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

Prostaglandins (PGs) play a critical role in porcine reproduction, of which prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 (CBR1) catalyzes the conversion of PGE2 to PGF2 alpha. A high ratio of PGE2: PGF2 alpha 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 kappa B (NF-kappa B) is involved in the process. Bioinformatic analysis has shown that NF-kappa B 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-kappa B 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-kappa B (p65) could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-kappa B was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.