Phospholipase D1 as a key enzyme for differentiation : Decidualization in human endometrial stromal cells

초록

Using a primary cell culture system of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which is a morphological and biological differentiation process. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Simultaneously, the activity and expression level of PLD1 increased also. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, accompanied with a decrease of PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas overexpression of PLD1 induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by 0.3% 1-butanol treatment decreased PRL/IGFBP1 mRNA expression, whereas overexpression of PLD1 increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological change, implying that PA as an end product of PLD activation induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and morphological change, whereas pretreatment with propranolol did not change, compared to cAMP-treated cells, suggesting that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that upregulation of PLD1 is essential for decidualization of ES cells