TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1

• DOI: 10.1007/s00011-015-0835-8
• Publication Date: 2015-06-05T05:25:53Z
• Authors: Mohanraj Sadasivam; Balamurugan Ramat...; Sivasangari Balak...; Chidambaram Praha...
• Keywords: Male; 0303 health sciences; DAX-1 Orphan Nuclear Receptor; MAP Kinase Kinase 4; MAP Kinase Signaling System; Tumor Necrosis Factor-alpha; Leydig Cells; Cell Line; Rats; 03 medical and health sciences; Gene Knockdown Techniques; Testis; Animals; Steroids; RNA, Messenger; Phosphorylation; RNA, Small Interfering; Signal Transduction

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. A detailed understanding of the mechanisms by which TNF-α regulates testicular steroidogenesis will be helpful in the design of novel clinical interventions for the treatment and prevention of male reproductive disorders. Here, we report that TNF-α-mediated activation of DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is involved in the inhibition of Leydig cell steroidogenesis.Rat testis Leydig tumor cells (LC-540) were treated with TNF-α (10 ng/ml) for different time intervals. To elucidate the pathways of intracellular signal transduction that regulate DAX-1 expression, we utilized specific inhibitors. The siRNA transfection of DAX-1 into LC-540 cells was performed by electroporation. The mRNA and protein levels were determined by RT-PCR and Western blotting, respectively.We found that the mRNA and protein levels of DAX-1 were increased by threefold approximately in TNF-α-treated cells when compared to controls. Staurosporine, JNK inhibitor SP600125 and ERK inhibitor PD98059 significantly decreased DAX-1 expression in TNF-α-treated Leydig cells when compared to their respective controls. Further, a siRNA-mediated knockdown of DAX-1 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells.These findings provide valuable information that TNF-α activates DAX-1 through JNK/ERK MAP kinase pathway which regulates the expression of steroidogenic enzyme genes in Leydig cells.