Induction of HIF–1α in response to hypoxia is instantaneous

Abstract

Despite the pivotal role the hypoxia-inducible factor-1α (HIF-1α) plays in physiological and pathological processes, little is known regarding the timeframe and mechanisms involved in its regulation. We determined the onset, accumulation, and degradation of HIF-1α and a number of redox-sensitive nuclear factors over a range of pathophysiological oxygen concentrations. Experiments were carried out on nonadherent human HeLaS3 cells placed in tonometers to achieve rapid equilibration between the cell suspension and the various hypoxic/reoxygenation conditions. Exposure to hypoxia for less than 2 min already revealed nuclear HIF-1α protein induction on Western blots and HIF-1 DNA binding in EMSAs. One hour after anoxic/hypoxic exposure, nuclear HIF-1α proteins reached maximal levels, which were maintained for 4 h. Reoxygenation reduced HIF-1 DNA binding within 2 min, and nuclear HIF-1α protein levels within 4 to 8 min, down to a level below the detection limit within 32 min. Western blot analysis of the redox sensitive nuclear factors NF-κB, c-Fos, c-Jun, Ref-1, and thioredoxin showed no alteration in their nuclear levels in response to anoxia/hypoxia, but reoxygenation rapidly caused a transient increase in nuclear NF-κB and thioredoxin protein levels. The instant initiation of HIF-1α accumulation shown here limits the hypoxic signaling pathway to below 2 min.Key words: anoxia • reoxygenation • redox factor • ROS issue hypoxia occurs during physiological and pathological processes such as embryogenesis, tumorigenesis, transplantation, and wounding and has recently attracted attention as a target for gene therapy since its correlation with tumor malignancy (1). The hypoxia-inducible factor-1 (HIF-1) is the molecular key player in the hypoxic response: It induces specific expression of several hypoxically regulated genes, such as vascular endothelial growth factor, transferrin, and glycolytic enzymes, and thereby restores blood supply and energy availability to the hypoxic tissue. HIF-1 is a heterodimeric transcription factor consisting of HIF-1α and HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator). Both ubiquitously expressed subunits are efficiently translated under normoxic and hypoxic conditions (2), they belong to the Per-ARNT/AhR-Sim subfamily of basic helix-loop-helix transcription factors (3), and they are capable of nuclear translocation (4). Inside the nucleus, the two subunits dimerize to form the HIF-1 complex, which binds to the conserved consensus sequence (A)CGTG within the hypoxia response element present in oxygen-regulated target genes (5) (personal T communications, Camenisch G. et al.). The DNA-binding domain of HIF-1α lies within the Nterminal region of the protein, whereas the C-terminal region holds the two transactivation domains. The central region of HIF-1α contains an oxygen-dependent degradation (ODD) domain located between amino acids 401 and 603 (6), which confers oxygen-sensitivity to the HIF-1α subunit. As a result, HIF-1α is rapidly degraded in normoxic conditions. Degr...

Properties of Switch-like Bioregulatory Networks Studied by Simulation of the Hypoxia Response Control System

“…The mechanism also implies a high rate of HIF␣ synthesis, consistent with the observed rapid increase in HIF-1␣ protein (detectable within 2 min) in response to hypoxia (Jewell et al, 2001). The rapid oxygen-independent synthesis and rapid oxygen-dependent degradation of HIF-1␣ permit rapid responses to changes in available oxygen.…”

Hif-1

“…As the level of oxygen is reduced toward approximately 0.5%, the activity of HIF-1 progressively increases (62). An uncertainty in many experiments is that un-stirred culture medium could limit oxygen diffusion, but a similar relationship between pO 2 and HIF activation results has been observed when cells are cultured on gas-permeable plastic or using tonometers (32,62,63).…”

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

“…For HIF-1a, nuclear-enriched protein extracts were prepared as described previously (Jewell et al, 2001). Total protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad, Reinach, CH) and equal amounts (20 mg) were separated by sodium dodecyl sulphate -polyacrylamide gel electrophoresis (10%).…”