Expression Of CREBZF In Testis And The Biological Functions In Testostrone Production

CREBZF is a member of the basic region-leucine zipper(b ZIP)transcription factors,which interacts with the host cell factor(HCF1)of the herpes simplex virus in order to inhibit viral replication proliferation.CREBZF gene expression produces two isoforms,arising from the alternative usage of initiation codons,SMILE and Zhangfei,which differ with regard to the presence or absence of 82 amino acids in the N-terminal regions.Previous studies have found that Zhangfei played a suppressive role in the unfolded protein response(UPR)by interaction with Luman,ATF4,Xbp1,HERP and GRP78.CREBZF is also involved in regulating apoptosis and proliferation.SMILE has been identified as a novel transcriptional co-regulator of a variety of nuclear receptors,and SMILE is expressed ubiquitously in mouse tissues and tumour-derived cells.Indeed,SMILE expressed in the mouse uterus and up-regulated by estrogen has been documented in our previous study.The leydig cells of the tesis are located between the testicular seminiferous tubules which are the only cells that has the capacity to synthesize and secrete testosterone.The synthesis and secretion of testosterone by leydig cells is regulated and influenced by a series of factors,and nuclear receptors can participate in the regulation of testosterone synthesis.Cholesterol is cleaved and isomerized to produce testosterone under the catalysis of steroid synthase,a process that occurs in the mitochondria and endoplasmic reticulum.Testosterone is a fat-soluble hormone that passes through the cell membrane,and is released immediately after testosterone production which is not stored.This experiment mainly studied the effect of CREBZF on testosterone synthesis and its mechanism of action.Immunohistochemistry,real-time quantitative PCR,siRNA interference,and Western blot were used to detect the expression of CREBZF in different stages of mouse testis,in order to study its effect on testosterone production and its mechanism of action.The test results are as follows:(1)Immunohistochemical localization indicated that CREBZF was detected specificlly in Leydig cells after sexual maturation and there was no obvious expression in the leydig cells of the mice before sexual maturation.mRNA expression of CREBZF was observed at all stages of mouse development and expression was significantly increased after puberty.SMILE protein isoforms were mainly expressed in mice testis,while zhangfei is rarely expressed.(2)Human chorionic gonadotrophin treatment significantly increased testosterone concentration in serum and Leydig cell culture supernatants,but did not affect the expression of CREBZF.Knockdown of CREBZF expression significantly inhibited hCG-stimulated testosterone production in vivo and in vitro.(3)CREBZF regulates testosterone synthesis by modulating steroid synthase.In primary leydig cells,knockdown of CREBZF significantly down-regulates the expression of the steroid synthase 3?-HSD,P450c17,and 17?-HSD,which in turn leads to low testosterone synthesis.The expression of StAR increased,which may be due to the decrease of testosterone synthesis,and then up-regulated StAR expression through negative feedback regulation.In leydig cells,overexpression of CREBZF can increase the expression of steroid synthase 3?-HSD,P450c17 and 17?-HSD.Knockdown and overexpression of CREBZF had no significant effect on P450 scc expression.These results suggest that CREBZF regulates the transcriptional expression of the steroid synthase gene in the endoplasmic reticulum.(4)CREBZF interacts with the promoters of nuclear receptor of Nur77 and SF1 to regulate their transcriptional expression.Knocking down of CREBZF expression in Leydig cells inhibits the transcriptional expressions of Nur77 and SF1,while over-expression of CREBZF can up-regulates the mRNA expression levels of there two genes.Further studies have shown that CREBZF can directly activate Nur77 and SF1 promoters and increase their transcriptional activation.In summary,CREBZF is specifically expressed in mouse Leydig cells after sexual maturation,and may indirectly regulate the transcription activity of steroid synthase by regulating the expression of nuclear receptors Nur77 and SF1 and play important role in the synthesis of testosterone.