GATA4 Is Required for Steroidogenesis in MA-10 Leydig Cells.

GATA4 is an essential transcription factor required for the development and function of multiple tissues derived from both mesoderm and endoderm. Among these tissues, Gata4 is highly expressed in the somatic cell lineages of the gonads in both sexes throughout development. Various knockout models in mice have demonstrated that GATA4 is essential for the onset of normal gonadal morphogenesis and is later recruited as an important regulator of genes required for the maintenance of normal male fertility. In spite of its essential role in reproduction, the genes and/or gene networks that are ultimately targeted and regulated by GATA4 remain to be fully understood. In males, testosterone production by testicular Leydig cells is essential for the initiation and maintenance of spermatogenesis. Leydig cells strongly express Gata4 at all stages of development. We and others have shown that GATA4 is an important activator of the promoters of several genes involved in the steroidogenic pathway such as steroidogenic acute regulatory protein (Star), P450scc (Cyp11a1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2). Our hypothesis is that GATA4 is required for male fertility in part through the regulation of Leydig cell steroidogenesis. To test this hypothesis and to identify new GATA4-regulated gene targets in Leydig cells, we performed a microarray screening analysis of MA-10 Leydig cells that were knocked down for GATA4 using siRNAs directed against the GATA4 coding sequence. MA-10 cells are an ideal model for this knockdown strategy since they are known to contain almost exclusively GATA4 protein (unlike other testicular somatic cell lines that contain multiple GATA factors). MA-10 cells were therefore treated with either a control siRNA sequence or siRNA against GATA4. The treated cells (i.e., each treatment group) were divided into two preparations for analysis: one-half to verify that GATA4 was indeed knocked down by Western blot and the other half was used to prepare RNA for the microarray analysis. The microarray analysis revealed that a decrease in GATA4 protein in MA-10 cells was associated with a sharp decrease in the expression of multiple steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and especially Star. The knockdown of GATA4 also led to an important and unexpected decrease in other steroidogenic targets including steroid 5 alpha-reductase type 1 (Srd5a1), 3β-hydroxysteroid dehydrogenase type 1 and type 6 (Hsd3b1 and Hsd3b6). Quantitative real-time PCR was used to confirm the validity of the targets identified through the microarray screen. Finally, the decrease in GATA4 protein was found to severely compromise the ability of MA-10 cells to produce steroids (progesterone) both basally and under hormonal stimulation. Taken together, our data demonstrate that GATA4 is an essential transcription factor that is required for the expression of multiple genes involved in the steroidogenic pathway. This suggests that aberrant GATA4 function might be associated with several human pathologies that involve reduced fertility and diminished steroid hormone synthesis. This research was supported by CIHR grant MOP-14796 to RSV.