HYPOXIA INDUCIBLE FACTOR-1 ALPHA IMPAIRS SYNCYTIALIZATION OF HUMAN TROPHOBLAST BEWO CELLS: ROLE OF ENDOCANNABINOID SYSTEM.

ETCHEVERRY, T; ACCIALINI, P; PALLIGAS, M; ABÁN, C; SARACO, N; MARTINEZ, N; FARINA, M

Congreso; 2019 IFPA - VIII SLIMP; 2019

Objectives: We have reported thathypoxia inducible factor-1 alpha (HIF-1α) deregulates the endocannabinoidsystem (ECS). The aim of the present work was to assess the impact of HIF-1αstabilization and ECS on BeWo cell line syncytialization.Methods: BeWo cell line wascultured with 25µM forskolin (FSK) to induce cell fusion. Stimulated cells weretreated with or without cobalt chloride (50µM-100µM CoCl2), ahypoxia-mimetic agent that stabilizes HIF-1α; or R-(+)-Methanandamide (0.01µM-10µMMet-AEA), a non-hydrolyzable anandamide analogous. AM251, SR141716 and AM630(0.01µM-1µM) were used to antagonizecannabinoid receptors (CBs). Cell viability and integrity were analyzed by MTTassay and LDH activity, respectively. HIF-1α stabilization was confirmed byWestern blot. Fatty acid amide hydrolase (FAAH), the enzyme that degradesanandamide, mRNA levels were measured by RT-qPCR. Several parameters were usedto study syncytialization: GCM1 mRNAlevels by RT-qPCR; human chorionic gonadotropin (hCG) release bychemiluminescence immunoassay; syncytin-1 protein expression, propidium iodide (PI)staining and cell size by flow cytometry; and E-cadherin distribution byimmunocytochemistry.Results: Cells treated with FSKshowed increased GCM1 mRNA levels, hCG secretion, syncytin-1 proteinexpression, DNA content (PI staining) and cell size compared to the non-treatedcontrol (p<0.05). Localization of E-cadherin confirmed the results above.Incubationwith Met-AEA significantly diminished GCM1 mRNA levels and hCG secretion(p<0.05). These findings were corroborated by E-cadherin distribution and flowcytometry: cells showed less syncytin-1 protein expression, less DNA contentand smaller size compared to forskolin-induced controls (p<0.05). Incubationwith CoCl2 significantly (p<0.05) diminished GCM1 mRNA levels andhCG secretion; and produced a reduction in cell fusion evidenced by immunocytochemistry.CoCl2 significantly decreased FAAH mRNA levels (p<0.05). Moreover,when CBs antagonists were added, GCM1 mRNA decrease was partially impaired(p<0.05).Conclusions: HIF-1α and Met-AEA decreaseforskolin-induced syncytialization. One of the mechanisms whereby HIF-1α disrupts syncytialization could be to increase endcannabinoidsignaling.