T. Hofer et al., Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1 alpha activation cascade: implications for HIF-1 alpha gene therapy, FASEB J, 15(12), 2001, pp. NIL_132-NIL_154
The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptio
nal regulator of oxygen homeostasis and a possible target for gene therapy
of ischemic disease. Although the role of oxygen concentration in HIF-1 alp
ha protein stabilization is well established, it is less clear whether and
how oxygen-regulated mechanisms contribute to HIF-1 alpha protein modificat
ions, nuclear translocation, heterodimerization with the beta -subunit, rec
ruitment of cofactors, and gene trans-activation. Because the HIF-1 alpha p
rotein is proteolytically degraded under normoxic conditions, we establishe
d two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress
high levels of HIF-1 alpha under normoxic conditions, allowing to distingui
sh hypoxia-dependent from hypoxia-independent activation mechanisms. Using
these cells, we found that normoxically induced HIF-1 alpha 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 in
hibitor PD98059 attenuated HIF-1 alpha protein modifications and trans-acti
vation ability but not protein stabilization and DNA-binding activity. Beca
use overexpressed HIF-1 alpha is fully localized to the nucleus but display
s only partial DNA-binding and trans-activation activity, mitogen-activated
protein kinase-dependent phosphorylation might be required for full HIF-1
activation. HIF-1 alpha protein was also overexpressed in vivo, following t
he transplantation of HT42 cells into nude mice, demonstrating the feasibil
ity of HIF-1 alpha gene transfer.