TGF-β1/ALK5 signaling represses cAMP-induced steroidogenic gene expression in Leydig cells.


<p>(<b>A and B</b>) The culture medium of purified mouse primary Leydig cells treated with 300 µM of 8-Br-cAMP and 5 ng/ml of TGF-β1 (A) and R2C cells treated with vehicle or 5 ng/ml of TGF-β1 (B) for 24 hours was collected for the measurement of testosterone levels by RIA. (<b>C and D</b>) The expression levels of steroidogenic genes in primary Leydig cells (C), which were treated with 300 µM of 8-Br-cAMP, 2.5 ng/ml of TGF-β1 and 10 µM SB431542 for 24 hours, and R2C cells (D), which were treated with 5 ng/ml of TGF-β1 for 24 hours, were analyzed by qRT-PCR. (<b>E</b>) The expression level of Tgfbr2 and Tgfbr1 was analyzed using total RNAs from primary Leydig, R2C and MA-10 cells by RT-PCR. (<b>F</b>) MA-10 cells were transiently transfected with the ALK5 (TD; constitutively active form) expression plasmid, along with an indicated reporter of the natural promoter, in medium containing 5% charcoal stripped FBS. Twenty four hours after transfection, the cells were treated with 300 µM of 8-Br-cAMP for 24 hours and harvested for luciferase assay. The pSV-β-gal expression plasmid was used as a control for transfection efficiency. The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

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