normoxic conditions and 6- to 10-fold under hypoxic conditions. Relative to the control, the longer 427 and 384 isoforms had no effect on promoter activity in C4 or 1C1C7 cells. However, the two shorter isoforms (202 and 159) enhanced promoter activity 2- to 3-fold in both C4 and 1C1C7 cells with no significant difference between cell lines (P <0.01).
Murine TEAD4 isoforms enhance VEGF promoter activity in human Muller glia, with increased effect under hypoxia. The lack of effect in C57M10 cells suggests that cell-type specific cofactors may be necessary for their activity. The results from C4/1C1C7 cells suggest that functional HIF1 is not required for the shorter TEAD4 isoforms to regulate VEGF promoter activity. The absence of activity of the two longer isoforms may indicate the involvement of YAP in these cells, as the two shorter isoforms lack the C-terminal YAP-binding domain.
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