Estrogen-related receptor γ (ERRγ) regulates oxygen-dependent expression of voltage-gated potassium (K+) channels and tissue kallikrein during human trophoblast differentiation

Abstract

Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in the differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikrein (KLK1) and voltage-gated K(+) channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, 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 short hairpin RNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1, and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements within the KLK1 and KCNE1 5'-flanking regions required for ERRγ-stimulated transcriptional activity. Binding of endogenous ERRγ to these ERRγ response elements increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced human chorionic gonadotropin 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 ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function, and vascular homeostasis.

Figure 1.

Expression of the K⁺ channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, and the kallikrein, KLK1, are induced in concert with ERRγ in human trophoblast cells during differentiation in culture and are inhibited by hypoxia. A, Freshly isolated human placental cytotrophoblasts were cultured in a 20% O₂ or 2% O₂ environment for 24, 48, and 72 hours. RNA was isolated from cells either before (Cyto) or after culture, and the expression of ERRγ, CYP19I.1, K_V channel α-subunits (KCNQ1, KCNQ3, and KCNQ5) and β-subunits (KCNE1, KCNE3, KCNE4, and KCNE5) and KLK1 mRNA was analyzed by qRT-PCR; RPLP0 was used as the internal reference. Data are the mean ± SEM of values from 3 independent experiments, each conducted in triplicate, and are expressed relative to expression levels in cytotrophoblasts before culture. *, Significantly different (P < .05) from values of cells cultured in 20% O₂. B, Total proteins extracted from cells cultured in a 20% O₂ or 2% O₂ environment for 72 hours were analyzed by immunoblotting using antisera to ERRγ, KCNQ1, KCNE1, and KLK1 or GAPDH, as loading control.

Figure 2.

Knockdown of endogenous ERRγ inhibits KCNQ1, KCNE1, KCNE3, and KLK1 expression in human placental cells. A, Freshly isolated human cytotrophoblasts were infected with lentiviral vectors carrying shRNA targeting ERRγ (shERRγ) or control shRNA (shcon). Nontreated cells (NTC) served as another negative control. RNA was isolated 72 hours later, and expression of ERRγ, CYP19I.1, K⁺ channel α-subunits (KCNQ1) and β-subunits (KCNE1, KCNE3, and KCNE5) and KLK1 mRNA was analyzed by qRT-PCR; RPLP0 was used as the internal reference. Data are the mean ± SEM of values from 3 independent experiments, each conducted in triplicate, and are expressed relative to expression levels in nontreated cells. *, Significantly different (P < .05) from values of cells transduced with control shRNA. B, Total proteins extracted from cells infected with lentiviral vectors carrying shRNA targeting ERRγ (shERRγ) or control shRNA (shcon) for 72 hours were analyzed by immunoblotting using antisera to ERRγ, KCNQ1, KCNE1, and KLK1 or GAPDH, as loading control.

Figure 3.

ERRγ directly upregulates KCNE1 and KLK1 promoter activity. A and B, COS-7 cells were transiently cotransfected with KCNE1-luciferase (A) and KLK1-luciferase (B) reporter plasmids, with or without mutation of putative ERREs (KCNE1m1, KCNE1m2, KCNE1m1,2, or KLK1m), along with either an ERRγ expression plasmid or empty control plasmid. All cells were cotransfected with β-gal expression plasmid as a control for transfection efficiency. The cells were lysed 48 hours after transfection and assayed for luciferase and β-gal activities. Data are the mean ± SEM of normalized values from 3 independent experiments, each conducted in triplicate, and are expressed as fold induction of relative luciferase activity upon ERRγ cotransfection over reporter alone. *, Significantly different (P < .05) from wild-type KCNE1-luc or KLK1-luc. C and D, Freshly isolated human placental cytotrophoblasts (0 hours) were cultured in 20% O₂ or 2% O₂ for 24 or 48 hours. The freshly isolated and cultured trophoblasts were treated with 1% formaldehyde and subjected to ChIP analysis using ERRγ IgG (ERRγ) or nonimmune IgG, as control. The immunoprecipitated ERRγ complexes bound to a 154-bp region (−914 to −761 bp) surrounding the KCNE1 ERRE-1 site, an 88-bp region (−107 to −20 bp) surrounding the KCNE1 ERRE-2 site (indicated by the arrows) (C) or a 107-bp region (−950 to −844 bp, indicated by the arrows) surrounding the KLK1 ERRE site (D) were quantified by qPCR using specific primers. The ChIP data were normalized to input control data and expressed relative to binding at the 0-hour time point. The data shown are the mean ± SEM of values from 3 independent experiments. Complexes immunoprecipitated with nonspecific IgG in the same experiment were quantified as a negative control. *, Significantly different (P < .05) from values of cells cultured in 20% O₂.

Figure 4.

Genomic analysis of ERRγ function. Freshly isolated human cytotrophoblasts were infected with a lentiviral vector carrying shRNA targeting ERRγ (shERRγ) or control shRNA (Con). RNA was isolated from cells either before (Cyto) or 72 hours after culture (Syncytio) from 3 independent experiments, and genome-wide analysis of gene expression was performed using Illumina whole-genome gene expression arrays to uncover processes that are regulated by ERRγ during trophoblast differentiation. A, Expression analysis detected altered expression of 1610 genes before and after syncytiotrophoblast differentiation (Cyto vs Con) (differentiation group). Altered expression of 303 genes was observed when we compared ERRγ knockdown v. control (shERRγ vs Con) (ERRγ knockdown group). There were 97 genes differentially regulated both by differentiation and ERRγ knockdown. B, These 97 genes were divided into 4 groups according to their expression during differentiation and ERRγ knockdown. C, Heat map of differentially expressed genes upregulated during differentiation and downregulated by ERRγ knockdown (group B) or downregulated during differentiation and upregulated by ERRγ knockdown (group C). The heat map shows in green the genes that are downregulated and in red the genes that are upregulated. D, Biological functions of differentially expressed genes. E, Gene expression of HSD11B2, HSD17B1, and PLAC1 was validated by qRT-PCR; RPLP0 was used as the internal reference. Data are the mean ± SEM of values from 3 independent experiments, each conducted in triplicate, and are expressed relative to expression levels in cytotrophoblasts before culture. *, Significantly different (P < .05) from values of cytotrophoblasts before culture; #, significantly different (P < .05) from values of cells transduced with control shRNA.

Figure 5.

Voltage-gated K⁺ channel blockers inhibit differentiation of human trophoblast cells. A, Freshly isolated human placental cytotrophoblasts were treated with voltage-gated K⁺ channel blocker 4-AP (0.01 mM, 0.1 mM, or 1 mM), K_V7 channel blocker LP (0.01 mM or 0.1 mM), or ethanol (con) for 72 hours. RNA was isolated from cells after treatment, and the expression of CYP19I.1 and hCGβ mRNA was analyzed by qRT-PCR; RPLP0 was used as the internal reference. *, Significantly different (P < .05) from values of cells treated with ethanol. B and C, Culture medium was collected 24 and 72 hours after the initiation of treatment with 4-AP (1 mM), LP (0.1 mM), or ethanol (control), and assayed for secreted hCG using ELISA (B) or LDH release using a cytotoxicity detection kit (C). Data are the mean ± SEM of values from 3 independent experiments, each conducted in duplicate, and are expressed relative to the 24-hour control. *, Significantly different (P < .05) from control. D, After 72 hours of treatment with 4-AP (1 mM), LP (0.1 mM), or ethanol (control), the cells were stained with hematoxylin and eosin and viewed by light microscopy (×200). ImageJ was used to quantify the percentage of clustered cells relative to total cell number in 8 different fields from 2 independent experiments performed in duplicate. *, Significantly (P < .05) decreased compared with control.

Figure 6.

Proposed mechanisms for ERRγ regulation of K⁺ channels and KLK1 during human trophoblast differentiation. In cytotrophoblasts and in cultured trophoblasts maintained in a hypoxic environment, decreased ERRγ levels prevent expression of selected K_V7 channels and KLK1. This prevents dilation of the vascular bed and inhibits vascularization, causing further hypoxia. The inhibition of K_V7 expression results in decreased hCG secretion, which suppresses trophoblast differentiation. On the other hand, increased placental vascularization and increased oxygen tension after 8–10 wk gestation promotes increased ERRγ expression, which enhances KLK1 expression, resulting in vasodilation and increased angiogenesis, further increasing O₂ tension and ERRγ expression. This, in turn, enhances expression of selected K_V7 channels resulting in increased hCG expression, which acts to further promote syncytiotrophoblast differentiation.