Regulation of the hypoxia inducible factor HIF-1a in the HTR8/SVneo cell line.

Mar del Plata

Congreso; Reunión Conjunta de Sociedades SAIC SAI & FAIC SAFIS; 2022

SAIC SAI&FAIC SAFIS

During pregnancy oxygen concentration is tightly regulated andalterations in its physiological levels are associated to pregnancydisorders such as preeclampsia. HIF-1α protein is rapidly degradedin the presence of oxygen and stabilized when oxygen levelsdecrease, regulating more than 50 genes in mammals thus influencingcell behavior. In this work, we identified a negative regulator ofHIF-1α under hypoxia in the extravillous trophoblast-derived HTR8/SVneo cell line. We have previously determined that the tumor suppressorKLF6 is transiently increased in hypoxia in a HIF-1α dependentmanner. Interestingly, here we established that KLF6 downregulationwith specific siRNAs further increases HIF-1α transcript andprotein levels under hypoxia, as evaluated through qPCR and westernblot respectively, suggesting a regulatory loop between thesetwo transcription factors. The scavenge of reactive oxygen species(ROS) with N-acetyl cysteine and the expression of negative dominantvariants of the NFκB pathway revealed that both signals areinvolved in the induction of HIF-1α in response to KLF6 silencing.Also, we determined that KLF6 downregulation under hypoxia inducesan active HIF-1α protein since in that context, the vascularendothelial growth factor (VEGF) mRNA, a classical HIF-1α target,is increased. Concomitant downregulation of KLF6 and HIF-1α usingspecific siRNAs restored VEGF transcript levels. On the otherhand, the overexpression of KLF6 under hypoxia led to a decreasein HIF-1α protein but its mRNA level remained unmodified. Moreover,treatment of KLF6-overexpressing HTR8/Svneo cells with 250μM of cobalt chloride (an inhibitor of HIF-1α degradation) preventsHIF-1α protein decrease indicating that KLF6 also regulates HIF-1αprotein stability. These results suggest a complex regulatory loopbetween HIF-1α and KLF6 that may be involved in the pathophysiologyof pregnancy diseases and other disorders in which oxygenplays an essential role.