A novel chimeric promoter that is highly responsive to hypoxia and metals.
To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.
Lee
J-Y
JY
Department of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Kangnam-ku, Seoul, Korea.
Lee
Y-S
YS
Kim
J-M
JM
Kim
K L
KL
Lee
J-S
JS
Jang
H-S
HS
Shin
I-S
IS
Suh
W
W
Jeon
E-S
ES
Byun
J
J
Kim
D-K
DK
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002678:Chimera; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005818:Genetic Engineering; D006367:HeLa Cells; D006614:Hindlimb / Q000098:blood supply; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D017078:Laser-Doppler Flowmetry; D008668:Metallothionein / Q000235:genetics; D008670:Metals / Q000378:metabolism*; D051379:Mice; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012039:Regional Blood Flow; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000379:methods; D000097809:Transcription Factor MTF-1
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008668:Metallothionein; D008670:Metals; D010735:Phosphoglycerate Kinase; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1; C400152:DNA enzyme ED5; C497442:HIF1A protein, human