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M. Labazi, D. M. Marcus, F. Lamoke, M. Varano, R. B. Caldwell, M. Bartoli; Signal Transducer and Activator of Transcription 1 (STAT1) is a Transcriptional Inhibitor of VEGF Gene Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5631.
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Enhanced expression and activity of vascular endothelial growth factor (VEGF) plays a pivotal pathogenic role in ischemic retinopathies. Our previous studies have implicated the transcription factor signal transducer and activator of transcription 3 (STAT3) in the induction of VEGF in the ischemic retina. Here we investigated the role of another member of the STATs family of proteins, STAT1, which has been shown to be anti-angiogenic and promote endothelial cell death.
Experiments were conducted by using retinal endothelial cells (REC) and human umbilical vein endothelial cells (HUVEC) exposed to hypoxic conditions (pO2= 2%±0.5) for different times (30min, 1h, 3h, 6h). Western blotting, immunoprecipitation and chromatin immunoprecipitation (ChIP) analysis were used to determine STAT3 and STAT1 protein levels, post-translational modifications (i.e.phosphorylation and acetylation) and binding to VEGF promoter region.
Hypoxia rapidly (within 30min) stimulated phosphorylation of STAT3 and STAT1 at tyrosine 705 and 701, respectively. ChIP analysis revealed that, in normoxic conditions, STAT1 is bound to VEGF promoter region while STAT3 is not. Within 30 minutes after hypoxia STAT3 was co-precipitated with VEGF promoter while STAT1 was not. The RNA-binding protein Sam68, used as negative control, was not immunoprecipitated with VEGF promoter in all the analyzed experimental conditions. Immunoprecipitation analysis determined that hypoxia induced STAT1 acetylation.
Our data reveal that STAT1 is a potential transcriptional repressor of VEGF expression in normoxic conditions by binding VEGF promoter in the same region of STAT3 binding sites. In addition, acetylation/deacetylation processes are involved in the regulation of STAT1 and STAT3 competitive binding to VEGF promoter region and its transcriptional regulation.
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