Abstract
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1αto the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.
Highlights
hypoxia inducible factor 1 (HIF-1) belongs to the family of hypoxia-inducible transcription factors (HIFs) involved in the regulation of cellular and molecular adaptation to hypoxia [1]. e three isoforms (HIF-1, HIF-2, and HIF-3) are all heterodimers consisting of a constitutively expressed, stable ββ-subunit and an inducible αα-subunit. e cellular level of the αα-subunit is regulated at the protein level where high cellular oxygen concentration results in hydroxylation and subsequent proteasomal degradation, whereas low cellular oxygen concentration results in repression of this degradation [2]
We modelled the biological activity of competitive and non-competitive HIF prolyl hydroxylase enzymes (HPHs) inhibitor compounds by quantifying their ability to (1) induce Hypoxia inducible factor 1 alpha HPH (HIF-1αα) stabilization and nuclear translocation, (2) protect against oxidative stress, and (3) to induce dopamine release
The two competitive compounds induced a lower and less sustained HIF-1αα response than what the non-competitive compounds DFO and FG41 did, whereas the downstream effects on protection against oxidative stress and dopamine release were comparable with the two types of inhibitor compounds
Summary
HIF-1 belongs to the family of hypoxia-inducible transcription factors (HIFs) involved in the regulation of cellular and molecular adaptation to hypoxia [1]. e three isoforms (HIF-1, HIF-2, and HIF-3) are all heterodimers consisting of a constitutively expressed, stable ββ-subunit and an inducible αα-subunit. e cellular level of the αα-subunit is regulated at the protein level where high cellular oxygen concentration results in hydroxylation and subsequent proteasomal degradation, whereas low cellular oxygen concentration results in repression of this degradation [2]. E hydroxylation and thereby stabilization of the HIFαα subunit are regulated by the HIF prolyl and asparagine hydroxylase enzymes, of which the prolyl hydroxylases, HPHs, are the focus of this work. E activity of the HPHs can be inhibited with small molecules either indirectly (noncompetitively) through a reduction in the cellular levels of oxygen, iron or, 2-oxoglutarate or directly (competitively) by a compound binding to and blocking the 2-oxoglutarate binding site of the enzyme. F 1: Cellomics images showing the algorithm used for quanti cation of nuclear and cytoplasmic levels of HIF-1αα. Circ is de ned by the outline of the Hoechst-staining and represents the nuclear region. Ring is de ned as a certain radius surrounding the Circ region and represents the cytoplasmic region of the cell. Ring is de ned as a certain radius surrounding the Circ region and represents the cytoplasmic region of the cell. e Hoechst uorescence and the HIF-1αα uorescence are recorded in two different channels
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