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Dissecting hypoxia‐dependent and hypoxia‐independent steps in the HIF‐1α activation cascade: implications for HIF‐1α gene therapy
Author(s) -
Hofer Thomas,
Desbaillets Isabelle,
Höpfl Gisele,
Gassmann Max,
Wenger Roland H.
Publication year - 2001
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.01-0546fje
Subject(s) - hypoxia inducible factors , hypoxia inducible factor 1 , kinase , reporter gene , transcription factor , microbiology and biotechnology , protein kinase a , phosphorylation , biology , transfection , protein subunit , transplantation , nuclear protein , chemistry , gene , gene expression , biochemistry , medicine , surgery
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‐1α protein stabilization is well established, it is less clear whether and how oxygen‐regulated mechanisms contribute to HIF‐1α protein modifications, nuclear translocation, heterodimerization with the β‐subunit, recruitment of cofactors, and gene fraws‐activation. Because the HIF‐1α 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‐1α under normoxic conditions, allowing to distinguish hypoxia‐dependent from hypoxia‐independent activation mechanisms. Using these cells, we found that normoxically induced HIF‐1α is localized to the nucleus, binds DNA, and fraws‐activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF‐1α protein modifications and fraws‐activation ability but not protein stabilization and DNA‐binding activity. Because overexpressed HIF‐1α is fully localized to the nucleus but displays only partial DNA‐binding and fraws‐activation activity, mitogen‐activated protein kinase‐dependent phosphorylation might be required for full HIF‐1 activation. HIF‐1α protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF‐1 α gene transfer.