Hypoxia is tightly associated with tumor angiogenesis, progression, and metastasis. It also is involved in cell proliferation and programmed cell death. Tumor hypoxia renders cells insensitive to apoptotic potential and resistant to chemo- and radio-therapy. Cells respond to hypoxia, at least in part, by transcriptional induction of a group of hypoxia responsive genes such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (NOS2), and glycolytic enzymes. The transcriptional up-regulation of these genes depends on the activation of hypoxia-inducible factor 1 (HIF1), an ab heterodimeric transcription factor of the basic helix-loop-helix PAS family. HIF1 is a crucial regulator in the cellular response to hypoxia and plays an essential role in embryogenesis, hematopoiesis, angiogenesis, and tumorigenesis. Our objective is to understand the molecular mechanisms underlying HIF1 activation, and the role of HIF1 in angiogenesis and tumorigenesis. HIF1 activity is regulated primarily by its a subunit (HIF1a), involving multiple hypoxia-induced posttranslational modifications that affect HIF1a stability and its transcriptional activity. The abundance of HIF1a is controlled by oxygen-dependent proteolysis through the ubiquitin-proteasomal pathway, and the oxygen-dependent degradation (ODD) domain within HIF1a is primarily responsible for the degradation via direct binding to the tumor suppressor gene pVHL, an E3 ubiquitin ligase. In addition to the identification of Pro564 required for pVHL binding, which was first reported by others to be a regulatory site through hydroxylation, we also have identified a destruction motif that contributes not only to HIF1a instability, but transcriptional activity as well. HIF1a-mediated transcription requires the co-activator, p300/CBP, at least in part, through the interaction with CH1 domain of p300/CBP. To understand the molecular basis of this interaction, we developed a random mutagenesis screen in yeast (RAMSY) approach for efficient identification of residues that are functionally critical for protein interactions. As a result, residues involved in the HIF1a-p300 interaction have been identified to be crucial for HIF1 transcriptional activity in mammalian systems. Moreover, data from residue substitution experiments indicate stringent necessity of leucines and hydrophobic cysteine for HIF1a transcriptional activity. Therefore we proposed that hypoxia-induced HIF1a-p300 interaction relies upon a leucine-rich hydrophobic interface that is regulated by the hydrophilic and hydrophobic sulfhydryls of HIF1a Cys800. These findings have prompted us to take advantage of genetically altered HIF1a to investigate the role of HIF1 in angiogenesis and tumorigenesis. In collaboration with the Arbeit Laboratory at the UCSF, we have made an intriguing observation that an overexpressed constitutively active HIF1a transgene induced hypervascularity in the mouse skin. The vasculature appeared to be mature without leak or inflammation, which is distinct from the phenotype of the VEGF transgenic mice. The data indicate the potential usage of HIF1a for gene therapy. Based on the residues directly involved in HIF1a transcriptional activity, we have engineered oxygen-insensitive HIF1a that is transcriptionally active, superactive, or inactive to test the role of HIF1a in cell growth, cell cycle, and tumorigenecity.
