Pregnancy Reprograms Large-Conductance Ca2+-Activated K+ Channel in Uterine Arteries: Roles of Ten-Eleven Translocation Methylcytosine Dioxygenase 1-Mediated Active Demethylation

Abstract

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is of critical importance in pregnancy-mediated increase in uterine artery vasodilation and blood flow. The present study tested the hypothesis that active DNA demethylation plays a key role in pregnancy-induced reprogramming and upregulation of BK_Ca channel β1 subunit (BKβ1) in uterine arteries. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Pregnancy significantly increased the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in uterine arteries. A half-palindromic estrogen response element was identified at the TET1 promoter, and estrogen treatment increased TET1 promoter activity and TET1 expression in uterine arteries. In accordance, pregnancy and steroid hormone treatment resulted in demethylation of BKβ1 promoter by increasing 5-hydroxymethylcytosine and decreasing 5-methylcytosine at the CpG in the Sp1_-380 binding site that is of critical importance in the regulation of the promoter activity and BKβ1 expression. Inhibition of TET1 with fumarate significantly decreased BKβ1 expression in uterine arteries of pregnant animals and blocked steroid hormone-induced upregulation of BKβ1. Functionally, fumarate treatment inhibited pregnancy and steroid hormone-induced increases in BK_Ca channel current density and BK_Ca channel-mediated relaxations. In addition, fumarate blocked pregnancy and steroid hormone-induced decrease in pressure-dependent myogenic tone of the uterine artery. The results demonstrate a novel mechanism of estrogen-mediated active DNA demethylation in reprogramming of BK_Ca channel expression and function in the adaption of uterine circulation during pregnancy.

Keywords: 5-hydroxymethylcytosine; 5-methylcytosine; RNA, small interfering; estrogens; pregnancy.

Figure 1. Pregnancy and steroid hormones increased TET1 expression in uterine arteries

A, Protein and mRNA abundance of TET1 in uterine arteries isolated from non-pregnant (NPUA) and near-term pregnant (PUA) sheep. Data are means ± SEM from 4 to 5 animals of each group. *P<0.05, PUA vs. NPUA. B, Protein and mRNA abundance of TET1 in NPUA treated with or without 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) for 48 hours. Data are means ± SEM from 4 animals of each group. *P<0.05, +E2β/P4 vs. −E2β/P4. C, Steroid hormones stimulate TET1 promoter activity determined using a dual-luciferase reporter assay system. Data are means ± SEM, n = 5. *P<0.05, TET1-E2β/P4 vs. TET1-control.

Figure 2. Pregnancy and steroid hormones promoted active demethylation at the Sp1_-380 site of KCNMB1 promoter

A, TET1 interacts with the Sp1_-380 binding site at the ovine KCNMB1 promoter. Chromatins were prepared from uterine arteries and immunoprecipitated by a TET1 antibody (ChIP: TET1 ab), followed by polymerase chain reaction (PCR) using primers flanking the Sp1_-380 binding site of the sheep KCNMB1 promoter (PCR: Sp1_-380 site). The arrow shows the PCR amplified product (97 bp) that was electrophoresed and visualized with ethidium bromide staining. M: molecular weight ruler. B, 5hmC abundance at the Sp1_-380 site in uterine arteries from non-pregnant (NPUA) and near-term pregnant (PUA) sheep, and in NPUA treated with or without 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) for 48 hours. C, Methylation of Sp1_-380 site of the KCNMB1 promoter. Data are means ± SEM from 5 animals of each group. *P<0.05, PUA vs. NPUA or +E2β/P4 vs. −E2β/P4.

Figure 3. Effects of fumarate on BK_Ca channel expression in uterine arteries

A, Protein and mRNA abundance of BK_Ca channel β1 subunit (BKβ1) in uterine arteries of pregnant sheep treated ex vivo with or without monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. Data are means ± SEM from 4 to 5 animals of each group. *P<0.05, Fumarate vs. Control. B, Protein abundance of BKβ1 in uterine arteries of non-pregnant sheep treated with 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) in the absence or presence of monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. Data are means ± SEM from 4 to 5 animals of each group.

Figure 4. Effects of fumarate on BK_Ca channel activity in uterine arteries

A, BK_Ca channel currents obtained in uterine arteries from pregnant animals treated ex vivo with or without monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. B and C, BK_Ca channel currents recorded in uterine arteries from non-pregnant animals treated ex vivo with or without 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) in the absence or presence of monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. BK_Ca currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA/pF; y axis), as a function of stepwise 10-mV depolarizing pulses (x axis). Data are means ± S.E.M. of 7–8 cells from 5 animals of each group. *P<0.05; −Fumarate vs. +Fumarate or +E2β/P4 vs. − E2β/P4.

Figure 5. Effect of knocking down TET1 and BKβ1 on expression of TET1 and BKβ1 expression and BK_Ca channel activity in uterine arteries

A, Protein and mRNA abundance of TET1 and BK_Ca channel β1 subunit (BKβ1) in uterine arteries of pregnant sheep treated with TET1 siRNAs for 48 hours. Data are means ± SEM from 5 animals of each group. *P<0.05, TET1 siRNA vs. Negative Control. B, Protein and mRNA abundance of BKβ1 in uterine arteries of pregnant sheep treated with BKβ1 siRNAs for 48 hours. Data are means ± SEM from 5 animals of each group. *P<0.05, BKβ1 siRNA vs. Negative Control. C, BK_Ca channel currents recorded in uterine arteries from pregnant animals treated with TET1 siRNAs or BKβ1 siRNAs for 48 hours. Data are means ± SEM of cells from 5 animals of each group. *P<0.05; TET1 siRNA or BKβ1 siRNA vs. Negative Control.

Figure 6. Effects of fumarate on NS1619-induced relaxations and pressure-dependent myogenic tone in uterine arteries

A, NS1619-induced relaxations of uterine arteries from pregnant animals treated ex vivo with or without monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. B and C, NS1619-induced relaxations of uterine arteries from non-pregnant animals treated ex vivo with or without 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) in the absence or presence of monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. D, Pressure-dependent myogenic tone in uterine arteries from pregnant animals treated ex vivo with or without monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. E and F, Pressure-dependent myogenic tone in uterine arteries from non-pregnant animals treated ex vivo with or without 17β-estradiol (E2β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) in the absence or presence of monoethyl fumarate (Fumarate, 3.0 mmol/L) for 48 hours. Data are means ± SEM of tissues from 5 animals of each group. *P<0.05, −Fumarate vs. +Fumarate or +E2β/P4 vs. − E2β/P4.