Pharmaceutical Research

, Volume 21, Issue 5, pp 736–741

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

Authors

  • Minhyung Lee
    • Clinical Research Center, College of MedicineInha University
  • Malavosklish Bikram
    • Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical ChemistryUniversity of Utah
  • Seungjoon Oh
    • Department of Internal Medicine, College of MedicineKyung Hee University
  • David A. Bull
    • Department of Surgery, Division of Cardiothoracic SurgeryUniversity of Utah Health Sciences Center
    • Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical ChemistryUniversity of Utah
Article

DOI: 10.1023/B:PHAM.0000026421.09367.b3

Cite this article as:
Lee, M., Bikram, M., Oh, S. et al. Pharm Res (2004) 21: 736. doi:10.1023/B:PHAM.0000026421.09367.b3

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.

hypoxiaRTP801Sp1transcriptional regulationvascular endothelial growth factor

Copyright information

© Plenum Publishing Corporation 2004