The Regulation Mechanism Of STAT1 On Cyp1b1 Expression In Mouse Granulosa Cells

Estradiol is an important factor for follicular growth and granulosa cells survival,which regulates the proliferation of granulosa cells and the differentiation of oocytes in ovary.The estradiol-producing capacity and metabolism levels are important for follicle health,and sufficient estradiol is necessary for follicle development and ovulation.Cyp1b1,a member of the cytochrome P450 1 subfamily,is responsible for the metabolism of a wide variety of halogenated and polycyclic aromatic hydrocarbons in diverse tissues.In mouse follicles,Cyplbl converts estradiol to 4-hydroxyestradiol,reducing the amount of estrogen in follicular.4-OH E2 is harmful for granulosa cells,which can induce apoptosis of granulosa cells.Therefore,to study the regulation mechanism of Cyplbl expression is of great significance for explanation the molecular mechanism of follicular development dysfunctions.There are a lot of transcription factors that regulate the expression of Cyp1b1,in this paper,we choose STAT1 as a candidate transcription factors to study the regulation mechanism of it on Cyplbl expression in mouse granulosa cells on the base of the previous studies in our laboratory.Related contents and conclusions are as follows:We constructed the vector of STAT1 to transfect into mouse granulosa cells,the transfected cells were collected to analysis the expression of STAT1 and Cyp1b1.The results showed:the vector of STAT1 could express efficiently in mouse granulosa cells,it increased the expression of Cyp1b1 both in mRNA and protein level.This indicated STAT1 could regulate the expression of Cyplbl in mouse granulosa cells.We found that there were two binding sites of STAT1 on Cyplbl promoter region,which were located-908 to-900(BS 1)and +486 to +494(BS 2),by predicting using www.cbrc.jp/research/db/TFSEARCH.html.Construction of wild type luciferase reporter plasmid of Cyp1b1 promoter region or binding sites mutant.We transfected these vectors into 3T3 cells with STATI over-expression vector together,the luciferase assay was performed 48 h after transfection.Compared with the WT vector,the luciferase activity of binding sites mutant vectors reduced significantly.This indicated the predicted binding sites of STAT1 on Cyplbl are correct,STAT1 directly regulated the expression of Cyplbl as a transcription factor.The binding sites mutant vectors and STAT1 over-expression vector were co-transfected into 3T3 cells to determine the importance of BS 1 and BS 2 on STAT1 regulation of Cyplbl.Compared with mutated BS 1,mutated BS 2 could further decreased the luciferase activity,but there is no significant difference on statistics.This result suggested that BS 1 and BS 2 may play an equally important role in STAT1 regulation of Cyp1b1transcription activity.