Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease

Pharm Res. 2004 May;21(5):736-41. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Enzyme-Linked Immunosorbent Assay
  • Gene Deletion
  • Humans
  • Hypoxia-Ischemia, Brain / metabolism*
  • Luciferases / genetics
  • Plasmids / genetics*
  • Promoter Regions, Genetic / genetics*
  • Sp1 Transcription Factor / physiology*
  • Transcription Factors / genetics*
  • Transfection
  • Vascular Endothelial Growth Factor A / biosynthesis*
  • Vascular Endothelial Growth Factor A / genetics*

Substances

  • DDIT4 protein, human
  • Sp1 Transcription Factor
  • Transcription Factors
  • Vascular Endothelial Growth Factor A
  • Luciferases