Linking Oxygen to Time: The Bidirectional Interaction Between the Hypoxic Signaling Pathway and the Circadian Clock

Abstract

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities.

Circadian clock regulates the host response to Salmonella

“…Nodes were much larger in WT mice compared with Clock mutant mice, thus indicating that many pathways regulated by HIF-1α also require a functional clock system. This observation is of particular interest because cross-talk between hypoxic and circadian pathways has been proposed, and Hif-1α is thought to be a clock-controlled gene (27).…”

Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish,Danio rerio

“…To compare between blots, all samples were normalized to a single sample (hypoxia exposed embryos at 24 hpf) ran on every gel. Western blot analysis of zebrafish fibroblast cells [26], 36 hpf zebrafish larvae and full-length recombinant zebrafish HIF-1a using anti-zebrafish HIF-1a antibody revealed specific binding at the expected molecular mass of 86 kDa as well as cross reaction to non-specific proteins (electronic supplementary material, figure S2). Densiometric analysis of the non-specific bands showed that the expression of these proteins was not affected by the low O 2 treatments.…”

Mitochondrial physiology and reactive oxygen species production are altered by hypoxia acclimation in killifish (Fundulus heteroclitus)

“…HIF-α proteins can be stabilized in response to both cellular hypoxia and ROS, and can ultimately lead to a variety of adaptive and maladaptive cellular responses to O 2 limitation (Prabhakar and Semenza, 2012). The HIF-1α and HIF-2α sequences of fish exhibit greater amino-acid divergence at functionally important sites than mammals (Rytkönen et al, 2011), but HIF-1α protein abundance increases as expected in zebrafish exposed to acute hypoxia (Kopp et al, 2011;Egg et al, 2013). Interestingly, genetic knockout of prolyl hydroxylase 1 (PHD1) -the oxygen-sensitive enzyme that hydroxylates HIF-α and targets it for degradation -stabilizes HIF-2α protein, reduces mitochondrial respiration and ROS production, and improves ischemia tolerance in skeletal muscle in mice (Aragonés et al, 2008).…”