Renin and renin-like activities have been detected in early gestation placentae1,2. To explore what regulates RAS expression in trophoblasts we studied the effects of cAMP and AZA (DNA demethylating agent) on the expression of mRNAs for prorenin receptor (ATP6AP2), prorenin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE2, angiotensin II type 1 receptor (AGTR1), and a downstream target of the prorenin receptor, cyclooxygenase-2 (PTGS2) in the early human trophoblast cell line HTR-8/SVneo. Cells were cultured for 24 or 48 h with vehicle, 0.5 mM cAMP or 15 mM AZA. Messenger RNA abundances were measured relative to Alien RNA using real-time qRT-PCR. All mRNAs were detected except for ACE and ACE2. REN mRNA abundance was increased by cAMP and AZA (P < 0.0001). However, cAMP was more effective than AZA at both 24 and 48 h (P < 0.0001; P < 0.05) at the concentrations employed. AZA significantly increased AGT mRNA levels at 24 h (P = 0.001) and AGTR1 mRNA levels at 48 h (P < 0.0001), while cAMP had no effect. P TGS2 mRNA expression was increased by cAMP at 24 h (P < 0.0001) and by AZA at 48 h (P < 0.0001). cAMP and AZA had no effect on ATP6AP2 abundance. Thus, prorenin may function directly through the prorenin receptor, since cAMP upregulated both REN and its downstream target PTGS2 in HTR-8/SVneo cells, especially considering that ACE and ACE2 were not expressed. Our findings also suggest that expression of the RAS in trophoblast is regulated by DNA methylation as there was sustained over expression of REN, AGT, AGTR1 and PTGS2 with AZA. The ATP6AP2 promoter possesses CpG islands; however, its expression was not affected by AZA. This may suggest that either DNA methylation is not involved in the regulation of this gene, or that the over expressed prorenin binding to its receptor is causing suppression of ATP6AP2 abundance via a previously identified negative feedback pathway.
(1) Pringle et al., Perinatal Society of Australia & New Zealand (PSANZ) 2010, Wellington, New Zealand (Abstract).(2) Itskovitz et al., Journal of Clinical Endocrinology and Metabolism, 1992, 75: 906–10.
Effect of steroids on transcription and secretion of Gal-1 by the human trophoblast cell line in vitro
