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 transcr iption factor that controls the induction of several genes involved in glyc olysis, 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 t rout cells. We have also cloned the full-length cDNA (3605 base pairs) of t he 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. Po lyclonal antibodies against the N-terminal part (amino acids 12-363) and th e C-terminal part (amino acids 330-730) of rainbow trout HIF-1 alpha protei n recognized rainbow trout and chinook salmon HIF-1 alpha protein in Wester n blot analysis. Also, the human and mouse HIF-1 alpha proteins were recogn ized by the N-terminal rainbow trout anti-HIF-1 alpha antibody but not by t he C-terminal HIF-1 alpha antibody. The accumulation of HIF-1 alpha was stu died by incubating rainbow trout and chinook salmon cells at different oxyg en 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 tha t the inhibitor is able to stabilize the protein, which normally is degrade d via the proteasome pathway both in normoxia and hypoxia. Notably, the hyp oxia response element of oxygen dependent degradation domain is identical i n mammalian, Xenopus, and rainbow trout HIF-1 alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1 alpha prot ein.