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

Mar del Plata

Congreso; Reunión Conjunta SAIC-SAI & FAIC-SAFIS; 2022

During pregnancy oxygen concentration is tightly regulated and alterations in its physiological levels are associated to pregnancy disorders such as preeclampsia. HIF-1α protein is rapidly degraded in the presence of oxygen and stabilized when oxygen levels decrease, regulating more than 50 genes in mammals thus influencing cell behavior. In this work, we identified a negative regulator of HIF-1α under hypoxia in the extravillous trophoblast-derived HTR8/SVneo cell line. We have previously determined that the tumor suppressor KLF6 is transiently increased in hypoxia in a HIF-1α dependent manner. Interestingly, here we established that KLF6 downregulation with specific siRNAs further increases HIF-1α transcript and protein levels under hypoxia, as evaluated through qPCR and western blot respectively, suggesting a regulatory loop between these two 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 are involved in the induction of HIF-1α in response to KLF6 silencing. Also, we determined that KLF6 downregulation under hypoxia induces an active HIF-1α protein since in that context, the vascular endothelial growth factor (VEGF) mRNA, a classical HIF-1α target, is increased. Concomitant downregulation of KLF6 and HIF-1α using specific siRNAs restored VEGF transcript levels. On the other hand, the overexpression of KLF6 under hypoxia led to a decrease in HIF-1α protein but its mRNA level remained unmodified.Moreover, treatment of KLF6-overexpressing HTR8/Svneo cells with 250 µM of cobalt chloride (aninhibitor of HIF-1α degradation) prevents HIF-1α protein decrease indicating that KLF6 also regulates HIF-1α protein stability. These results suggest a complex regulatory loop between HIF-1α and KLF6 that may be involved in the pathophysiology of pregnancy diseases and other disorders in which oxygen plays an essential role.