Repression of the rat steroidogenic acute regulatory (StAR) protein gene by PGF2alpha is modulated by the negative transcription factor DAX-1.

Abstract

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 that StAR gene expression is enhanced by gonadotropins, whereas prostaglandin F2alpha (PGF2alpha) appears to suppress both basal and gonadotropin-stimulated StAR mRNA levels. While studies have demonstrated that steroidogenic factor 1 (SF-1) mediates transcriptional activation of the StAR gene, the mechanism for the reduction in StAR expression requires analysis. Recent studies have shown that DAX-1 (Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X-chromosome, gene-1), a negative transcription factor, inhibits transcription of reporter genes in vitro. To determine whether DAX-1 could negatively regulate expression of the StAR gene, approx 2 kb of the rat StAR promoter was linked to a luciferase reporter gene (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 promoter, Y1 adrenal and HTB9 cell luciferase activities were reduced to levels that were 57% and 24% of basal promoter levels, respectively. Furthermore, when dibutyryl-cAMP (dbcAMP) was added to the DAX-1 expressing cells, cAMP responsiveness was repressed 50% and 75% in Y1s and HTB9s respectively, relative 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 PGF2alpha regulates ovarian StAR expression via DAX-1, Western blot analysis indicated a three- and fivefold increase in rat ovarian DAX-1 levels at 2 and 4 h following PGF2alpha injection (250 microg). The increase in DAX-1 protein corresponded 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-terminal 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, did 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-fold induction in luciferase activity demonstrating that these proteins interact in the two-hybrid assay. This study is the first to demonstrate that the rat StAR promoter is regulated by DAX-1 and that DAX-1 reduces StAR promoter responsiveness to cAMP. The enhanced level of DAX-1 following PGF2alpha administration is consistent with DAX-1 having a role in controlling both basal, gonadotropin-stimulated, and PGF2alpha-mediated StAR gene expression. These results imply that DAX-1 has an important role in regulating ovarian steroidogenesis by repressing StAR transcription.