Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease.
OBJECTIVE
Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro.


METHODS
The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay.


RESULTS
In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia.


CONCLUSIONS
With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.
Lee
Minhyung
M
Clinical Research Center, College of Medicine, Inha University, Inchon 400-711, Korea.
Bikram
Malavosklish
M
Oh
Seungjoon
S
Bull
David A
DA
Kim
Sung Wan
SW
D002460:Cell Line; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D017353:Gene Deletion; D006801:Humans; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics*
C469991:DDIT4 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D008156:Luciferases