Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1α activation cascade:: implications for HIF-1α 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-1 alpha protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1 alpha protein modifications, nuclear translocation, heterodimerization with the beta -subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1 alpha 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-1 alpha under normoxic conditions, allowing to distinguish 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 inhibitor PD98059 attenuated HIF-1 alpha protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1 alpha 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-1 alpha protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1 alpha gene transfer.