Regulation of HIF1 alpha in hyperoxia and hyperglycaemia
Authors: Iwuagwu Benedicta Ugochi 
Published: June 2021

Hypoxia inducible factor 1 alpha (HIF‐1α) is the master regulator of cell response to hypoxia. Increasing evidence demonstrates the existence of non‐hypoxic mediated HIF‐1α induction/activation1,2. Reactive oxygen species (ROS) production and increased levels of interleukin (IL)‐6 and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) are linked with hypoxia‐induced expression of HIF1a. Hyperoxia is known to stimulate ROS production and the concomitant effect of high glucose concentrations causes increases of ROS with known involvement of the nuclear factor (erythroid derived 2)‐like 2 (Nrf‐2), the principal regulator of Redox status in cells2. There is clearly a merit in understanding the effect of hyperoxia on HIF1α protein synthesis and stabilisation as it has been reported to result in both an upregulation and a downregulation of HIF1α from different groups2, 3.Here, primary human dermal microvascular endothelial cells (HDMECs) were maintained in growth media containing either 5.5 or 20mmol/L glucose and incubated at either 18–20% or 95% oxygen. RNA was extracted using Trizol and cDNAs prepared using a kit (Bioline UK), sub‐cellular fractions were prepared using lysis buffer and differential centrifugation and followed by Western blotting (WB) or the qRT‐PCR. Cells were fixed in 100% ice cold methanol and stained according to standard immunocytochemistry (ICC) protocol. HIF1α and Nrf2 expression were examined with ICC using HIF1a (1:20) (Thermo Fischer Scientific) and Nrf2 (1:20) (Abcam) and visualized with immunofluorescence (DayLight 488)(Thermo Fischer Scientific). WB was used to confirm the presence of the respective proteins; HIF1α (1:100) (Thermo Fischer Scientific), Nrf2 (1:100)(Santa Cruz), NFκB (1:100)(Santa Cruz) and gene expression was examined by qRT‐PCR to detect and quantify HIF1α using β2‐microglobulin as the internal control.Results showed an absence of HIF1a protein in the nuclear fraction and no change in the level of cytosolic HIF1α protein in basal or treated conditions. However, an increase in HIF1α mRNA level in hyperoxia/hyperglycemia relative to control was evident We also found an acute increase IL‐6 mRNA level and NFκB protein in normoglycemia/hyperoxia, which was further enhanced in hyperglycemia/hyperoxia with an increased expression of Nrf2 protein.This study demonstrates a likely transcriptional link between HIF1α expression and hyperoxia in HDMECs. The possibility of the existence of a transcriptional effect was further strengthened when we examined the gene and protein expression levels of IL‐6 and NFκB respectively as both IL‐6 and NFκB are transcriptional activators of HIF1α mRNA. The hyperoxia‐mediated expression of HIF1α protein via accumulation of ROS2 is supported with the corresponding increased level of nrf‐2. The relevance of this data points to the role of hyperglycemia/hyperoxia, highlighting associated cell response to redox imbalance and inflammation and the convergence on HIF1α regulation at a transcriptional level. HIF1α signalling is involved in several functions and the understanding of its regulation is warranted.Support or Funding InformationThe first author, Benedicta U. Iwuagwu received an academic scholarship for her PhD (3 years) and is at the last stage of PhD (4th year) finalising data collection and writing which is nonfunded.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.