HIF-1α stabilization promotes cell survival by reducing ROS in SHED

Abstract

Objectives: To promote cellular adaptation under stress condition through HIF-1α stabilization in stem cells from human exfoliated deciduous teeth (SHED). Methods: Hypoxia-inducible factor-1α (HIF-1α) stabilization in SHED was achieved by knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using premade target shRNA lentiviral particles. Knockdown of PHD2 (PHD2kd) expression and stabilization of HIF-1α expression were confirmed by RT-PCR and western blot assay. Cell viability in vitro was tested by MTT assay after exposing cells to different concentrations of exogenous H2O2 (0, 100, 200, 300, 400 and 500μM) for 24 hours. Oxygen Consumption Rate (OCR) kit and ATP detection kit were used to evaluate cellular O2 consumption and ATP production. Expressions of reactive oxygen species (ROS) related genes were examined by RT-PCR. Total glutathione level was detected using glutathione fluorometric assay kit. Expressions of AMP-activated protein kinase (AMPK) and phosphorylated AMP-activated protein kinase (p-AMPK) were tested by western blot assay. Results: HIF-1α expression was significantly increased in PHD2kd-SHED as shown by mRNA and protein levels compared to that of control-SHED (p<0.05). PHD2kd-SHED demonstrated significantly higher cell viability under stress condition compared to that of SHED that did not express HIF-1α (p<0.05). OCR, ATP and p-AMPK levels were significantly decreased in PHD2kd-SHED (p<0.05) indicating less O2 consumption by the cells. PCR results showed that mRNA expressions of Cox4-1 and Cox4-2 were down-regulated, which signified a reduced ROS generation; whereas expressions of genes involved in ROS such as scavenging glucose transporter 1 (Glut1), hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) were upregulated following PHD2kd (p<0.05). The total level of glutathione, a mitochondrial superoxide scavenger, was increased in PHD2kd-SHED illustrating enhanced ROS scavenging (p<0.05). Conclusions: HIF-1α stabilization promotes cellular adaptation under stress condition through down-regulating the intracellular ROS levels in SHED, which can be attributed to increased ROS scavenging, reduced oxygen consumption and ROS production.