The transcriptional activator Kruppel-like factor 6 is involved in trophoblast redox homeostasis

KOURDOVA, L; ROJAS, MARIA L.; PANZETTA-DUTARI, G.M.; MIRANDA, AL; CRUZ DEL PUERTO, MARIANO; RACCA, A.C.; GENTI-RAIMONDI, S.

Buenos Aires

Congreso; 8th Latin American Symposium on Maternal-Fetal Interaction and Placenta (VIII SLIMP); 2019

OBJECTIVEDysregulated ROS signalling is associated with multiple diseases including placental pathologies. We have previously demonstrated that KLF6, an early response gene induced by several stress stimuli, is involved in trophoblast syncytialization and that hypoxia induces its expression in the placenta. Herein, we aimed to evaluate whether KLF6 is required to maintain redox balance in trophoblast cells.METHODSHuman HTR8/SVneo cells were transfected with a negative control siRNA or with KLF6-specific siRNAs. Reactive oxygen species (ROS) were measured by flow cytometry using the H2DCFDA probe. Cell proliferation was evaluated by bromodeoxyuridine incorporation and cell cycle by flow cytometry with propidium iodide staining. Mitochondrial membrane potential was analyzed by flow cytometry with the fluorescent JC-1 dye. Protein and transcript level of relevant molecules were determined through western blot and qRT-PCR, respectively. Activation of the Nrf2 pathway was assessed by immunofluorescence and HO-1 expression. Catalase activity was evaluated by the decomposition of H2O2. RESULTSIntracellular ROS levels were higher in KLF6-silenced cells compared to scramble and mock transfected, as measured 48 hours after transfection. Surprisingly, ROS levels induced by KLF6 silencing were similar to those detected in cells treated with 100 µM H2O2 for 3 h. Catalase expression and activity, as well as peroxiredoxin levels were similar in all experimental conditions. In addition, the Nrf2 pathway was not activated and mitochondrial membrane potential was not impaired by KLF6 silencing. On the contrary, cells with downregulated KLF6 expression showed reduced proliferation, cell cycle inhibition and increased p21 protein levels independently of p53 phosphorylation.CONCLUSIONDownregulation of KLF6 expression leads to an increase in intracellular ROS levels which is not due to a diminished antioxidant catalase activity or peroxiredoxin expression. Dysregulated ROS level is concomitant with the inhibition of cell growth. Altogether these results support the notion that KLF6 is involved in trophoblast cell homeostasis.