Figure 1.
Expression of Inhibin-α was significantly reduced in Wt1-deficient Sertoli cells.
Wt1 was inactivated in Sertoli cells by Tamoxifen induction. The expression of Wt1 and Inhibin-α was examined by Real-time PCR and Western blot. A, Wt1 expression was dramatically reduced in Wt1−/flox; Cre-ER™ sertolic cells after tamoxifen treatment. B, Compared to controls, the expression of inhibin-α was significantly decreased in Tamoxifen treated Wt1−/flox; Cre-ER™ Sertoli cells.
Figure 2.
The inhibin alpha promoter is transactivated by the Wt1 A and B isoforms (−KTS), but not the C and D isoforms (+KTS).
A, Schematic drawing of inhibin-α promoter. The open and filled ovals indicate the putative Wt1 binding sites. B, Real-time PCR results showed the elevated endogenous inhibin-α expression in primary Sertoli cells after transfection with Wt1A. C, The inhibin-α promoter was transactivated by the Wt1 A and B isoforms (−KTS), but not the C and D isoforms (+KTS). TM4 cells were transiently transfected with a luciferase reporter plasmid termed pGL3-Inhibin alpha (−745) and a Renilla luciferase reporter plasmid along with Wt1-expressing vectors. Luciferase activity was examined 36 h later. Compared to control cells, the luciferase activity was significantly increased in Wt1 A and B transfected TM4 cell, not in C and D isoforms transfected cells. (* p<0.05, Wt1A: −Exon5, −KTS; Wt1B: +Exon5, −KTS; Wt1C: −Exon5, +KTS; Wt1D: +Exon5, +KTS).
Figure 3.
Identification of the DNA sequence within the mouse inhibin-α promoter required for Wt1A-dependent transcriptional activation.
A, TM4 cells were transiently transfected with luciferase reporter constructs bearing different lengths of the mouse Inhibin alpha promoter sequence, and Renilla luciferase reporter plasmids together with an empty plasmid (pCMV-tag2b) or Wt1A expression vector. The filled and open ovals indicated putative Wt1 response elements (Wt1RE). B, The Wt1-dependent transactivational activity of inhibin-α promoter was significantly decreased after deletion of the ten base pairs (5′- GGC GGG AGT G -3′) sequence including part of the Wt1 consensus binding motif. C, Western blot showed the endogenous Wt1 protein expression in TM4 cells and primary Sertoli cells. TM4 cells were transiently transfected with the empty plasmid or with the expression plasmid for Flag-Wt1A. 48 h after transfection, cells were cross-linked with formaldehyde and cross-linked chromatin was sonicated followed by immunoprecipitation with anti-Flag antibody. Genomic DNA was purified from the immunoprecipitates and subjected to PCR using the primers as indicated in schematic drawing of Inhibin-α promoter and distal primers as negative control. A specific PCR product (black arrow) was amplified in Flag-Wt1A transfected cells with proximal primers, not with the distal primers. Normal mouse IgG served as a negative control.
Figure 4.
The inhibin-α promoter is synergistically activated by Sf1 and Wt1.
A, Schematic drawing of the inhibin-α promoter region. The filled oval and box indicate the putative Wt1 and Sf1 responsive elements. B, Co-transfection of Wt1 and Sf1 significantly activated the inhibin-α promoter compared to Wt1 or Sf1 alone; luciferase activity increased about 4-fold. In contrast, when the inhibin-α promoter luciferase reporter vector with a mutated Sf1 responsive element sequence (5′-TCA GGG CCA-3′ to 5′- TCA GTT CCA-3′), Sf1REM Inh-α Luc, was co-transfected with Wt1 and Sf1, the synergistic action between Wt1 and Sf1 was completely abolished.
Figure 5.
The inhibin-α promoter is synergistically activated by Sf1 and the Wt1 A and B, but not C and D isosforms, and Wt1 synergizes with Sf1 in a dose-independent manner.
A. Luciferase activity indicated that the inhibin-α promoter is significantly transactivated by Wt1 A and B and Sf1 co-transfection, but transfection of Sf1 and Wt1 C and D isoforms did not change the luciferase activity compared to the control. B. No difference in luciferase activity was noted when Sf1 was co-transfected with different doses of Wt1 A (10, 50, 100, 300, 500 ng).
Figure 6.
The Sf1 ligand-binding domain (residues 235–238) is essential for Wt1 and Sf1 synergistic activation of the inhibin-α promoter, but does not affect the interaction between Wt1 and Sf1.
A. Mutations of the Sf1 ligand-binding domain (residues 235–238) completely abolish the synergistic action of Wt1 and Sf1 on the inhibin-α promoter. B. Interactions between Wt1 and Sf1 proteins were assessed by co-immunoprecipitation; both wild type Sf1 and Sf1 (235–238 4A) interacted with Wt1 protein. Normal mouse IgG served as a negative control.
Figure 7.
Synergistic activation of the inhibin-α promoter by Wt1 and Sf1 is enhanced by Wnt signaling, but the inhibin-α promoter is not synergistically activated by Wt1 and Wnt signaling.
A, Compared to Wt1 and Sf1 co-transfected TM4 cells, co-transfection of constitutively activated β-catenin, S37A could significantly induced the luciferase activity. In contrast, S37A β-catenin alone did not promote the activity of inhibin-α promoter induced by Wt1 only. B, The same results were obtained when TM4 cells were treated with 40 mM LiCl treatment.