Growth Hormone-induced STAT5B RegulatesStarGene Expression Through a Cooperation With cJUN in Mouse MA-10 Leydig Cells

Abstract

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star mRNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (EMSA and supershift) and in vivo (ChIP) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function

“…NUR77 regulates the expression of these genes by cooperating with CAMKI ( 29 ), cJUN ( 9 ), KLF6 ( 34 ), and SF1 ( 34 ). In Leydig cells, Nur77 expression is controlled by distinct regulatory elements for both basal and hormone-induced expression ( 77 ), through mechanisms involving MEF2 ( 52 ), STAT5B ( 8 ), CREB ( 77 ), cJUN ( 9 ), C/EBPβ ( 20 ), and NF-κβ p50 ( 20 ).…”

“…So far, STAT5B is the only STAT factor identified in Leydig cells ( 114 ). In these cells, STAT5B is activated by growth hormone, an important regulator of steroidogenesis ( 8 ). STAT5B activates Star expression directly by binding to a GAS element and in cooperation with cJUN ( 8 ).…”

“…In these cells, STAT5B is activated by growth hormone, an important regulator of steroidogenesis ( 8 ). STAT5B activates Star expression directly by binding to a GAS element and in cooperation with cJUN ( 8 ). STAT5B also activates the Nur77 promoter ( 8 ).…”

“…STAT5B activates Star expression directly by binding to a GAS element and in cooperation with cJUN ( 8 ). STAT5B also activates the Nur77 promoter ( 8 ).…”

Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti‐Müllerian hormone receptor type 2 transcription in mouse MA‐10 Leydig cells

“…Expression plasmids for mouse COUP-TFI, COUP-TFII, GATA1, GATA3, and rat GATA4 and GATA6 have been previously described. 4,8,24,45 To measure promoter activation, MA-10, MLTC-1, and CV-1 cells were transiently transfected using polyethylenimine hydrochloride (Sigma-Aldrich Canada, Ontario, Canada) as previously described 8,42 or using the calcium phosphate co-precipitation method as described in. [46][47][48] Briefly, the cells were plated in 24-well plates and co-transfected with 400 ng of reporter vector along with 100 ng of expression vectors (empty expression vector pcDNA3.1 as control, COUP-TFs, GATAs, or COUP-TFs+GATAs).…”