ABSTRACT: Estrogen-related receptor M-NM-3 (ERRM-NM-3) serves a critical O2-dependent regulatory role in differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikreins (KLK1) and voltage-gated K+ channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRM-NM-3 and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRM-NM-3, expression of KLK1, KCNQ1, KCNE1, KCNE3 and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with shRNAs for endogenous ERRM-NM-3, KLK1, KCNQ1, KCNE1 and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRM-NM-3 response elements (ERREs) within the KLK1 and KCNE1 5'-flanking regions required for ERRM-NM-3 -stimulated transcriptional activity. Binding of endogenous ERRM-NM-3 to these ERREs increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced hCG secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRM-NM-3 knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRM-NM-3 mediates O2-dependent expression of genes involved in human trophoblast differentiation, function and vascular homeostasis. Illumina whole genome gene expression array analysis was performed on human trophoblasts before culture, or 72 h after infection with recombinant lentiviruses expressing ERRM-NM-3 shRNA, or a control, nonsilencing shRNA. RNA from three independent experiments using placental tissues from three abortuses was extracted by miRNeasyM-BM-. Mini Kit (Qiagen, Maryland, USA). RNA was labeled and hybridized to an Illumina HumanHT-12 v4 BeadChip, according to the manufacturerM-bM-^@M-^Ys protocol.
