REV-ERBα inhibits the PTGS2 expression in bovine uterus endometrium stromal and epithelial cells exposed to ovarian steroids

Abstract

The nuclear receptor REV-ERBα (encoded by NR1D1) has a critical role in metabolism and physiology as well as circadian rhythm. Here, we investigated the possible contribution of clock genes including NR1D1 to the secretion of prostaglandin F2α (PGF2α) from bovine uterine stromal (USCs) and epithelial cells (UECs) by modulating the expression of PTGS2. The circadian oscillation of clock genes in the cells was weak compared with that reported in rodents, but the expression of BMAL1, PER1, and NR1D1 was changed temporally by treatment with ovarian steroids. Significant expression of clock genes including NR1D1 was detected in USCs exposed to progesterone. NR1D1 was also significantly expressed in UECs exposed to estradiol. The expression of PTGS2 was suppressed in USCs exposed to progesterone, while the expression was initially suppressed in UECs exposed to estradiol and then increased after long-term exposure to estradiol. BMAL1 knockdown with specific siRNA caused a significant decrease in the transcript levels of NR1D1 and PTGS2 in USCs, but not in UECs. The production of PGF2α also decreased in USCs after BMAL1 knockdown, while its level did not significantly change in UECs. The transcript level of PTGS2 was increased by treatment with the antagonist of REV-ERBα in both cell types, but the agonist was ineffective. In these two cell types treated with the agonist or antagonist, the PGF2α production coincided well with the PTGS2 expression. Collectively, these results indicate that REV-ERBα plays an inhibitory role in the expression of PTGS2 in both bovine USCs and UECs treated with ovarian steroids.

Fig. 1.

Generation of bioluminescence oscillations by rat and bovine USCs transfected with pGL3 vector containing the mouse Per1 promoter region after synchronization with forskolin. (A) Schematic of the pGL3 vector containing the mouse Per1 promoter region (upper). Black bars indicate the location of E-box motifs (–146 to –151, –509 to –514, and –1255 to –1260) and a cyclic-AMP response element (CRE, –1725 to –1732). (B) Bioluminescence activity was induced in bovine and rat USCs transfected with 1 μg of the constructed vector by synchronization with forskolin. Bioluminescence was monitored in real time in serum-free medium DMEM/F12 supplemented with 0.1 mM luciferin, 0.1% BSA, 1% ITS, 1×AA and 100 nM P₄. Each value represents the means of three independent determinations.

Fig. 2.

Expression profiles of core clock gene transcripts over the course of 48 h in bovine USCs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from cells cultured with (bottom) or without (upper) the presence of P₄. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to GAPDH and expressed relative to the first time point (0 h). Each value represents the mean ± SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison tests. * P<0.05 vs. 0 h.

Fig. 3.

Expression profiles of core clock gene transcripts over the course of 48 h in bovine UECs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from cells cultured with (bottom) or without (upper) the presence of estradiol. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to GAPDH and expressed as relative to the first time point (0 h). Each value represents the mean ± SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison tests. * P<0.05 vs. 0 h.

Fig. 4.

Expression of the PTGS2 gene in bovine USCs and UECs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from USCs (A) and UECs (B) cultured with or without the presence of ovarian steroids. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to GAPDH and expressed as relative to the first time point (0 h). Each value represents the mean ± SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison tests. * P<0.05 vs. 0 h.

Fig. 5.

Expression of the BMAL1, NR1D1 and PTGS2 gene transcripts in bovine USCs and UECs transfected with BMAL1-specific siRNA or non-silencing RNA. USCs (A) and UECs (B) were separately treated with BMAL1-specific siRNA (siRNA) or non-silencing RNA (CONT) according to the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30 h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2 transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to GAPDH and expressed relative to the non-silencing RNA group. Each value represents the means ± SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.

Fig. 6.

Expression of the PTGS2 gene transcript in bovine USCs and UECs treated with the agonist or antagonist of REV-ERBα. USCs (A) and UECs (B) were separately treated with the agonist (heme) or antagonist (SR8278) of REV-ERBα according to the indicated protocols. Cells were then synchronized with forskolin. Total RNA samples were collected at 30 h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2 transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to GAPDH and expressed as relative to the CONT group. Each value represents the means ± SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.

Fig. 7.

Production of PGF₂α by bovine USCs and UECs treated with BMAL1-specific siRNA and the agonist or the antagonist of REV-ERBα. USCs and UECs were treated with BMAL1-specific siRNA (A) and the agonist or antagonist of REV-ERBα (B) as described in Figs. 4 and 5. The culture media were collected at 48 h after synchronization with forskolin and assayed for PGF₂α. Each value represents the means ± SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.