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.
Mesh Terms:
Animals, Biological Transport, Cell Hypoxia, Cell Nucleus, Endothelial Growth Factors, Enzyme Inhibitors, Flavonoids, Gene Expression, Gene Expression Regulation, Genetic Therapy, Glucose Transporter Type 1, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Lymphokines, MAP Kinase Kinase 1, Mice, Mice, Nude, Mitogen-Activated Protein Kinase Kinases, Monosaccharide Transport Proteins, Neoplasm Transplantation, Protein-Serine-Threonine Kinases, RNA, Messenger, Transcription Factors, Transcriptional Activation, Transplantation, Heterologous, Tumor Suppressor Protein p53, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
Animals, Biological Transport, Cell Hypoxia, Cell Nucleus, Endothelial Growth Factors, Enzyme Inhibitors, Flavonoids, Gene Expression, Gene Expression Regulation, Genetic Therapy, Glucose Transporter Type 1, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Lymphokines, MAP Kinase Kinase 1, Mice, Mice, Nude, Mitogen-Activated Protein Kinase Kinases, Monosaccharide Transport Proteins, Neoplasm Transplantation, Protein-Serine-Threonine Kinases, RNA, Messenger, Transcription Factors, Transcriptional Activation, Transplantation, Heterologous, Tumor Suppressor Protein p53, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
FASEB J.
Date: Dec. 01, 2001
PubMed ID: 11606485
View in: Pubmed Google Scholar
Download Curated Data For This Publication
211965
Switch View:
- Interactions 1