Phthalates Impair Estrogenic Regulation of HIF2α and Extracellular Vesicle Secretion by Human Endometrial Stromal Cells

Abstract

Di(2-ethylhexyl) phthalate (DEHP), a known endocrine-disrupting chemical, is a plasticizer found in many common consumer products. High levels of DEHP exposure have been linked to adverse pregnancy outcomes, yet little is known about how it affects human uterine functions. We previously reported that the estrogen-regulated transcription factor hypoxia-inducible factor 2 alpha (HIF2) promotes the expression of Rab27b, which controls the trafficking and secretion of extracellular vesicles (EVs). EVs facilitate communication between multiple cell types within the pregnant uterus, ensuring reproductive success. In this study, we report that exposure of differentiating primary human endometrial stromal cells (HESC) to an environmentally relevant concentration (1 g/mL) of DEHP or its primary metabolite mono(2-ethylhexyl) phthalate (MEHP) markedly reduces the expression of HIF2. We also observed a concomitant decrease in RAB27B expression, reducing EV secretion from HESC. Interestingly, we found that DEHP or MEHP exposure disrupts estrogenic regulation of the HIF2/Rab27b signaling pathway. Estrogen receptor alpha (ER) could no longer bind to the HIF2 regulatory region following phthalate treatment, and epigenetic analysis suggested that this may be due to hypermethylation of nearby CpG islands. Further investigation revealed a potential interaction between ER and the transcription factor Sp1 within the HIF2 regulatory region, which is affected by the inhibition of Sp1 binding to the phthalate-induced hypermethylated DNA. Additionally, our results suggest that the abnormal DNA methylation is likely due to increased expression of the DNA methyltransferase 1 (DNMT1) gene in response to phthalate exposure. Overall, this study provides valuable mechanistic insights into how phthalate-induced differential DNA methylation disrupts estrogenic regulation of the HIF2 gene and, consequently, EV secretion during HESC differentiation. This knowledge is crucial for our understanding of how phthalates may cause adverse reproductive outcomes by disrupting the hormonal regulation of cell-to-cell communication within the pregnant uterus.