This study tested the hypothesis that inhibins act in an autocrine manner on Leydig cells using a pre-pubertal Leydig cell line, TM3, as a model of immature Leydig cells. The expression of Inha, Inhba, and Inhbb in TM3 cells was determined by RT-PCR and the production of the inhibin-alpha subunit was confirmed by western blot. Knockdown of Inha expression resulted in significant decreases in the expression of Leydig cell markers Cyp17a1, Cyp11a1, Nr5a1, and Insl3. Western blot showed that activin A, TGFβ1 and TGFβ2 activated SMAD2, and that knockdown of Inha expression in TM3 cells enhanced both activin A- and TGFβ-induced SMAD2 activation. SB431542, a chemical inhibitor of the TGFβ/activin type I receptors, blocked ligand-induced SMAD2 activation and the downregulation of Cyp17a1 expression. Our findings demonstrate that TGFβs and activin A negatively regulate steroidogenic gene expression in TM3 cells via ALK4/5 and SMAD2 and endogenous inhibins can counter this regulation.
Keywords: Inha knockdown; Leydig; SMAD2; Steroidogenesis; TM3 cells.
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