We assessed the response of primary cultures of placental villous mononucleated trophoblasts and multinucleated syncytiotrophoblast to calcitriol, the most biologically active form of vitamin D. Whole-genome microarray data showed that calcitriol modulates the expression of many genes in trophoblasts within 6 hours of exposure and RT-qPCR revealed similar responses in cytotrophoblasts, syncytiotrophoblasts and villous explants. Both cytotrophoblasts and syncytiotrophoblasts expressed genes for the vitamin D receptor, for LRP2 and CUBN that mediate internalization of calcidiol, for CYP27B1 that encodes the enzyme that converts calcidiol into active calcitriol, and for CYP24A1 that encodes the enzyme that modifies calcitriol and calcidiol to inactive calcitetrol. Notably, we found an inverse effect of calcitriol on expression of CD14 and CD180/RP105, proteins that differentially regulate toll-like receptor 4-mediated immune responses. Supported by gene ontology analysis, we tested the hypothesis that CD14 and CD180 modulate the inflammatory response of syncytiotrophoblast to bacterial lipopolysaccharide (LPS). These cells showed a robust response to a wide range of LPS concentrations, with induction of active NF-κB and increased secretion of IL-6 and IL-8. SiRNA-mediated knockdown of CD14 reduced the secretion of IL-6 and IL-8 in response to LPS. Collectively, our data showed that calcitriol has a rapid and widespread effect on villous trophoblast gene expression in general, and a specific effect on the innate immune response by syncytiotrophoblast.
Mark S Longtine, Silvija Cvitic, Bryanne N Colvin, Baosheng Chen, Gernot Desoye and D Michael Nelson
Bryanne N Colvin, Mark S Longtine, Baosheng Chen, Maria Laura Costa and D Michael Nelson
Pre-pregnancy obesity is increasingly common and predisposes pregnant women and offspring to gestational diabetes, pre-eclampsia, fetal growth abnormalities and stillbirth. Obese women exhibit elevated levels of the two most common dietary fatty acids, palmitate and oleate, and the maternal blood containing these nutrients bathes the surface of trophoblasts of placental villi in vivo. We test the hypothesis that the composition and concentration of free fatty acids modulate viability and function of primary human villous trophoblasts in culture. We found that palmitate increases syncytiotrophoblast death, specifically by caspase-mediated apoptosis, whereas oleate does not cause enhanced cell death. Importantly, exposure to both fatty acids in equimolar amounts yielded no increase in death or apoptosis, suggesting that oleate can protect syncytiotrophoblasts from palmitate-induced death. We further found that palmitate, but not oleate or oleate with palmitate, increases endoplasmic reticulum (ER) stress, signaling through the unfolded protein response, and yielding CHOP-mediated induction of apoptosis. Finally, we show that oleate or oleate plus palmitate both lead to increased lipid droplets in syncytiotrophoblasts, whereas palmitate does not. The data show palmitate is toxic to human syncytiotrophoblasts, through the induction of ER stress and apoptosis mediated by CHOP, whereas oleate is not toxic, abrogates palmitate toxicity and induces fat accumulation. We speculate that our in vitro results offer pathways by which the metabolic milieu of the obese pregnant woman can yield villous trophoblast dysfunction and sub-optimal placental function.