Construction Of Screening Model For Selective Aromatase Modulators Basis On Reporter Gene

Recent research indicated that estrogen played important physiological and pathophysiological roles more than reproduction in both females and males. These include actions on the carbohydrate and lipid metabolism, bone growth and maintenance, and the maintenance of cognitive function. Estrogen biosynthesis is catalyzed by aromatase cytochrome P450 (P450arom). The aromatase P450 gene is expressed in a number of tissues, including ovary, testis, adipose tissue, skin, brain and bone. Aromatase expression in these different tissue sites is mediated by tissue-specific promoters. Various tissue-specific promoter regions contain different cohorts of response elements, thus employ entirely different signaling pathways and cohorts of transcription factors. These selective first exons downstream of tissue-specific promoters splice at a common 3' -splice junction that is upstream of the translation start site in exon â…¡.This means that although transcripts in different tissues have different 5' terminal, the coding region and thus the protein expressed in these various tissue sites is always the same. Furthermore, in men and in postmenopausal women when the ovaries cease to produce estrogens, extragonadal sites of estrogen biosynthesis are the major sources of estrogen. At these sites, estrogen is produced and acts locally as a paracrine or evenintracrine factor. Thus the total amount of estrogen synthesized by these extragonadal sites may be small but the local tissue concentrations achieved are probably high and exert significant biological influence locally without significant affecting circulating levels. The concept of selective aromatase modulators is based on these considerations, namely the characteristics of tissue-specific aromatase expression and extragonadal estrogen biosynthesis. It is to development tissue-specific regulation of aromatase expression and estrogen biosynthesis. Aromatase promoter I . 4? promoter II and 1.3 sequence were cloned and ligated to a luciferase reporter gene into the pGL3-Basic vector. Aromatase expression in bone tissue> adipose tissue and adipose stromal cells is predominantly directed by promoter I . 4. While in breast cancer and surrounding adipose tissue, promoter II and I. 3 predominant. The recombinant reporter gene plasmid containing aromatase promoter I . 4 sequence was transfected into ROS17/2.8 cells. To identify and optimize the assay condition, the effects of some factors on the assay were examined, such as cell number in each welK incubation time and the dosage of luciferase substrate. The extracts of about 500 traditional Chinese medicines were screened and results indicated that a reliable method for tissue-specific aromatase modulators screening were established.