Effects of cannabinoids on trophoblast cell growth and syncytialization

Background Recent statistics indicates a ~65% increase in pregnancy‐associated marijuana abuse in the US affecting ~4% of all pregnant women. Low birth weight, pre‐term birth and other fetal abnormalities have been linked to marijuana abuse during pregnancy. Cannabinoids (CB) are the active constituents of marijuana in which delta‐9‐tetrahydrocannabinol (Δ 9 ‐THC), cannabidiol (CBD) and cannabinol (CBN) are most active and interact with cannabinoid receptors (CNR) such as CNR1 and CNR2. Placenta is a transient organ which develops during pregnancy. Cytotrophoblast (CT) are predominant cell type of the placenta, which syncytialize/fuse to form multinucleated syncytiotrophoblast (ST) at the maternal‐fetal interface. Any interruption in this process can adversely affect placental development and fetal growth. Human placenta and the model human trophoblast cell line, BeWo, highly express CNR1 and CNR2 (Fig 1C). However, a clear mechanistic understanding of the adverse effects of CB on pregnancy is lacking. We hypothesize that CB compromise CT syncitialization and ST formation, thus affecting placental and fetal development and leading to adverse pregnancy outcomes. We tested this hypothesis using the Forskolin (FK)‐induced BeWo cell syncitialization model. Methods Effects of Δ 9 ‐THC, CBD and CBN at 0.2, 2 and 20 μM on BeWo cell viability in the presence and absence of 25 μM FK were evaluated using the MTT assay. Non‐cytotoxic CB concentration of 2 μM was used to treat a confluent monolayer of BeWo cells in the presence or absence of FK for 72 h to evaluate the effects of Δ 9 ‐THC, CBD or CBN on syncytialization. Changes in mRNA levels of syncitialization marker genes Dysferlin ( DYSF ) and Syncytin‐1 ( SYNC‐1 ) were determined using real‐time PCR. ELISA was used to quantify changes in hCG secretion by syncytializing BeWo cells using the above conditions. E‐cadherin immunostaining was used to assess changes in syncytialization upon CB treatment. Short‐hairpin RNA (shRNA) was used to knock‐down of specific CNRs in BeWo cells. Results and Discussion Δ 9 ‐THC, CBD and CBN all decreased BeWo cell viability with IC 50 values of ~10 μM. FK induced syncytialization as evidenced by induction of DYSF and SYNC‐1 mRNA (Fig 1A, B), appearance of multinuclear syncytia and increased secretion of hCG all of which were significantly decreased by 2 μM of Δ 9 ‐THC, CBD or CBN treatment. Δ 9 ‐THC treatment decreased phosphorylation of protein kinase A (PKA) substrates (Fig 1C). PKA signaling pathway plays a pivotal role in mediating trophoblast syncytialization. It is possible that CB potentially exert their toxicity by altering PKA‐mediated signaling in placental trophoblasts. Additionally, Δ9‐THC at 20 μM significantly increased cellular ceramide production (Fig 1D). We are currently evaluating if the effects of CB on BeWo cell proliferation and syncytialization are mediated through CNR by knocking down CNR individually or in combination. These studies will provide the first mechanistic insights into the adverse effects of marijuana abuse on pregnancy that are important for developing intervention strategies to address CB toxicity to the placenta and fetus. Support or Funding Information National Institutes of Health (NIH) Institute on Drug Abuse [Grant DA032507], University of Washington‐National Institute of Environmental Health Sciences (NIEHS)‐sponsored Interdisciplinary Center for Exposures, Diseases, Genomics, and Environment [Grant ES007033]. University of Washington, Alcohol and Drug Abuse Institute‐Small Grant Award. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .