THE TRANSCRIPTIONAL ACTIVATOR KRÜPPEL-LIKE FACTOR 6 (KLF6) IS INVOLVED IN TROPHOBLAST REDOX HOMEOSTASIS

Buenos Aires

Congreso; IFPA 2019 - VIII SLIMP; 2019

IFPA - SLIMP

Objectives: Dysregulated ROS signalling is associated with multiple diseasesincluding placental pathologies. We have previously demonstratedthat KLF6, an early response gene induced by several stress stimuli, isinvolved in trophoblast syncytialization and that hypoxia induces itsexpression in the placenta. Herein, we aimed to evaluate whether KLF6 isrequired to maintain redox balance in trophoblast cells.Methods: Human HTR8/SVneo cells were transfected with a negativecontrol siRNA or with KLF6-specific siRNAs. Reactive oxygen species (ROS)were measured by flow cytometry using the H2DCFDA probe. Cell proliferationwas evaluated by bromodeoxyuridine incorporation and cell cycleby flow cytometry with propidium iodide staining. Mitochondrial membranepotential was analyzed by flow cytometry with the fluorescent JC-1dye. Protein and transcript level of relevant molecules were determinedthrough western blot and qRT-PCR, respectively. Activation of the Nrf2pathway was assessed by immunofluorescence and HO-1 expression.Catalase activity was evaluated by the decomposition of H2O2.Results: Intracellular ROS levels were higher in KLF6-silenced cellscompared to scramble and mock transfected, as measured 48 hours aftertransfection. Surprisingly, ROS levels induced by KLF6 silencing weresimilar to those detected in cells treated with 100 mMH2O2 for 3 h. Catalaseexpression and activity, as well as peroxiredoxin levels were similar in allexperimental conditions. In addition, the Nrf2 pathway was not activatedand mitochondrial membrane potential was not impaired by KLF6silencing. On the contrary, cells with downregulated KLF6 expressionshowed reduced proliferation, cell cycle inhibition and increased p21protein levels independently of p53 phosphorylation.Conclusion: Downregulation of KLF6 expression leads to an increase inintracellular ROS levels which is not due to a diminished antioxidantcatalase activity or peroxiredoxin expression. Dysregulated ROS level isconcomitant with the inhibition of cell growth. Altogether these resultssupport the notion that KLF6 is involved in trophoblast cell homeostasis.