Docosahexaenoic Acid Induces Adipose Differentiation-Related Protein through Activation of Retinoid X Receptor in Human Choriocarcinoma BeWo Cells
Biological and Pharmaceutical Bulletin
Intracellular lipid droplet (LD) is a cellular organelle observed ubiquitously in many cell types including chorionic cells. LDs are storage sites for neutral lipids and may play a central role in whole body energy homeostasis and distribution of lipids. 1,2) Adipose differentiation-related protein (ADRP; also called as ADFP or adipophilin) is a major LDforming protein expressed in a wide variety of cells and tissues including placenta. 3-7) Addition of high concentrations of fatty acids to
... fatty acids to various cells in culture up-regulates both Adrp mRNA and ADRP protein, 8) while ADRP is degraded via the ubiquitin-proteasome mechanism when cellular triacylglycerol (TG) is reduced. 9,10) These observations suggest that ADRP has a role in cellular fatty acid uptake and storage. Placental transport of fatty acids to the fetus is essential for proper fetal growth and development. Fetal demand for fatty acids increases during the latter stages of pregnancy, and long chain n-3 polyunsaturated fatty acids (LCPUFA) are supplied from the maternal circulation. 11) Docosahexaenoic acid (DHA: 22 : 6, n-3) plays a particularly important role in fetal development because it is a major lipid constituent in the brain and retina. 12,13) The blood DHA concentration is higher in the fetus than in maternal circulation, suggesting an active placental transfer. 12) In BeWo cells, a human placental choriocarcinoma cell line, DHA was preferentially incorporated into TG. 14) From postmortem studies of fetuses, stillbirths, and preterm infants, it was estimated that the fetus consumes Ͻ50-60 mg/d of n-3 LCPUFA during the last trimester of pregnancy, which are probably transferred from the placenta. 15) Peroxisome proliferator-activated receptor-g (PPARg) is a nuclear receptor that acts as a transcription factor by forming a heterodimer with its partner, retinoid X receptor (RXR). Both PPAR-g and RXR are essential for proper placental development, since disruption of these genes in mice resulted in embryonic death, with embryos exhibiting placental defects, including improper labyrinth development and reduced LD in trophoblasts. 16, 17) Sadovsky and colleagues demonstrated that PPARg enhances the differentiation of cultured primary term human trophoblasts, and that PPARg increases LDs in syncytiotrophoblasts. 18) Since it has been suggested that DHA is a natural ligand of RXR, 19, 20) we assumed that DHA plays a role in placental development through ADRP expression and LD formation. In this study, we examined DHA-dependent ADRP expression in BeWo cells. BeWo cells, a human placental choriocarcinoma cell line, maintain several characteristics of natural trophoblasts. We showed that DHA induces Adrp mRNA expression and ADRP protein accumulation in BeWo cells. The DHA-dependent ADRP accumulation was inhibited by RXR-antagonists, indicating that DHA acts as a natural RXR-agonist to induce ADRP. MATERIALS AND METHODS Cell Culture BeWo cells were cultured in Ham's F12 medium with 15% fetal bovine serum (FBS) supplemented with 2 mM L-glutamine and 1% antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin). The cells were maintained at 37°C in a 5% CO 2 atmosphere. Cells were supplemented with 100 mM DHA or oleic acid (OA) to induce LD formation. Stock solutions of 4 mM DHA and OA were prepared as Adipose differentiation-related protein (ADRP) is associated with intracellular lipid droplets that accumulate neutral lipids. Here we report that ADRP expression in a human choriocarcinoma cell line, BeWo, is regulated through activation of retinoid X receptor (RXR) and peroxisome proliferator-activated receptor-g g (PPARg g). Incubation with docosahexaenoic acid (DHA) or oleic acid (OA) induced accumulation of triacylglycerol (TG) and ADRP in BeWo cells. DHA-induced ADRP expression was suppressed by RXR-antagonists, PA452 and HX531. However, oleic acid-induced ADRP expression was not blocked by the RXR-antagonists but by a PPARg gantagonist. Treatment of the cells with RXR-agonists, HX630 and PA024, increased Adrp transcripts, however, they alone did not change the levels of ADRP protein and TG in BeWo cells. Induction of ADRP protein was observed in the presence of a proteasome inhibitor, suggesting that ADRP is degraded under lipid-poor conditions. These results suggest that expression of ADRP is in part regulated by RXR and PPARg g transcription factors, and DHA induces ADRP by acting as an endogenous agonist of RXR.