Forskolin Induced Syncytialization in Placental Trophoblast Induces Changes in Glycocalyx Composition

Abstract

The placenta is known to function as a bidirectional selectively permeable barrier between maternal and fetal circulatory systems during pregnancy. The fetal placental villi are surrounded by a syncytiotrophoblast‐cell layer, which is the first functional layer of the placental barrier. Syncytiotrophoblasts possess an apical extracellular matrix (ECM) which functions similarly to the endothelial glycocalyx; acting as a mechanical barrier for large particles and by regulating permeability and transport of molecules. Among the most important ECM proteins are the syndecan (SDC) family. The immortalized trophoblast‐like BeWo cell line is a commonly used in vitro model of placental trophoblasts, however, they do not spontaneously syncytialize and thus do not represent an accurate model to study placental barrier function. However, they can be induced to syncytialize by addition of forskolin, an adenylate cyclase stimulator. Here we hypothesized that forskolin‐induced syncytialization of BeWos upregulates ECM protein expression and produces a morphological phenotype through which placental barrier function can be studied in the future. To test our hypothesis, we treated BeWos with 25 uM Forskolin or DMSO as a control for 48 hours and measured SDC gene expression and protein levels. After treatment, media and cells were collected for analysis. Because SDC has four non‐allelic variants in rodents, SDC‐1, ‐2, ‐3, and ‐4, we measured the expression of all four via qRT‐PCR and normalized them to β‐actin expression. SDC‐1, ‐2, and ‐3 expression were all increased in the forskolin treated group compared to controls (p<0.0001 for all comparisons). Interestingly, SDC‐4 expression was decreased by treatment with forskolin (p=0.004). Next, we wished to verify our results and also to identify the predominant SDC isoforms in BeWos. In order to do this we utilized digital droplet PCR to determine absolute copy number. Again, we saw increases in SDC‐1, SDC‐2, and SDC‐3 expression in the forskolin treated group compared to controls (p<0.0001 for all comparisons). We also observed decreased SDC‐4 expression in the forskolin treated group compared to controls using this method (p<0.0001). Additionally, we observed that SDC‐1 (2255 ± 119 copies/ng RNA) and SDC‐4 (2881 ± 136.6 copies/ng RNA) were the predominant isoforms expressed in BeWos under control conditions, while SDC‐2 (15 ± 0.7 copies/ng RNA) and SDC‐3 (124 ± 5.4 copies/ng RNA) were expressed at very low levels. We then measured cellular and media SDC‐1 protein levels via ELISA. Compared to controls, forskolin treatment significantly increased cellular levels of SDC‐1, which were normalized to total protein (1.23 pg/ug ± 0.05 vs 2.56 pg/ug ± 0.22, respectively; p=0.002). Forskolin treatment also significantly increased media SDC‐1 compared to controls (1189 ± 10 pg/mL vs 555.5 ± 19 pg/mL, respectively; p<0.0001). From this data we concluded that forskolin‐induced syncytialization of BeWos regulates the composition and quantity of the placental glycocalyx. Future studies of placental barrier function in vitro should first induce syncytialization with forskolin treatment for 48 hours before additional protocols are carried out.Support or Funding InformationP01HL51971, P20GM104357, T32HL105324, and R01HL137791