Functional characterization of the promoter of carbonyl reductase 1 gene in porcine endometrial cells

Functional characterization of the promoter of carbonyl reductase 1 gene in porcine endometrial cells CBR1 基因启动子在猪子宫内膜细胞的功能研究 1 first-author †E-mail:zhangmeixial@163.comzhanghao@scau.edu.cn †E-mail:zhangmeixial@163.comzhanghao@scau.edu.cn zhangmeixial@163.com 2 2 2 2 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhangmeixial@163.comzhanghao@scau.edu.cn †E-mail:zhangmeixial@163.comzhanghao@scau.edu.cn zhangmeixial@163.com Guangdong Development Center of Applied Ecology and Ecological Engineering in Universities, Biology and Food Engineering Institute, Guangdong University of Education, Guangzhou 510310, China Guangdong Development Center of Applied Ecology and Ecological Engineering in Universities, Biology and Food Engineering Institute, Guangdong University of Education, Guangzhou 510310, China Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, National Engineering Research Center for Breeding Swine Industry, National & Local Joint Engineering Research Center for Livestock and Poultry Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, China Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, National Engineering Research Center for Breeding Swine Industry, National & Local Joint Engineering Research Center for Livestock and Poultry Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, China Prostaglandins (PGs) play a critical role in porcine reproduction, of which prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 (CBR1) catalyzes the conversion of PGE2 to PGF2α. A high ratio of PGE2:PGF2α is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor κB (NF-κB) is involved in the process. Bioinformatic analysis has shown that NF-κB is a possible factor bound to two cis -regulatory elements in CBR1 promoter. In this study, we cloned the 2997 bp (−2875/+122) of the promoter, and constructed six 5'-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 (−1640/+7) exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 (−2089/+7). The activity of P1, P2, and P3 (−1019/+7) was highly significantly higher than that of others ( P < 0.01), suggesting that two positive regulatory elements were likely present in the regions of −1640/−1019 and −1019/−647. The results also showed that the −1640/−647 region was indispensable for the promoter. The results of chromatin immunoprecipitation (ChIP) demonstrated that the NF-κB subunit p65 binds to one site around −1545/−1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 ( P < 0.05) in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBR1 expression. These results indicated that NF-κB (p65) could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-κB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression. 目的 2 研究 CBR1 基因启动子在猪子宫内膜细胞的表达调控机制。 创新点 2 发现 CBR1 基因启动子在猪子宫内膜受到炎性因子核转录因子kappa B（NF-κB）成员p65调控。p65对该启动子具有正向调节作用，但是对于 CBR1 基因的表达并不是必需的。 方法 2 通过双荧光素酶报告基因载体确定 CBR1 基因启动子转录活性区，通过染色质免疫沉淀（ChIP）技术确定p65能够结合 CBR1 基因启动子，通过超表达和干扰表达实验证实p65对 CBR1 基因启动子的调控作用。 结论 2 猪 CBR1 基因启动子−1640/−647区对于其转录活性是必需的，在−1545/−1531区存在p65的结合位点。p65在猪子宫内膜细胞中促进了 CBR1 基因mRNA的表达，但是干扰p65则不会造成 CBR1 基因mRNA表达量下降，推断p65不是 CBR1 基因表达的必需因素。 Pig Carbonyl reductase 1 (CBR1) Promoter NF-κB Endometrium 猪 CBR1 基因 启动子 核转录因子kappa B（NF-κB） 子宫内膜 Introduction 1 Prostaglandins (PGs) play a critical role in porcine reproduction. Prostaglandin F2α (PGF2α) is a luteolysis factor (Christenson et al., 1994 1994 ), while prostaglandin E2 (PGE2) helps to establish and maintain early pregnancy by preventing luteolysis (Gadsby et al., 1993 1993 ; Ziecik, 2002 2002 ). Because of their opposite functions, the ratio of PGE2 to PGF2α is regulated accurately, which is helpful to maintain the porcine normal estrous cycle and pregnancy (Weems et al., 2006 2006 ; Waclawik and Ziecik, 2007 2007 ). Carbonyl reductase 1 (CBR1), known as prostaglandins 9-ketoreductase in pigs, catalyzes the conversion of PGE2 to PGF2α and modulates the PGE2:PGF2α ratio (Schieber et al., 1992 1992 ; Tanaka et al., 1992 1992 ; Ghosh et al., 2001 2001 ; Waclawik and Ziecik, 2007 2007 ). A low level of endometrial CBR1 facilitates a high PGE2:PGF2α ratio which is essential for the establishment and maintenance of porcine pregnancy (Waclawik and Ziecik, 2007 2007 ). The higher ratio of PGE2:PGF2α may be a beneficial factor for sows that have large litter sizes (Bazer et al., 1991 1991 ; Zhang et al., 2013 2013 ). During the early pregnancy before implantation (11–12 d) of pig, elevated amounts of estrogen from conceptus decreased CBR1 protein expression and then increased PGE2 secretion and the PGE2: PGF2α ratio (Waclawik et al., 2009 2009 ). Recent research showed that CBR1 exhibited a lower expression level in peripheral blood from pregnant sows (14 d after insemination) than from non-pregnant sows, indicating the importance of the gene in early pregnancy (Shen et al., 2014 2014 ). In the mid-secretory phase of the estrous cycle, proinflammatory responses occur in the endometrium for preparation of implantation of the fertilized conceptus. Implantation resembles an inflammatory event and can be mediated via activation of the nuclear factor kappa B (NF-κB)-dependent gene transcription (Ross et al., 2010 2010 ). In the porcine uterine endometrial epithelium, numerous genes including interleukin-6 (IL-6) and leukemia inhibitory factor are regulated by NF-κB (Geisert and Yelich, 1997 1997 ). In humans, the promoter of prostaglandin E2 synthase ( PGES ) gene, which encodes the catalyzing enzyme for the conversion of PGH2 to PGE2, was reported to contain a functional estrogen response element (ERE) upstream of the transcription start site (Frasor et al., 2008 2008 ), and estrogen and IL-1 (a proinflammatory cytokine) interact positively to enhance ERE activity in the endometrial epithelium and to up-regulate the expression of PGES (King et al., 2010 2010 ). All these indicated that higher expression of PGES was mediated by NF-κB-dependent genes and lower CBR1 was helpful to keep a higher PGE2 synergistically for gestation establishment during the implantation window. NF-κB is a heterogeneous collection of dimers consisting of five family members: NF-κB1 (p105/p50), NF-κB2 (p100/52), Rel A (p65), Rel B, and c-Rel (Lindström and Bennett, 2005 2005 ). The heterodimer of p50 (NF-κB1):p65 (RelA) is the most abundant form (Ghosh et al., 1998 1998 ). NF-κB was reported to be activated during peri-implantation in pigs (Mathew et al., 2011 2011 ). Previous studies showed that the expression of p65 and p50 increased during the mid-secretory phase (Laird et al., 2000 2000 ), and the mRNAs for p65 and p105 NF-κB subunits were reported to be maximally expressed in the human endometrium from the putative implantation window (King et al., 2010 2010 ). In porcine ovarian cells, p50/p50 promoted the release of PGF2α but did not influence the release of PGE2, while p65/p65 enhanced the release of PGE2 and PGF2α (Pavlová et al., 2011 2011 ). The above results showed that the members of NF-κB played roles in different porcine tissues. We found that there was a putative binding site of NF-κB in the promoter of CBR1 gene and speculated that the expression of the gene might be mediated by NF-κB. Considering the importance of CBR1 in gestation establishment in early pregnancy, we aimed to detect the core region of the CBR1 gene promoter and discover whether NF-κB might be involved in mediating the expression of CBR1 in porcine endometrial cells. Materials and methods 1 Sample collection and isolation of genomic DNA 2 All animal procedures in the present research were approved by the Animal Care and Use Committee of Guangdong Province, China. The ear tissues from Erhualian pigs were collected and preserved in 70% ethanol for DNA extraction. Genomic DNA was extracted from the ear tissues using the phenol method. Cloning of porcine CBR1 gene promoter and construction of 5'-deleted recombinant vectors 2 Primers are listed in Table 1 1 . Based on the porcine CBR1 gene sequence (Gene ID 397143), CBR1- 5' primers were used to amplify the CBR1 promoter of 2997 bp (−2875/+122). The fragment was cloned into the pMD18-T vector (Promega, USA) and sequenced. The cloned fragment was used as a template to amplify the series of CBR1 promoter fragments. With the primers of p1, p2, p3, p4, p5, and p6, the series of fragments of the CBR1 promoter, P1 (−2089/+7), P2 (−1640/+7), P3 (−1019/+7), P4 (−647/+7), P5 (−334/+7), and P6 (−80/+7) were respectively amplified. The fragments were cloned into the pMD18-T vector and sequenced, and the positive vectors were digested with Mlu I and Xho I restrictive enzymes. The pGL-Basic vector (Promega, USA) was also digested with the same restrictive enzymes. Each digested promoter and vector fragment were linked with the T4 DNA ligation enzyme. The recombinants were detected by the above restrictive enzymes and sequenced. The endotoxin free plasmids were extracted with OMEGA GenExtract (USA). Italic letters show the protection bases; lowercase letters show Mlu I (acgcgt) and Xho I (ctcgag) sites for p1, p2, p3, p4, p5, and p6, and Kpn I (gatatc) and Eco RV (ggtacc) for p-p65 Name Sequence * * Location Size (bp) CBR1 -5' F: 5' GGAAAGTGGAATGGATGTCA 3' −2875/+122 2997 R: 5' AGTGTTGGAAGACATGGCTG 3' p1 F: 5' CG acgcgtCGTCGTCATGTGGAAAGGA 3' −2098/+7 2106 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' p2 F: 5' CG acgcgtCGAGACTGAGGAAAACTA 3' −1640/+7 1648 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' p3 F: 5' CG acgcgtCGAGATGTCCTGGCTACAC 3' −1019/+7 1027 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' p4 F: 5' CG acgcgtCGATAGGCATTTTCAGCAA 3' −647/+7 655 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' p5 F: 5' CG acgcgtCGAAGACAATGAATCTGC 3' −334/+7 342 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' p6 F: 5' TT acgcgtAGGGAGGGCAGAAGGC 3' −80/+7 88 R: 5' CCG ctcgagCGGAGCCAGGTAGACCAGA 3' ChIP-1539 F: 5' CAGCGCAAACATGGAAAT 3' −1570/−1389 180 R: 5' CCTAACCAAGGAAGGGAG 3' ChIP-869 F: 5' GAAGCTACTGTTTACCCTCAA 3' −972/−753 220 R: 5' ACAATCCCTGGCACCCT 3' p-p65 F: 5' CGG gatatcATGGACGACCTCTTCCC 3' 2880 R: 5' CGG ggtaccTTAGGAGCTGATCTGACTCA 3' p-RPS20 F: 5' CGCTCCTGGCTCACCGCTGTT 3' 148 R: 5' TGCGGCTGGTGAGGGTGATCC 3' 18s F: 5' AGGCCCTGTAATTGGAATGA 3' 150 R: 5' CTCCCAAGATCCAACTACGA 3' GADPH F: 5' CAGGTTGTGTCCTGTGACTT 3' 90 R: 5' CTTGACGAAGTGGTCGTTGA 3' β-actin F: 5' TGTTCGAGACCTTCAACACC 3' 285 R: 5' AGCACAGCTTCTCCTTGATG 3' siRNA1 Sense: 5' GCACCGGAUUGAGGAGAAA dTdT 3' Antisense: 3' dTdT CGUGGCCUAACUCCUCUUU 5' siRNA2 Sense: 5' CCAACACUGCAGAGCUCAA dTdT 3' Antisense: 3' dTdT GGUUGUGACGUCUCGAGUU 5' siRNA3 Sense: 5' GAAGAAGAGUCCUUUCAAU 3' Antisense: 3' dTdTCUUCUUCUCAGGAAAGUUA 5' CBR1 promoter alignments were performed using ClustalW2 ( http://www.ebi.ac.uk http://www.ebi.ac.uk ). Putative transcription factor binding sites in the promoter region of the porcine CBR1 gene were predicted using MatInspector ( http://www.genomatix.de http://www.genomatix.de ). The CpG islands in the promoter were analyzed using the tool available from http://www.urogene.org/methprimer/index1.html http://www.urogene.org/methprimer/index1.html . Culture of porcine endometrial epithelial cells 2 The culture of endometrial epithelial cells was based on the procedures in Zhang et al. ( 1991 1991 ), Blitek and Ziecik ( 2004 2004 ), and Blitek et al. ( 2007 2007 ). The uterus of sows collected from a local abattoir was ligatured in the incision, placed in an ice box, and transported to the laboratory within an hour. The tissue was washed with 75% (v/v) ethanol and phosphate buffer solution (PBS) containing 100 IU/ml penicillin and 100 μg/ml streptomycin. The uterine horn was cut longitudinally, separated from the myometrium, and washed with PBS. The tissue was minced into 1 mm 3 and digested by 0.1% (1 g/L) collagenase I at 37 °C under shaking at 100 r/min for 2 h. Cell suspension was filtered to remove undigested tissue fragments, and pelleted at 500 r/min for 10 min. Red blood cells were lysed by a lysing buffer. The obtained epithelial cells were washed twice with PBS, and re-suspended in 1 ml of a culture medium (DMEM/F12 containing 10% (v/v) fetal bovine serum with 100 IU/ml penicillin and 100 μg/ml streptomycin). Cells were counted in a haemocytometer, plated in 25-cm 2 culture flasks at a density of 2×10 5 cells/ml, and incubated at 37 °C in a humidified incubator with 5% CO 2 . After reaching confluence, cells were seeded onto 24-well plates for transfection and luciferase assays. Transient transfections and luciferase activity assays 2 When epithelial cells were approaching confluence with 50%–80%, they were transfected by the constructed 5'-deleted recombinant vectors. The transfections were carried out using the Lipo-fectamine reagent (Invitrogen, USA) according to the manufacturer’s instructions. Luciferase activity was determined with the dual-luciferase reporter assay system (Promega, USA). Chromatin immunoprecipitation for the binding of NF-κB to the promoter 2 Chromatin immunoprecipitation (ChIP) procedures were performed with endometrial epithelial cells with the EZ-ChIP kit (Millipore, USA). The transcription factor p65 was immunoprecipitated by anti-p65 antibody (Abcam, UK). The anti-RNA polymerase II antibody and normal mouse IgG antibody were used as controls. Recovery and purification of DNA fragments from degradation of cross-linking DNA were used as the template for polymerase chain reaction (PCR). PCR was performed with primers ChIP-1539 and ChIP-869 for 180 and 220 bp, respectively, which contain the putative binding sites for NF-κB (−1545/−1531 and −875/−861 regions) (Table 1 1 ). Over-expression of p65 on the acceleration CBR1 gene promoter 2 With the primer of p-p65 (Gene ID: 100135665, Table 1 1 ), the 2880 bp of p65 coding sequence (CDS) was cloned and was linked to pcDNA3.1/mcy-His(−)B. The over-expression vector of p65 was constructed with restrictive enzymes of Kpn I and Eco RV. The vector was transfected into endometrial epithelial cells. After 48 h, the cells were washed twice with PBS. The cell mRNA was extracted with TRIzol (TaKaRa, Japan). The expression of CBR1 and p65 was detected by quantitative real-time PCR (qRT-PCR) using the genes of 18s (AY265350.1), β-actin (DQ452569.1), GAPDH (KJ786424.1), and ribosomal protein S20 ( RPS20 ) (Wang et al., 2011 2011 ) (NM_001129954, p-RPS20) as the reference genes (Table 1 1 ). Knockdown of p65 on the expression of CBR1 2 Three siRNAs (Ambion, UK) in Table 1 1 were designed to knockdown p65 to further reveal the effect of NF-κB on the expression of the CBR1 gene. The siRNAs were transfected into endometrial epithelial cells with Lipofectamine LTX and PLUS reagents (Invitrogen, USA) following the manufacturer’s instructions. After the transfection, the expression of CBR1 and p65 was measured by qRT-PCR using the reference genes as above. Statistical analysis 2 Data were analyzed by one-factor analysis of variance (ANOVA) and means were separated using the Tukey’s test. Results 1 Functional analysis of porcine CBR1 promoter 2 The sequence of the 2997-bp fragment was amplified, and was submitted to GenBank (JQ743647), spanning −2875/+122 of the porcine CBR1 promoter and containing a region of 1067 bp (−2370/−1304) which was not part of the reference sequence (Gene ID: 397143). A search of putative cis -acting elements of porcine CBR1 gene using MatInspector showed some typical regulatory elements, including GC-Box (−359/−353) and E-Box (−882/−860) identified, and putative binding sites for NF-κB (−1545/−1531, −875/−861), cAMP-response element-binding protein (CREB) (−617/−597), CCAAT/enhancer-binding protein (C/EBP) (−584/−560), and octamer transcription factor 1 (OCT-1) (−396/−382) predicted. No typical CpG islands were found in the sequence. Based on these results, the luciferase activity driven by the six constructed 5'-deleted plasmids of the promoter was measured in endometrial cells (Fig. 1 1 ). P2 (−1640/+7) was the greatest at driving luciferase expression in all the constructs. The activity of P1 (−2098/+7) was the second highest, but not significantly different from P2 ( P > 0.05). P2 and P3 (−1019/+7) differed significantly ( P < 0.05), suggesting that a positive cis -regulatory element was likely present in the −1640/−1019 region. The activity of P1–P3 was highly significantly higher than that of others ( P < 0.01), which suggested that another positive regulatory element was likely present in the region of −1019/−647. Together, it was shown that the −1640/−647 region was indispensable for the CBR1 promoter. Basic is the negative control with transfection of pGL3-basic vector, and Blank is the blank control without the transfection. Data were expressed as mean±standard error of mean (SEM) ( n =3). Different uppercase and lowercase letters above the bar represent the difference at P < 0.01 and P < 0.05, respectively Interaction of NF-κB on porcine CBR1 core promoter 2 With MatInspector, two putative NF-κB binding sites were predicted in −1545/−1531 and −875/−861 regions in the CBR1 core promoter. The ChIP results showed that p65 was bound to a site in the −1545/−1531 region but not in −875/−861 region (Fig. 2 2 ). Thus, it is highly likely that CBR1 gene expression is directly mediated by NF-κB in porcine endometrial epithelial cells in vivo. Effect of NF-κB on porcine CBR1 promoter 2 Gain and loss of p65 in endometrial cells were examined to further detect the effect of NF-κB on CBR1 expression. After the transfection into the cells for 48 h, the p65 was over-expressed strongly, which significantly enhanced the expression of CBR1 ( P < 0.05) compared with the negative control and the blank with RPS20 as the reference gene (Fig. 3 3 ). Basic is the negative control with transfection of empty vector; Blank is the blank control without the transfection. Data are expressed as mean±SEM ( n =3). Different letters above the bar show significant difference at P < 0.05 Then, three designed siRNAs were transfected into the cells’ expression and the endogenous p65 was nearly completely silenced by siRNA3 (Fig. 4 4 ). Under the silence of siRNA3, the mRNA of CBR1 did not decrease in the cells compared with the four reference genes ( RPS20 , 18s , β-actin , and GADPH ) (Fig. 5 5 ). Data are expressed as mean±SEM ( n =3). Different letters above the bar show significant difference at P < 0.01 Data are expressed as mean±SEM ( n =3) Discussion 1 Many hormones are involved in the establishment and maintenance of porcine pregnancy. PG is an important hormone for its variety and multifunction. As two important members of PG, PGE2 is an antiluteolytic factor (Gadsby et al., 1993 1993 ; Ziecik, 2002 2002 ), while PGF2α exerts luteolytic action (Christenson et al., 1994 1994 ). Thus, the ratio of PGE2 to PGF2α is regulated accurately. As a rate-limiting enzyme, CBR1 catalyzes the conversation of PGE2 to PGF2α, and regulates the ratio according to physiologic conditions (Waclawik and Ziecik, 2007 2007 ). The catalytic extent of CBR1 will influence the ratio of the two members. So the regulation of CBR1 gene expression is involved (Miura et al., 2013 2013 ; Guo et al., 2014 2014 ). The establishment and maintenance of porcine pregnancy are dependent on the corpora luteum function. These events resemble proinflammatory responses. As an important inflammatory factor, NF-κB is a transcription factor that controls the expression of many reproduction-associated genes. The binding sites of NF-κB have also been found in the human CBR1 promoter (Lakhman et al., 2007 2007 ). In human endometrial stromal and amnion mesenchymal cells, NF-κB was able to promote PGF2α production through oxygen radicals (Sugino et al., 2004 2004 ) and prostaglandin E syntheses (PTGES) (Ackerman et al., 2008 2008 ). On the contrary, NF-κB negatively regulated progesterone (P4) catabolism in rat corpus luteum (Telleria et al., 2004 2004 ). NF-κB exhibited opposing effects on different genes. In the PG synthesis pathway, the conversion of PGE2 to PGF2α is changed with estrous phases or tissues and regulated by CBR1 (Waclawik et al., 2009 2009 ). However, whether NF-κB is a vital regulator for porcine CBR1 is not well determined. It is known that NF-κB binds specific sites (κB sites, 5'-GGGRNNYYCC-3', where R is purine, Y is pyrimidine, and N is any kind of nucleotide) of various genes and enhances gene expression (Inoue et al., 2007 2007 ). In the region of the porcine CBR1 promoter, two putative similar binding sites were found in −1545/−1531 and −875/−861 regions, which locate in the core promoter region (−1640/+7; Fig. 1 1 ). We found that p65 bound with one assumed site of −1545/−1531 but another site of −875/−861 through ChIP in endometrial cells (Fig. 2 2 ). This showed that NF-κB may participate in the transcription of CBR1 . For target gene transcription activation, NF-κB usually exerts its roles with p50/p65 heterodimer (Inoue et al., 2007 2007 ). In our work, over-expression of p65 enhanced the CBR1 mRNA, which showed that p65 played a promotion role on CBR1 (Fig. 3 3 ). When p65 was silenced by the siRNA3, we first detected that the CBR1 gene expression was not influenced using the reference gene of RPS20 which had been shown to be the best reference gene in the study of endometrial gene expression (Wang et al., 2011 2011 ). Then the other three common reference genes were used to verify the CBR1 gene expression when the p65 was silenced and the same results were obtained (Fig. 5 5 ). We concluded that the knockdown of p65 will not decrease the mRNA of CBR1 . From our results, it seemed that NF-κB would enhance the expression of CBR1 and then decrease the ratio of PGE2 to PGF2α which would not be helpful to the establishment and maintenance of porcine pregnancy. However, the data showed that the levels of endometrial CBR1 on gestation day 12 (GD12) and GD14 did not differ (Waclawik and Ziecik, 2007 2007 ), and that the ratio of PGE2 to PGF2α on GD14 was higher than that on GD12 (Bazer et al., 1991 1991 ). It was speculated that other factors, such as CBR2, would play a role in the conversion of PGE2 to PGF2α ( http://www.kegg http://www.kegg . jp/pathway/hsa00590). Methylation is mainly found in the CpG islands and decreases the transcription extent. Although the core CBR1 promoter of humans had a typical CpG island (AP001724), we did not find CpG islands in the region of the porcine CBR1 promoter. The NF-κB was expressed during the porcine per-implantation period (Ross et al., 2010 2010 ), but the CBR1 level was not increased (Waclawik and Ziecik, 2007 2007 ). This may be related to post-transcription regulation. It is noted that hsa-miR-574-5p can effectively down-regulate human CBR1 expression in the human lymphoblastoid cell (Kalabus et al., 2012 2012 ). Ssc-miR-574 expressed in porcine endometrial cells may reduce the CBR1 level in porcine endometriun, and hence the expression p50:p65 did not differ significantly between GD10–GD17 ( P > 0.05) (Ross et al., 2010 2010 ), whereas CBR1 mRNA and protein during GD10–GD13 were significantly lower than those of other days ( P < 0.05) (Waclawik and Ziecik, 2007 2007 ). This showed that the stable expression p50:p65 did not keep the CBR1 mRNA higher during GD10–GD17. During GD10–GD13, porcine CBR1 gene may be decreased by negative factors or miRNAs. In conclusion, we have shown that the porcine CBR1 promoter region contained multiple cis -regulatoty elements. NF-κB enhances the CBR1 expression but does not influence its basal expression in porcine endometrial epithelial cells. It seemed that NF-κB was not essential to the basal expression of CBR1 . However, as a transcription factor, NF-κB does have an enhancing effect on CBR1 expression. References WE Ackerman , TLS Summerfield , DD Vandre , 等 . Nuclear factor-kappa B regulates inducible prostaglandin E synthase expression in human amnion mesenchymal cells . Biol Reprod , 2008 . 78 ( 1 ): 68 - 76 . 17928629 DOI: 10.1095/biolreprod.107.061663 http://doi.org/10.1095/biolreprod.107.061663 . Nuclear factor-kappa B regulates inducible prostaglandin E synthase expression in human amnion mesenchymal cells FW Bazer , WW Thatcher , F Matinat-Botte , 等 . Composition of uterine flushings from Large White and prolific Chinese Meishan gilts . Reprod Fertil Dev , 1991 . 3 ( 1 ): 51 - 60 . 1957014 DOI: 10.1071/RD9910051 http://doi.org/10.1071/RD9910051 . Composition of uterine flushings from Large White and prolific Chinese Meishan gilts A Blitek , AJ Ziecik . Prostaglandins F 2α and E 2 secretion by porcine epithelial and stromal endometrial cells on different days of the oestrous cycle . Reprod Domest Anim , 2004 . 39 ( 5 ): 340 - 346 . 15367267 DOI: 10.1111/j.1439-0531.2004.00523.x http://doi.org/10.1111/j.1439-0531.2004.00523.x . Prostaglandins F2α and E2 secretion by porcine epithelial and stromal endometrial cells on different days of the oestrous cycle A Blitek , AU Mendrzycka , MK Bieganska , 等 . Effect of steroids on basal and LH-stimulated prostaglandins F 2α and E 2 release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells . Reprod Biol , 2007 . 7 ( 1 ): 73 - 88 . 17435834 . Effect of steroids on basal and LH-stimulated prostaglandins F2α and E2 release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells LK Christenson , DB Farley , LH Anderson , 等 . Luteal maintenance during early pregnancy in the pig: role for prostaglandin E2 . Prostaglandins , 1994 . 47 ( 1 ): 61 - 75 . 8140263 DOI: 10.1016/0090-6980(94)90075-2 http://doi.org/10.1016/0090-6980(94)90075-2 . Luteal maintenance during early pregnancy in the pig: role for prostaglandin E2 J Frasor , AE Weaver , M Pradhan , 等 . Synergistic up-regulation of prostaglandin E synthase expression in breast cancer cells by 17β-estradiol and proinflammatory cytokines . Endocrinology , 2008 . 149 ( 12 ): 6272 - 6279 . 18703630 DOI: 10.1210/en.2008-0352 http://doi.org/10.1210/en.2008-0352 . Synergistic up-regulation of prostaglandin E synthase expression in breast cancer cells by 17β-estradiol and proinflammatory cytokines JE Gadsby , JA Lovdal , JH Britt , 等 . Prostaglandin F2α receptor concentrations in corpora lutea of cycling, pregnant, and pseudopregnant pigs . Biol Reprod , 1993 . 49 ( 3 ): 604 - 608 . 8399855 DOI: 10.1095/biolreprod49.3.604 http://doi.org/10.1095/biolreprod49.3.604 . Prostaglandin F2α receptor concentrations in corpora lutea of cycling, pregnant, and pseudopregnant pigs RD Geisert , JV Yelich . Regulation of conceptus development and attachment in pigs . J Reprod Fertil Suppl , 1997 . 52 133 - 149 . 9602725 . Regulation of conceptus development and attachment in pigs D Ghosh , M Sawicki , V Pletnev , 等 . Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases . J Biol Chem , 2001 . 276 ( 21 ): 18457 - 18463 . 11279087 DOI: 10.1074/jbc.M100538200 http://doi.org/10.1074/jbc.M100538200 . Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases S Ghosh , MJ May , EB Kopp . NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses . Annu Rev Immunol , 1998 . 16 225 - 260 . 9597130 DOI: 10.1146/annurev.immunol.16.1.225 http://doi.org/10.1146/annurev.immunol.16.1.225 . NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses C Guo , W Wang , C Liu , 等 . Induction of PGF2α synthesis by cortisol through GR dependent induction of CBR1 in human amnion fibroblasts . Endocrinology , 2014 . 155 ( 8 ): 3017 - 3024 . 24654784 DOI: 10.1210/en.2013-1848 http://doi.org/10.1210/en.2013-1848 . Induction of PGF2α synthesis by cortisol through GR dependent induction of CBR1 in human amnion fibroblasts J Inoue , J Gohda , T Akiyama , 等 . NF-κB activation in development and progression of cancer . Cancer Sci , 2007 . 98 ( 3 ): 268 - 274 . 17270016 DOI: 10.1111/j.1349-7006.2007.00389.x http://doi.org/10.1111/j.1349-7006.2007.00389.x . NF-κB activation in development and progression of cancer JL Kalabus , Q Cheng , JG Blanco . MicroRNAs differentially regulate carbonyl reductase 1 ( CBR1 ) gene expression dependent on the allele status of the common polymorphic variant rs9024 . PLoS ONE , 2012 . 7 ( 11 ): e48622 23133646 DOI: 10.1371/journal.pone.0048622 http://doi.org/10.1371/journal.pone.0048622 . MicroRNAs differentially regulate carbonyl reductase 1 (CBR1) gene expression dependent on the allele status of the common polymorphic variant rs9024 AE King , F Collins , T Klonisch , 等 . An additive interaction between the NFκB and estrogen receptor signalling pathways in human endometrial epithelial cells . Hum Reprod , 2010 . 25 ( 2 ): 510 - 518 https://dxdoiorg/101093/humrep/dep421 . 19955102 . An additive interaction between the NFκB and estrogen receptor signalling pathways in human endometrial epithelial cells SM Laird , EM Tuckerman , BA Cork , 等 . Expression of nuclear factor kappa B in human endometrium; role in the control of interleukin 6 and leukaemia inhibitory factor production . Mol Hum Reprod , 2000 . 6 ( 1 ): 34 - 40 . 10611258 DOI: 10.1093/molehr/6.1.34 http://doi.org/10.1093/molehr/6.1.34 . Expression of nuclear factor kappa B in human endometrium; role in the control of interleukin 6 and leukaemia inhibitory factor production SS Lakhman , X Chen , V Gonzalez-Covarrubias , 等 . Functional characterization of the promoter of human carbonyl reductase 1 ( CBR1 ). Role of XRE elements in mediating the induction of CBR1 by ligands of the aryl hydrocarbon receptor . Mol Pharmacol , 2007 . 72 ( 3 ): 734 - 743 . 17569794 DOI: 10.1124/mol.107.035550 http://doi.org/10.1124/mol.107.035550 . Functional characterization of the promoter of human carbonyl reductase 1 (CBR1). Role of XRE elements in mediating the induction of CBR1 by ligands of the aryl hydrocarbon receptor TM Lindström , PR Bennett . The role of nuclear factor kappa B in human labour . Reproduction , 2005 . 130 ( 5 ): 569 - 581 . 16264088 DOI: 10.1530/rep.1.00197 http://doi.org/10.1530/rep.1.00197 . The role of nuclear factor kappa B in human labour DJ Mathew , EM Sellner , JC Green , 等 . Uterine progesterone receptor expression, conceptus development, and ovarian function in pigs treated with RU 486 during early pregnancy . Biol Reprod , 2011 . 84 ( 1 ): 130 - 139 . 20864645 DOI: 10.1095/biolreprod.110.086843 http://doi.org/10.1095/biolreprod.110.086843 . Uterine progesterone receptor expression, conceptus development, and ovarian function in pigs treated with RU 486 during early pregnancy T Miura , A Taketomi , T Nishinaka , 等 . Regulation of human carbonyl reductase 1 (CBR1, SDR21C1) gene by transcription factor Nrf2 . Chem Biol Interact , 2013 . 202 ( 1-3 ): 126 - 135 . 23247010 DOI: 10.1016/j.cbi.2012.11.023 http://doi.org/10.1016/j.cbi.2012.11.023 . Regulation of human carbonyl reductase 1 (CBR1, SDR21C1) gene by transcription factor Nrf2 S Pavlová , K Klucska , D Vašíček , 等 . Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions . Anim Reprod Sci , 2011 . 128 ( 1-4 ): 73 - 84 . 21996578 DOI: 10.1016/j.anireprosci.2011.09.005 http://doi.org/10.1016/j.anireprosci.2011.09.005 . Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions JW Ross , MD Ashworth , D Mathew , 等 . Activation of the transcription factor, nuclear factor kappa-B, during the estrous cycle and early pregnancy in the pig . Reprod Biol Endocrinol , 2010 . 8 39 20426870 DOI: 10.1186/1477-7827-8-39 http://doi.org/10.1186/1477-7827-8-39 . Activation of the transcription factor, nuclear factor kappa-B, during the estrous cycle and early pregnancy in the pig A Schieber , RW Frank , S Ghisla . Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase . Eur J Biochem , 1992 . 206 ( 2 ): 491 - 502 . 1597188 DOI: 10.1111/j.1432-1033.1992.tb16952.x http://doi.org/10.1111/j.1432-1033.1992.tb16952.x . Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase J Shen , C Zhou , S Zhu , 等 . Comparative transcriptome analysis reveals early pregnancy-specific genes expressed in peripheral blood of pregnant sows . PLoS ONE , 2014 . 9 ( 12 ): e114036 25479131 DOI: 10.1371/journal.pone.0114990 http://doi.org/10.1371/journal.pone.0114990 . Comparative transcriptome analysis reveals early pregnancy-specific genes expressed in peripheral blood of pregnant sows N Sugino , A Karube-Harada , T Taketani , 等 . Withdrawal of ovarian steroids stimulates prostaglandin F2α production through nuclear factor-κB activation via oxygen radicals in human endometrial stromal cells: potential relevance to menstruation . J Reprod Dev , 2004 . 50 ( 2 ): 215 - 225 . 15118249 DOI: 10.1262/jrd.50.215 http://doi.org/10.1262/jrd.50.215 . Withdrawal of ovarian steroids stimulates prostaglandin F2α production through nuclear factor-κB activation via oxygen radicals in human endometrial stromal cells: potential relevance to menstruation M Tanaka , S Ohno , S Adachi , 等 . Pig testicular 20β-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity. cDNA cloning of pig testicular 20β-hydroxysteroid dehydrogenase . J Biol Chem , 1992 . 267 ( 19 ): 13451 - 13455 . 1377683 . Pig testicular 20β-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity. cDNA cloning of pig testicular 20β-hydroxysteroid dehydrogenase CM Telleria , AA Goyeneche , CO Stocco , 等 . Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20α-hydroxysteroid dehydrogenase . J Mol Endocrinol , 2004 . 32 ( 2 ): 365 - 383 . 15072545 DOI: 10.1677/jme.0.0320365 http://doi.org/10.1677/jme.0.0320365 . Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20α-hydroxysteroid dehydrogenase A Waclawik , AJ Ziecik . Differential expression of prostaglandin (PG) synthesis enzymes in conceptus during peri-implantation period and endometrial expression of carbonyl reductase/PG 9-ketoreductase in the pig . J Endocrinol , 2007 . 194 ( 3 ): 499 - 510 . 17761889 DOI: 10.1677/JOE-07-0155 http://doi.org/10.1677/JOE-07-0155 . Differential expression of prostaglandin (PG) synthesis enzymes in conceptus during peri-implantation period and endometrial expression of carbonyl reductase/PG 9-ketoreductase in the pig A Waclawik , HN Jabbour , A Blitek , 等 . Estradiol-17β, prostaglandin E2 (PGE2), and the PGE2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium . Endocrinology , 2009 . 150 ( 8 ): 3823 - 3832 . 19359378 DOI: 10.1210/en.2008-1499 http://doi.org/10.1210/en.2008-1499 . Estradiol-17β, prostaglandin E2 (PGE2), and the PGE2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium S Wang , J Li , A Zhang , 等 . Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12 . Biochem Biophys Res Commun , 2011 . 408 ( 2 ): 265 - 268 . 21501585 DOI: 10.1016/j.bbrc.2011.04.010 http://doi.org/10.1016/j.bbrc.2011.04.010 . Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12 CW Weems , YS Weems , RD Randel . Prostaglandins and reproduction in female farm animals . Vet J , 2006 . 171 ( 2 ): 206 - 228 . 16490704 DOI: 10.1016/j.tvjl.2004.11.014 http://doi.org/10.1016/j.tvjl.2004.11.014 . Prostaglandins and reproduction in female farm animals H Zhang , S Wang , M Liu , 等 . Differential gene expression in the endometrium on gestation day 12 provides insight into sow prolificacy . BMC Genomics , 2013 . 14 ( 1 ): 45 23339594 . Differential gene expression in the endometrium on gestation day 12 provides insight into sow prolificacy Z Zhang , BC Paria , DL Davis . Pig endometrial cells in primary culture: morphology, secretion of prostaglandins and proteins, and effects of pregnancy . J Anim Sci , 1991 . 69 ( 7 ): 3005 - 3015 . 1885410 DOI: 10.2527/1991.6973005x http://doi.org/10.2527/1991.6973005x . Pig endometrial cells in primary culture: morphology, secretion of prostaglandins and proteins, and effects of pregnancy AJ Ziecik . Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in pig . Domest Anim Endocrinol , 2002 . 23 ( 1-2 ): 265 - 275 . 12142243 DOI: 10.1016/S0739-7240(02)00162-5 http://doi.org/10.1016/S0739-7240(02)00162-5 . Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in pig Compliance with ethics guidelines Ai-ling ZHANG, Xian-yue SUN, Qi YIN, Jian-hua ZENG, Zhe ZHANG, Jia-qi LI, and Hao ZHANG declared that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on animal research. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1600225 Project supported by the National Natural Science Foundation of China (No. 31201771) and the Earmarked Fund for China Agriculture Research System (No. CARS-36) 2016-05-18 2017-06-16 2016-09-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 18