REGULATORY TRANSCRIPTIONAL DOMAIN OF KRÜPPEL-LIKE FACTOR 6 (KLF6) IS NECESSARY FOR PLACENTAL TROPHOBLAST CELLS FUSION

KOURDOVA L. T; CRUZ DEL PUERTO M.M; PANZETTA-DUTARIA GM; MIRANDA A.L.; RODRIGUEZ-LOMBARDI G.; GENTI-RAIMONDI S; RACCA A.; ROJAS L

Mar del Plata

Congreso; Anual meeting of S A I C . S A F E . S A B . SAP 2 0 1 9; 2019

Placental development and maintenance require the fusion of villous cytotrophoblasts (vCTB) to generate the syncytiotrophoblast layer involved in the placental barrier function, synthesis of critical proteins and nutrient exchange between the mother and the fetus. The Krüppel-like factor 6 (KLF6) is highly expressed in placenta and Klf6-/- mice die at day E12.5 showing impaired placenta development. Previously, we have demonstrated that KLF6 modulates the expression of the cell cycle inhibitory protein p21, of Syn-1, and β-hCG, and it is required for cell?cell fusion in human primary vCTB as well as in the BeWo trophoblast-derived cell line. More importantly, KLF6 is sufficient to trigger cell fusion in BeWo cells. Herein we analysed the KLF6-dependent mechanisms involved in KLF6-induced fusion. Stable BeWo cell lines containing a KLF6 mutant that lacks the acidic domain required for its transcriptional activity (KLF6∆ac-BeWo), the full-length KLF6 protein (KLF6-BeWo), or the Empty-vector (EV-BeWo) were generated. KLF6∆ac-BeWo cells were treated or not with 30 µM forskolin and BeWo-KLF6 cells were transduced with KLF6∆ac lentivirus particles and cell fusion was analysed by immunofluorescence after 72 h. Transcript and/or protein levels of Syn-1, β-hCG, Cx43, ABCG2, Gal-1, and GRP78 were measured by qRT-PCR and western blot, respectively. KLF6-BeWo cells were treated with a specific p21 siRNA or a scramble siRNA as control. KLF6 overexpression induced the formation of syncytial structures and increased the expression of molecules involved in this process. While, KLF6∆ac did not promote syncytialization, impaired forskolin-induced cell fusion, and decreased Syn-1 and β-hCG expression. Moreover, cell fusion was reduced in KLF6-BeWo cells transduced with the deletion mutant lentivirus or silenced for p21. These results suggest that KLF6 induces vCTB syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.