Article

Pharmaceutical Research

, Volume 21, Issue 5, pp 736-741

First online:

Sp1-Dependent Regulation of the RTP801 Promoter and Its Application to Hypoxia-Inducible VEGF Plasmid for Ischemic Disease

  • Minhyung LeeAffiliated withClinical Research Center, College of Medicine, Inha University
  • , Malavosklish BikramAffiliated withCenter for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah
  • , Seungjoon OhAffiliated withDepartment of Internal Medicine, College of Medicine, Kyung Hee University
  • , David A. BullAffiliated withDepartment of Surgery, Division of Cardiothoracic Surgery, University of Utah Health Sciences Center
  • , Sung Wan KimAffiliated withCenter for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah Email author 

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Abstract

Purpose. 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.

hypoxia RTP801 Sp1 transcriptional regulation vascular endothelial growth factor