A constitutively active hypoxia-inducible factor-1alpha/VP16 hybrid factor activates expression of the human B-type natriuretic peptide gene.
Hypoxia-inducible factor-1 (HIF-1) is a primary regulator of the physiological response to hypoxia. A recombinant adenovirus expressing a constitutively active hybrid form of the HIF-1alpha subunit (Ad2/HIF-1alpha/VP16) is being evaluated as a gene therapy for the treatment of peripheral vascular disease. Ad2/HIF-1alpha/VP16 up-regulates known HIF-1-responsive genes, including those involved in angiogenesis. Expression profile analysis revealed that the brain natriuretic peptide (BNP) gene was significantly up-regulated in response to HIF-1alpha/VP16 in human fetal cardiac cells. Real-time reverse transcription-polymerase chain reaction analyses confirmed transcriptional activation of the BNP gene by HIF-1alpha/VP16 in human but not rat cardiac cells. Because hypoxia itself did not increase human BNP gene expression in these analyses, the mechanism of the HIF-1alpha/VP16 effect was determined. Analyses of promoter deletion mutants suggested that the cis-acting sequence in the human BNP promoter mediating activation by HIF-1alpha/VP16 was a putative HIF-1 responsive element (HRE) located at -466. An SV40 basal promoter-luciferase plasmid containing a minimal BNP HRE was up-regulated by HIF-1alpha/VP16, whereas a similar construct carrying a mutation within the HIF-1 binding site was not. Mutation of an E-box motif within the BNP HRE reduced HIF-1alpha/VP16-mediated transcriptional activation by 50%. Gel-shift analyses showed that both the native HIF-1alpha and HIF-1alpha/VP16 are able to bind to a probe containing the HIF-1 binding site. These experiments demonstrate the existence of a functional HRE in the BNP promoter and further define the scope and mechanism of action of Ad2/HIF-1alpha/VP16.
Luo
Yuxia
Y
Genzyme Corporation, 31 New York Avenue, P.O. Box 9322, Framingham, MA 01701-9322, USA.
Jiang
Canwen
C
Belanger
Adam J
AJ
Akita
Geoffrey Y
GY
Wadsworth
Samuel C
SC
Gregory
Richard J
RJ
Vincent
Karen A
KA
D001483:Base Sequence; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D008969:Molecular Sequence Data; D020097:Natriuretic Peptide, Brain / Q000235:genetics*; D016491:Peripheral Vascular Diseases / Q000628:therapy*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D015533:Transcriptional Activation
C511617:Ad2-HIF-1-VP16 protein; D011993:Recombinant Fusion Proteins; D020097:Natriuretic Peptide, Brain