Cdna Cloning Of Grass Carp Brain Aromatase, Isolation And Transcriptional Activity Of Promoter Regulation

As cytochrome P450 aromatase can catalyze the conversion of androgens to estrogens, it is considered as a key enzyme in the hormonal steroidogenic pathway. In teleosts, two isoforms of aromatase are expressed mainly in ovary and brain, which are named as aromA and aromB respectively. Interestingly, teleost fish aromB expression is 100-1000 times greater than that found in other vertebrates, but the mechanism responsible for this phenomenon is unclear. In the present study, the full-length cDNA of aromB was cloned from grass carp by RT-PCR coupled to rapid amplification of cDNA ends, named as gcAromB. The cDNA contained an open reading frame of 1527bp encoding a predicted protein of 509 amino acid residues, which shared 66-93% sequence identity with its counterparts in other teleosts, but only showed about 50% identity to mammalian aromatase. RT-PCR analysis revealed that gcAromB only expressed in brain from 10 selected tissues. Subsequently, aromB mRNA was detected in the partial areas of grass carp brain, including the optic tectum, hypothalamus and pituitary. Furthermore, A PCR-based genomic walking strategy was used to isolate the 5'-flanking region of grass carp aromB. The grass carp aromB 5'flanking region (2.3 kb) contained a consensus TATA box, a CCAAT box and some potential transcription factor binding sites including some GATAs, four AP1, an ERE, two CRE, four C/EBP, two NF-1, two Sp1, a PPARalpha/RXR-alpha, a SREBP-1, a GP, a NF-kappaB, an AhR/Arnt, three CRE-BP1/c-Jun and three STATx elements. The transcription activity of gcAromB was evaluated in vitro using luciferase as the reporter gene. In the glioma cells (C6) transfected by constructs with decreasing length of gcAromB promoter (-2290/+164), unexpectively, the basal levels of luciferase activity could not be detected. In contrast, all reporter constructs with intron I transiently transfected into glioma C6 showed differentical basal activity. To test effects of hypothalamus factor and pituitary hormones on the transcription activity of gcAromB, C6 cells transfected by constructs were exposed to PACAP, GTH, GH and activin. In this case, both PACAP and GTH significantly reduced the promoter activity from pGL3-159/+2822 to pGL3+1855/+2822, and this effect was blocked by the following deletion, and then reversed due to the absence of +861/+1855 region. Consistently, activin exhibited similar effects on promoter activity of gcAromB as GH.