Characterization of a hypoxia-inducible factor (HIF-1 alpha) from rainbow trout - Accumulation of protein occurs at normal venous oxygen tension

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1 alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1 alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1 alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1 alpha protein recognized rainbow trout and chinook salmon HIF-1 alpha protein in Western blot analysis. Also, the human and mouse HIF-1 alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1 alpha antibody but not by the C-terminal HIF-1 alpha antibody. The accumulation of HIF-1 alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O-2 for 1 h. The greatest accumulation of HIF-1 alpha protein occurred at 5% O-2 (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1 alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1 alpha protein.