title: TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry, Bharathidasan University, Tiruchirappalli, 620 024, India.
authors.forename: Mohanraj
authors.initials: M
authors.lastname: Sadasivam
authors.forename: Balamurugan
authors.initials: B
authors.lastname: Ramatchandirin
authors.forename: Sivasangari
authors.initials: S
authors.lastname: Balakrishnan
authors.forename: Chidambaram
authors.initials: C
authors.lastname: Prahalathan
mesh_terms: D000818:Animals; D002460:Cell Line; D057137:DAX-1 Orphan Nuclear Receptor / Q000187:drug effects* / Q000235:genetics*; D055785:Gene Knockdown Techniques; D007985:Leydig Cells / Q000187:drug effects* / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D008297:Male; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013256:Steroids / Q000096:biosynthesis*; D013737:Testis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D057137:DAX-1 Orphan Nuclear Receptor; C539967:Nr0b1 protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013256:Steroids; D014409:Tumor Necrosis Factor-alpha; D048670:MAP Kinase Kinase 4