Connecting with Mom -Trophoblast and Placenta
Session: Poster Session A
Haijun Gao, PhD
Assistant Professor
Howard University College of Medicine
Washington, District of Columbia, United States
Albert Gao1, Bin Wu2, Bin He3, Haijun Gao1
1. Department of Physiology & Biophysics, Howard University College of Medicine, Washington DC, USA
2. Department of Reproductive Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, China
3. Department of Cell Biology, National Institute for Family Planning, Beijing, China
Abstract Text: The placental plays a critical role in maintaining pregnancy, nurturing the fetus, mediating the communication between the mother and the fetus, and predisposing adult health and disease in offspring. Our recent study suggested the impaired mitophagy in the placentas from pregnant subjects with gestational diabetes mellitus (GDM) and the regulatory role of BNIP3 in mitophagy of trophoblast cells in response to AMPK signaling, however, what’s the potential role of BNIP3 mediated mitophagy in the functions of placenta and trophoblast cells remains unclear. In this study, we hypothesized that BNIP3 can regulate mitophagy and mitochondrial functions in human trophoblast cells. In Study One, placental tissues were collected from pregnant subjects carrying a male fetus without (NGT) or with gestational diabetes mellitus (GDM) and the abundance of BNIP3 in mitochondrial fraction of placental tissues was measured by Western blotting; In Study Two, we knocked down BNIP3 expression in human trophoblast cell line BeWo using Lentiviral shRNA transfection (BKD), then analyzed the protein abundance of mitophagy mediators (LC3II, SQSTM1) in mitochondrial fractions by Western blotting, mitochondrial membrane potential by TMRM staining, ATP production by Seahorse Cell Stress Test; In Study Three, to explore genes which interact with BNIP3 in mitochondrial regulation, next generation RNAseq was conducted in BKD and CT cells; In Study Four, to investigate the effect of BKD on syncytiotrophoblast formation, BKD and CT cells were treated with forskolin (20µM) for 48 hours, and the expression of syncytialization related genes (CGB, ERVW1, ERVFRD-1, HSD11B2, CDH1) was analyzed by q-PCR and normalized to that of ribosomal 18S. All numerical parameters between the NGT and GDM groups, between BKD and CT groups were analyzed by ANOVA (n=5). The main findings include: 1) The protein abundance of BNIP3 in the male placental tissues was reduced by 1.29-fold in GDM compared to NGT group (P< 0.05); 2) The protein abundance of LC3II M1 in mitochondrial fraction was 1.35-fold lower (P< 0.001) in BKD compared to CT cells, while that SQSTM1 was increased by 1.88-fold (P< 0.05); 3) Mitochondrial membrane potential presented in TMRM signals was 1.21-fold higher (P< 0.001) in BKD compared to CT cells; 4) The basal and maximal respiration rates, ATP-production coupled respiration and spare respiratory capacity were 1.22-, 1.39-, 1.26- and 1.74-fold lower (P< 0.05) in BKD compared to CT cells, respectively; 5) mRNA levels of mitochondria related genes TOMM6, MT-ATP6, NOL3 and IFI6, were reduced by 9.1-, 2.2-, 1.6-, and 1.5-fold in BKD cells, respectively, while the expression of MCUB was increased by 1.5-fold; 6) With the treatment of Forskolin, mRNA levels of syncytiotrophoblast marker genes (CGB, ERVW1, ERVFRD-1, HSD11B2) in BKD cells were reduced by 1.33-, 1.26-, 1.36 and 1.20-fold, respectively (P< 0.05), compared to CT cells. These results suggest that BNIP3 and BNIP3 mediated mitophagy pathway may play an important role in mitochondrial homeostasis in trophoblast cells, and the reduced BNIP3 expression in the placenta may lead to mitochondrial structural and functional defects, thus affecting placental functions and contributing to the development of GDM (Supported by NIH research grants R16HD116702, R03HD95417, U54MD007597, DC CFAR Pilot Award).