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

Isayama K, Chen H, Yamauchi N, Hattori MA - J. Reprod. Dev. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.

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.

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Expression of the BMAL1, NR1D1 and PTGS2 gene transcripts inbovine 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) accordingto the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specificprimers. The relative transcript level was normalized to GAPDH and expressed relative to thenon-silencing RNA group. Each value represents the means ± SEM of three independent determinations. The statisticalanalyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.
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fig_005: Expression of the BMAL1, NR1D1 and PTGS2 gene transcripts inbovine 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) accordingto the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specificprimers. The relative transcript level was normalized to GAPDH and expressed relative to thenon-silencing RNA group. Each value represents the means ± SEM of three independent determinations. The statisticalanalyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.

Mentions: We used BMAL1-specific siRNA to investigate whether the PTGS2 expression is controlledunder BMAL1 transcriptional regulation in the two bovine cell types. BMAL1 associated with CLOCK or NPAS2 promotes thetranscription of genes such as NR1D1 through binding to the E-box at the promoter region. The transfectionof BMAL1-specific siRNA caused a significant decrease in the BMAL1 transcript level of boththe USCs (P<0.01) and the UECs (P<0.05) (Fig. 5Fig. 5.


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

Isayama K, Chen H, Yamauchi N, Hattori MA - J. Reprod. Dev. (2014)

Expression of the BMAL1, NR1D1 and PTGS2 gene transcripts inbovine 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) accordingto the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specificprimers. The relative transcript level was normalized to GAPDH and expressed relative to thenon-silencing RNA group. Each value represents the means ± SEM of three independent determinations. The statisticalanalyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219993&req=5

fig_005: Expression of the BMAL1, NR1D1 and PTGS2 gene transcripts inbovine 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) accordingto the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30h for the BMAL1 transcript and 48 h for the NR1D1 and PTGS2transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specificprimers. The relative transcript level was normalized to GAPDH and expressed relative to thenon-silencing RNA group. Each value represents the means ± SEM of three independent determinations. The statisticalanalyses were performed by one-way ANOVA with the Student’s t test. ** P<0.01; * P<0.05.
Mentions: We used BMAL1-specific siRNA to investigate whether the PTGS2 expression is controlledunder BMAL1 transcriptional regulation in the two bovine cell types. BMAL1 associated with CLOCK or NPAS2 promotes thetranscription of genes such as NR1D1 through binding to the E-box at the promoter region. The transfectionof BMAL1-specific siRNA caused a significant decrease in the BMAL1 transcript level of boththe USCs (P<0.01) and the UECs (P<0.05) (Fig. 5Fig. 5.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.

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.

Show MeSH
Related in: MedlinePlus