Repression of the rat steroidogenic acute regulatory (StAR) protein gene by PGF2 alpha is modulated by the negative transcription factor DAX-1

The steroidogenic acute regulatory protein (StAR) is thought to mediate the rapid increase in steroid hormone biosynthesis by facilitating cholesterol transport to the inner mitochondrial membrane. Recent studies indicate tha t StAR gene expression is enhanced by gonadotropins, whereas prostaglandin F2 alpha (PGF2 alpha) appears to suppress both basal and gonadotropin-stimu lated StAR mRNA levels. While studies have demonstrated that steroidogenic factor 1 (SF-1) mediates transcriptional activation of the StAR gene, the m echanism for the reduction in StAR expression requires analysis. Recent stu dies have shown that DAX-1 (Dosage-sensitive sex reversal adrenal hypoplasi a congenita critical region on the X-chromosome, gene-1), a negative transc ription factor, inhibits transcription of reporter genes in vitro. To deter mine whether DAX-1 could negatively regulate expression of the StAR gene, a pprox 2 kb of the rat StAR promoter was linked to a luciferase reporter gen e (creating p-1862 StAR) and cotransfected into Y1 adrenal tumor cells and HTB9 human bladder carcinoma cells with vectors which encode DAX-1 and SF-1 . Luciferase levels were significantly increased in both cell types when SF -1 was present. In contrast, when DAX-1 was cotransfected with the StAR pro moter, Y1 adrenal and HTB9 cell luciferase activities were reduced to level s that were 57% and 24% of basal promoter levels, respectively. Furthermore , when dibutyryl-cAMP (dbcAMP) was added to the DAX-1 expressing cells, cAM P responsiveness was repressed 50% and 75% in Y1s and HTB9s respectively, r elative to the non-DAX-1 expressing dbcAMP-treated cells. The inhibition of StAR gene transcription by DAX-1 was dose-dependent reducing transcription to 6% of control levels. Consistent with the possibility that PGF2a regula tes ovarian StAR expression via DAX-1, Western blot analysis indicated a th ree- and fivefold increase in rat ovarian DAX-1 levels at 2 and 4 h followi ng PCF2 alpha injection (250 mu g) The increase in DAX-1 protein correspond ed to a 50% reduction in StAR mRNA levels concomitant with a 39% reduction in serum progesterone levels. Truncation of the DAX-1 protein at the C-term inal end caused a loss of inhibition of transcriptional activity. Deletion of bp -95 to -50 within the StAR promoter, a proposed DAX-1 binding site, d id not alter the ability of wild-type DAX-1 to inhibit transcription. In a mammalian two-hybrid system, cotransfection of DAX-1 and SF-1 caused a 25-f old induction in luciferase activity demonstrating that these proteins inte ract in the two-hybrid assay. This study is the first to demonstrate that t he rat StAR promoter is regulated by DAX-1 and that DAX-1 reduces StAR prom oter responsiveness to cAMP. The enhanced level of DAX-1 following PCF2a ad ministration is consistent with DAX-1 having a role in controlling both bas al, gonadotropin-stimulated, and PGF2 alpha-mediated StAR gene expression. These results imply that DAX-1 has an important role in regulating ovarian steroidogenesis by repressing StAR transcription.