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Identification of novel alternatively spliced isoforms of RTEF-1 within human ocular vascular endothelial cells and murine retina


, : Identification of novel alternatively spliced isoforms of RTEF-1 within human ocular vascular endothelial cells and murine retina. Investigative Ophthalmology & Visual Science 48(8): 3775-3782

PURPOSE. Identification of transcription factors that regulate the transcription of the vascular endothelial growth factor ( VEGF) gene may facilitate understanding of the etiology and progression of ocular neovascular diseases. The purpose of this study was to determine whether transcriptional enhancer factor 1-related (RTEF-1) was present within ocular vascular endothelial cells and whether it played a role in the control of the transcription of the VEGF gene.METHODS. Primary cultures of human retinal vascular endothelial cells (RVECs) were maintained under normoxic or hypoxic conditions before isolation of mRNA. RT-PCR was performed to detect RTEF-1 transcripts. Amplified products were cloned into an expression plasmid. Human VEGF promoter and deletion constructs were cloned into a pSEAP reporter vector. Various RTEF-1 isoforms and VEGF promoter constructs were coelectroporated into human cells, and reporter expression levels were determined. Retinal tissue from a mouse model of retinopathy of prematurity (ROP) was analyzed by RT-PCR for the presence of RTEF-1 transcripts.RESULTS. Full-length 1305-bp and novel 936-bp RTEF-1 transcripts were identified in cultured human RVECs under normoxic conditions. A novel 447-bp isoform was present in cells maintained in a hypoxic environment. Four of the 11 translated exons predicted to code for the 1305-bp product were spliced out of the 936-bp transcript. The 1305-bp product enhanced expression from the VEGF promoter 4-fold greater than background, whereas the 936-bp and the 447-bp isoforms enhanced expression 3X and 12X, respectively. Analysis with deletion promoter constructs determined that all isoforms required the presence of Sp1 elements for efficient activation and that the hypoxia response element (HRE) was not essential for enhancement. Transcripts for novel RTEF-1 isoforms were also identified in neural retinal tissue of mice. Different murinespecific isoforms were present at different stages of postnatal development.CONCLUSIONS. Novel RTEF-1 transcripts are present within human ocular vascular endothelial cells and mouse neural retina during normal and ROP development, and alternatively spliced products are produced under hyperoxic and hypoxic conditions. Alternative spliced variants of human RTEF-1 transcripts are able to potentiate expression from the VEGF 5' proximal promoter region.

US$19.90

PMID: 17652751

DOI: 10.1167/iovs.06-1172


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