Inhibition of Cyclic Nucleotide Efflux by Placental MRP Transporters Enhances Trophoblast Syncytialization

Abstract

In the placenta, multinucleated syncytiotrophoblasts arise from the fusion of cytotrophoblasts, a process regulated by cyclic nucleotide (cAMP and cGMP) signaling. Syncytiotrophoblasts secrete hormones and express efflux transporters, including the multidrug resistance‐associated proteins (MRP) 1 and 5. Understanding the interplay of cyclic nucleotides, efflux transporters, and syncytialization is important because aberrant cytotrophoblast functioning contributes to the pathogenesis of placental diseases and toxicities. In the present study, we sought to determine whether MRP transporters regulate the syncytialization of human placental cells. BeWo b30 trophoblasts were treated with vehicle, MK‐571 (25 μM), 8‐Bromo‐cAMP (100 μM), forskolin (25 μM), IBMX (200 μM), or their combination for up to 24 hr. Markers of trophoblast cell fusion (glial cells missing homolog 1, GCM1; syncytin 2) and hormone secretion (human chorionic gonadotropin, hCGa and hCGb) were quantified by qPCR. Treatment of BeWo cells with MK‐571, a pharmacological inhibitor of MRPs, increased intracellular concentrations of cAMP and cGMP by 15‐ and 19‐fold, respectively. Moreover, MK‐571 treatment up‐regulated cell fusion and hormone secretion markers (GCM1, hCGa, hCGb, syncytin 2) by 20–200% under basal conditions. Similarly, MRP inhibition with MK‐571 further enhanced syncytialization and hormone markers following stimulation with the permeable cAMP analog 8‐Bromo‐cAMP (2‐ to 6‐fold), activation of cAMP synthesis with forskolin (20–160%), or inhibition of cAMP/cGMP breakdown by IBMX (50–130%). In addition, shRNAs were used to generate a stable BeWo B30 trophoblast cell line with reduced MRP5 expression. Control and MRP5‐knockdown cells were treated with vehicle or 8‐Bromo‐cAMP for 24 hr. In response to treatment with 8‐Bromo‐cAMP, the induction of GCM1, hCGa, and hCGb mRNA expression was higher in MRP5 knockdown cells as compared to control cells. Furthermore, MRP5‐knockdown cells were larger in size (including greater numbers of multinucleated cells) and exhibited decreased staining of the tight junction protein E‐cadherin, pointing to enhanced syncytialization. In conclusion, MRP transporters participate in trophoblast cell fusion, which likely represents a novel mechanism regulating placentation and placental toxicities.Support or Funding InformationSupported by F31ES029794, R01ES029275, T32ES007148, P30ES005022.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.