Testicular GATA-1 factor up-regulates the promoter activity of rat inhibin alpha-subunit gene in MA-10 Leydig tumor cells.

Abstract

We have previously demonstrated that the basal transcription of rat inhibin alpha-subunit gene in a mouse testicular Leydig tumor cell line, MA-10, depends upon a 67-bp DNA fragment at the position of -163 to -97. Within this promoter region two GATA motifs were observed. In this study, we investigated the possible role of GATA-binding proteins in the regulation of inhibin alpha-subunit gene transcription in testicular cells. Northern blot and RT-PCR analyses showed that mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis and in MA-10 and rat Sertoli cells. Testis-specific GATA-1 mRNA, which is transcribed from a promoter 8 kb upstream to the erythroid exon I of mouse GATA-1 gene, was also identified in MA-10 cells. Mutations of GATA sequences in alpha-subunit promoter markedly decreased the transcriptional activity of alpha-subunit gene when measured by their ability of transient expression of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), in MA-10 cells. Cotransfection of alphaCAT chimeric construct with cDNA expression plasmid coding for mouse GATA-1 or GATA-4 protein revealed that GATA-1 but not GATA-4 can transactivate alpha-subunit promoter in a dose-dependent manner. The transactivation by GATA-1 was inhibited if GATA sequences in alpha-subunit promoter were mutated. Furthermore, electrophoretic mobility shift assay demonstrated that GATA-binding proteins present in nuclear extracts of MA-10 cells and rat testis interacted with the GATA motifs in alpha-subunit promoter, and the GATA-1 in these nuclear extracts formed a supershifted immunocomplex with antibody raised against mouse GATA-1 protein. We therefore concluded that the basal transcription of inhibin alpha-subunit gene in testicular MA-10 cells is up-regulated by testicular GATA-1 but not GATA-4 through its interaction with the GATA motifs in alpha-subunit promoter. In summary, we have provided the first evidence of the functional role of a GATA-binding protein in the regulation of testicular gene expression.