Identification of novel alternatively spliced isoforms of RTEF-1 within human ocular vascular endothelial cells and murine retina.
OBJECTIVE
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 murine-specific 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.
Appukuttan
Binoy
B
Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239, USA.
McFarland
Trevor J
TJ
Davies
Michael H
MH
Atchaneeyasakul
La-ongsri
LO
Zhang
Yi
Y
Babra
Bobby
B
Pan
Yuzhen
Y
Rosenbaum
James T
JT
Acott
Ted
T
Powers
Michael R
MR
Stout
J Timothy
JT
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000502:physiology; D006801:Humans; D018496:Hyperoxia / Q000235:genetics / Q000503:physiopathology; D000860:Hypoxia / Q000235:genetics / Q000503:physiopathology; D007231:Infant, Newborn; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology / Q000502:physiology*; D012178:Retinopathy of Prematurity / Q000235:genetics / Q000503:physiopathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000235:genetics*; D015854:Up-Regulation / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D000091262:TEA Domain Transcription Factors; C486309:TEAD4 protein, human; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A