Phospholipase D1 as a key enzyme for decidualization in human

27 28 Using a primary cell culture system of human endometrial stromal cells (ES cells), we investigated 29 the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which is a 30 morphological and biological differentiation process. When treated with 0.5 mM 8-Br-cAMP for 12 31 days, ES cells were transformed into a decidualized morphology and produced significant amounts 32 of prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Simultaneously, the 33 activity and expression level of PLD1 increased also. In addition, removal of 8-Br-cAMP from 34 decidualized ES cells restored the undifferentiated state, accompanied with a decrease of PLD1 35 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 36 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas overexpression of 37 PLD1 induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, 38 knockdown of PLD1 by siRNA and blockage of PLD by 0.3% 1-butanol treatment decreased 39 PRL/IGFBP1 mRNA expression, whereas overexpression of PLD1 increased PRL/IGFBP1 mRNA 40 expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA 41 expression and morphological change, implying that PA as an end product of PLD activation 42 induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased 43 PRL/IGFBP1 expression and morphological change, whereas pretreatment with propranolol did not 44 change, compared to cAMP-treated cells, suggesting that PA induces decidualization through 45 phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br46 cAMP-induced decidualization through PLA2G1B, and that upregulation of PLD1 is essential for 47 decidualization of ES cells. 48