Regulation of the Murine Endozepine Gene by the Forkhead Transcription Factor FoxL2.

Abstract

The transcription factor Foxl2 is required for ovarian function and female sex determination. In this study we make use of PCR select (Clontech) to identify genes that are differentially expressed upon the introduction of a FoxL2-VP16 expression vector into KK1 granulosa cells. The potential targets discovered through this analysis were Tgfb1i4 (TSC22: Transforming Growth Factor-β-Stimulated Clone 22), Oaz (ornithine decarboxylase antizyme), Anxa1 (Annexin A1), Nap1 (Nck associated protein 1), Pygo (Pygopus), Skp1, and Dbi (diazepam binding inhibitor). Dbi encodes a 10-kDa protein that is known as diazepam binding inhibitor, acyl-CoA-binding protein, and endozepine. Endozepine has been characterized as a ligand for central benzodiazepine receptors, for peripheral benzodiazepine receptors (PBR) which is involved in the regulation of steroidogenesis, and as an intracellular acyl-CoA transporter. We have performed further studies to confirm the FoxL2 regulation of the endozepine gene. First, we have made use of Real Time RT PCR to determine the effect of FoxL2 over-expression on endozepine gene expression. FoxL2 was over-expressed in KK1 cells through transfection of a Foxl2 expression vector and resulted in a decrease in endozepine mRNA levels. We have also cloned the endozepine promoter and created a luciferase fusion. Over-expression of Foxl2 caused a decrease in luciferase expression in transient co-transfections. These results suggest that Foxl2 is a negative regulator of the mouse endozepine gene in KK1 granulosa cells.