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

ABSTRACT

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 PGE2PGF2α 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.