Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1alpha activation cascade: implications for HIF-1alpha gene therapy.
The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.
Hofer
T
T
Institutes of, Physiology and, Veterinary Physiology, University of Zürich, CH-8057 Zürich, Switzerland.
Desbaillets
I
I
Höpfl
G
G
Gassmann
M
M
Wenger
R H
RH
D000818:Animals; D001692:Biological Transport; D015687:Cell Hypoxia / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D051272:Glucose Transporter Type 1; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D051379:Mice; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D009368:Neoplasm Transplantation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D051272:Glucose Transporter Type 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one