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

SUBMITTER: Zhang AL

PROVIDER: S-EPMC5498843 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Zhang Ai-Ling AL Sun Xian-Yue XY Yin Qi Q Zeng Jian-Hua JH Zhang Zhe Z Li Jia-Qi JQ Zhang Hao H

Journal of Zhejiang University. Science. B 20170701 7

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 facto ...[more]

PMID: 28681587

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Project description:Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Dataset Information

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

Publications

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

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