(W1130-04-22) Valproic Acid Elevates HIF-1α-mediated CGB Expression and Suppresses Glucose Uptake in BeWo Cells

Purpose: Pregnant women and fetuses are vulnerable to many chemicals. Chemical exposure can be a risk factor for adverse outcomes such as spontaneous abortion and developmental disorders. Therefore, it is necessary to understand how chemicals can cause reproductive toxicity. The reproductive toxicity of chemicals is determined mainly from the presence or absence of toxic signs, such as maternal general toxicity, maternal reproduction, and the development of the next generation. On the other hand, the evaluation of the placenta is limited. The placenta plays essential roles in the formation and maintenance of pregnancy. However, placental function can be harmed through exposure to various chemicals. Although a relationship between impairment of placental function due to chemical exposure and adverse pregnancy outcomes has been suggested, how chemical exposure causes reproductive toxicity by affecting placental function is still obscure. Within the placenta, chorionic villi mediate material exchange between the maternal blood and the fetal blood. They consist of two types of cell layers, made of cytotrophoblasts and syncytiotrophoblasts. Syncytiotrophoblast cells are formed in early pregnancy by the syncytialization process, by which mononuclear cytotrophoblast cells fuse with each other and play a central role in maintaining placental function, such as producing placental hormones to maintain pregnancy and supplying nutrients to the fetus. To investigate the aspect, we used valproic acid (VPA) as a model chemical. VPA has been used from the late 1900s clinically for the treatment of epilepsy and bipolar disorder. However, VPA presents a risk of reproductive and developmental toxicity. Here, we examined the effects of VPA on production of hormones and on glucose uptake in human choriocarcinoma cell line BeWo.

Methods: Cells were treated with forskolin to differentiate into syncytiotrophoblasts, which were then treated with VPA for 72 h. The mRNA expression of chorionic gonadotropin β (CGB), a hormone that is produced by the placenta in the first trimester of pregnancy, was analyzed by real-time RT-PCR. We evaluated its effect on glucose uptake in Frk-treated BeWo cells as the amount of intracellular fluorescence as an index when tagged glucose was added.

Results: Frk treatment significantly increased CGB expression relative to the untreated cells, and subsequent VPA treatment further increased it concentration-dependently. VPA increased hCGβ production concentration-dependently. These results show that VPA can disrupt placental hormone production in differentiated BeWo cells. Moreover, Frk increased the intracellular uptake of fluorescent glucose relative to the untreated group, but VPA decreased it concentration-dependently relative to Frk alone. Since VPA decreased glucose uptake, we evaluated its effect on the level of GLUT1 by western blotting analysis. Frk increased the production of GLUT1 and VPA decreased it. These results suggest that the suppression of glucose uptake by VPA in differentiated BeWo cells involves VPA-induced downregulation of GLUT1. To elucidate how VPA harms placental function, we analyzed genes whose expression it altered in BeWo cells. Frk increased the expression of 450 genes and VPA decreased it. Pathway analysis of these genes identified the “HIF-1 transcriptional activity in hypoxia” pathway, as having the most significant q-value. Thus, to evaluate the production of molecules involved in that pathway, we assessed the effect of VPA on HIF-1α protein production. Frk increased the production of HIF-1α and VPA decreased it concentration-dependently. Then, we evaluated placental dysfunction induced by VPA after HIF-1α activity was increased by co-treatment with VPA and CoCl₂, a HIF-1α inducer, in Frk-treated BeWo cells. Co-treatment with VPA + CoCl₂ inhibited the increase in CGB expression induced by VPA. Furthermore, VPA suppressed glucose uptake relative to Frk alone, while VPA + CoCl₂ increased it to the same extent as Frk alone. Besides, VPA decreased the production of GLUT1 relative to Frk alone, while VPA + CoCl₂ increased it. These results suggest that VPA modulates CGB expression and inhibits glucose uptake and GLUT1 production through inhibition of HIF pathway activity in differentiated BeWo cells.

Conclusion: Collectively, we found that in Frk-treated BeWo cells, VPA increased CGB expression and suppressed glucose uptake under mediation by HIF pathway activity. However, how VPA inhibits HIF pathway activity, and the relationship between HIF pathway activity and CGB expression have not been elucidated. Understanding these will lead to an understanding of the reproductive toxicity of VPA.

Acknowledgements: This study was supported by a Health Labor Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan. The authors declare that they have no competing interests.