TESTICULAR GATA-1 FACTOR UP-REGULATES THE PROMOTER ACTIVITY OF RAT INHIBIN ALPHA-SUBUNIT GENE IN MA-10 LEYDIG TUMOR-CELLS

We have previously demonstrated that the basal transcription of rat in hibin 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 testicul ar 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 erythr oid exon I of mouse GATA-1 gene, was also identified in MA-10 cells, M utations of GATA sequences in alpha-subunit promoter markedly decrease d the transcriptional activity of alpha-subunit gene when measured by their ability of transient expression of a bacterial reporter gene, ch loramphenicol acetyltransferase (CAT), in MA-10 cells, Cotransfection of alpha CAT chimeric construct with cDNA expression plasmid coding fo r mouse GATA-1 or GATA-4 protein revealed that GATA-1 but not GATA-4 c an transactivate alpha-subunit promoter in a dose-dependent manner, Th e transactivation by GATA-1 was inhibited if GATA sequences in cu-subu nit promoter were mutated. Furthermore, electrophoretic mobility shift assay demonstrated that GATA-binding proteins present in nuclear extr acts of MA-10 cells and rat testis interacted with the GATA motifs in alpha-subunit promoter, and the GATA-1 in these nuclear extracts forme d a supershifted immunocomplex with antibody raised against mouse GATA -1 protein, We therefore concluded that the basal transcription of inh ibin alpha-subunit gene in testicular MA-10 cells is up-regulated by t esticular GATA-1 but not GATA-4 through its interaction with the GATA motifs in alpha-subunit promoter, In summary, we have provided the fir st evidence of the functional role of a GATA-binding protein in the re gulation of testicular gene expression.