Developmentally regulated SMAD2 and SMAD3 utilization directs activin signaling outcomes

Abstract

Activin is required for testis development. Activin signals via phosphorylation and nuclear accumulation of SMAD2 and SMAD3. We present novel findings of developmentally regulated activin signaling leading to specific transcriptional outcomes in testicular Sertoli cells. In immature, proliferating, Sertoli cells, activin A induces nuclear accumulation of SMAD3, but not SMAD2, although both proteins become phosphorylated. In postmitotic differentiating cells, both SMAD proteins accumulate in the nucleus. Furthermore, immature Sertoli cells are sensitive to activin dosage; higher concentrations induce maximal SMAD3 nuclear accumulation and a small increase in nuclear SMAD2. Microarray analysis identified distinct transcriptional outcomes correlating with differential SMAD utilization and new activin target genes, including Gja1 and Serpina5, which are essential for Sertoli cell development and male fertility. In transgenic mice with altered activin bioactivity that display fertility phenotypes, Gja1 and Serpina5 are significantly altered. Thus, differential SMAD utilization in response to activin features during Sertoli cell maturation.

Figure 1. A: SMAD3, but not SMAD2, is detected in Sertoli cell nuclei of 6 dpp mouse testes

Immunohistochemical localization of SMAD2 and SMAD3 in 6 dpp mouse testes, indicated by brown staining and counterstained with Harris haematoxylin to stain chromatin blue. SMAD2 and SMAD3 were visible within the Sertoli cell cytoplasm (asterisk) and SMAD3 was readily detectable within the nuclei of Sertoli cells (white arrowheads) however little to no SMAD2 was apparent in Sertoli cell nuclei. The dotted black squares in each image are enlarged as insets. Spermatogonia: black arrowhead; Sertoli cell cytoplasm: white asterisk; Sertoli cell nucleus: white arrowhead; interstitial cells: I; peritubular cells: open black arrow. Scale bar represents 50 μm. B,C: SMAD2 and SMAD3 utilization by Sertoli cells is developmentally regulated. Detection of SMAD2 and SMAD3 by immunofluorescence in 6 and 15 dpp mouse Sertoli cells (Fig 1B and 1C respectively). Cells were cultured for 24 hours in serum-free medium then either left untreated or stimulated with activin A for 45 minutes. In 6 dpp Sertoli cells, SMAD2 sub-cellular localization did not change visibly following activin A treatment, whereas SMAD3 showed increasing nuclear localization with increasing activin concentrations. In 15 dpp Sertoli cells, both SMAD2 and SMAD3 proteins accumulated in the nucleus with 5 ng/ml activin A treatment. Scale bar = 25 μm. B′, C′: Quantitation of SMAD2 and SMAD3 nuclear accumulation in Sertoli cells Changes in subcellular localization of SMAD2 and SMAD3 in 6 dpp (n=3; Fig 1B′) and 15 dpp (n=2; Fig 1C′) Sertoli cells following activin A treatment (from Figure 1B and 1C respectively) was measured by analysis of confocal images, with an increase in the ratio of nuclear to cytoplasmic fluorescence (Fn/c) indicating accumulation of protein in the nucleus. In 6 dpp Sertoli cells, SMAD3 (black bars) exhibited nuclear accumulation at all concentrations of activin tested, to a point of saturation (50 ng/ml). SMAD2 distribution did not change at lower concentrations of activin A tested (grey bars), yet a small but significant increase in SMAD2 nuclear accumulation was measured when SMAD3 nuclear accumulation was saturated. In 15 dpp Sertoli cells, both SMAD2 and SMAD3 showed similar profiles of nuclear accumulation, with significant and maximal increases in SMAD2 and SMAD3 nuclear accumulation observed with 5 ng/ml activin A. Statistical significance was determined using one-way ANOVA and Tukey's post hoc test (P<0.05). Different letters signify significant differences between samples; error bars indicate SEM. ***P<0.001.

Figure 2. A: Activin A increased the amount of SMAD3, but not SMAD2, in nuclear lysates

Western blot analysis of SMAD2 and SMAD3 in nuclear and cytoplasmic extracts of 6 dpp Sertoli cells cultured for 24 hours in serum-free medium and either left untreated or stimulated with activin A at 5 or 50 ng/ml for 45 minutes (n=2). α-TUBULIN and LAMIN B1 signals indicate highly purified cytoplasmic and nuclear lysates, respectively. An increase in SMAD3 protein in the nucleus of cells treated with 5 and 50 ng/ml activin A was evident whereas there was no change in the amount of SMAD2 present in nuclei of treated cells. B: Both SMAD2 and SMAD3 are phosphorylated upon stimulation of immature Sertoli cells with activin A: Western blot analysis of SMAD2 (n=5) and SMAD3 (n=3) phosphorylation in 6 dpp Sertoli cells cultured for 24 hours in serum-free medium and either left untreated or stimulated with activin A at 5, 10 or 25 ng/ml for 45 minutes. ALPHA-TUBULIN was used as a loading control. Increased phosphorylation of SMAD2 and SMAD3 was readily detected upon treatment of cells with activin A, each exhibiting the same profile of phosphorylation across the range of concentrations tested. C: SMAD2 is phosphorylated but is not increased in nuclear lysates from Sertoli cells treated with 5 ng/ml activin A: Western blot analysis of phospho-SMAD2 and phospho-SMAD3 in nuclear and cytoplasmic extracts of 6 dpp Sertoli cells cultured for 24 hours in serum-free medium and either left untreated or stimulated with activin A at 5 or 50 ng/ml for 45 minutes (n=4). ALPHA-TUBULIN and LAMIN B1 indicate purity of cytoplasmic and nuclear lysates, respectively. Phosphorylated SMAD2 and phosphorylated SMAD3 were detected in cytoplasmic fractions of cells treated with both 5 and 50 ng/ml activin A. Phosphorylated SMAD2 was absent from nuclear fractions of cells treated with 5 ng/ml activin A but is readily detected in nuclear fractions from cells treated with 50 ng/ml activin A. Intense signals for phosphorylated SMAD3 were detected in nuclear fractions of 5 and 50 ng/ml activin A treated samples. D: Summary of developmentally regulated SMAD usage in Sertoli cells in response to activin A: Immature Sertoli cells exhibited a dose-dependent response to activin A stimulation in vitro, with nuclear accumulation of SMAD3, but not SMAD2 at lower concentrations of activin. At higher concentrations, both SMAD2 and SMAD3 accumulated in the nucleus. Nuclear accumulation of both SMAD2 and SMAD3 was observed in post-mitotic Sertoli cells at all concentrations of activin tested.

Figure 3

Candidate genes identified by microarray exhibiting a minimum two-fold change in expression in response to activin A were grouped according to the conditions under which they were regulated. Most genes were upregulated or downregulated under specific conditions consistent with the hypothesis that SMAD2 and SMAD3 have distinct activities in the regulation of gene expression in Sertoli cells at different developmental stages.

Figure 4. Quantitative analysis of several candidate activin A target genes in 6 dpp and 15 dpp Sertoli cells

Real Time PCR was used to validate the activin-regulation of several candidate activin target genes. 6 dpp Sertoli cells were treated for 6.5 hours with 5 (grey bars) or 50 ng/ml (black bars; bars span lowest and highest values measured) activin A and 15 dpp Sertoli cells were treated with 5 ng/ml activin A (white bars). Two independent cultures were analyzed and gene expression was compared to that measured in untreated cells (white bars), which was set at 1. Normalization to beta-actin or 18S generated equivalent results. Copy number of target genes was determined by the comparative threshold cycle method (ΔΔCT) using the Pfaffl method (Pfaffl, 2001). Fold-change in expression relative to untreated cells is depicted under bars (* P<0.05, ** P<0.01, ***P<0.001). A: mRNAs increased by activin A. Gja1, Pdgfb, Serpina5 and Tesc were all significantly upregulated in 6 dpp cells treated with 50 ng/ml activin A, but not in cells treated with 5 ng/ml. Pdgfb, Serpina5 and Tesc were significantly upregulated in 15 dpp cells following treatment with just 5 ng/ml; Gja1 exhibited a 1.9-fold increase relative to untreated cells but this did not reach significance. B: mRNAs decreased by activin A. Dhcr24, Cryl1 and Rarres1 were all significantly downregulated in 6 dpp cells treated with 5 ng/ml activin A, with greater downregulation observed upon treatment with 50 ng/ml. In 15 dpp cells, no change in expression of Dhcr24 was observed in response to activin whereas Cryl1 and Rarres1 were both significantly downregulated.

Figure 5. Tightly regulated activin production and Sertoli cell responsiveness to activin are required for normal testis develoment

Immature Sertoli cells preferentially transduce activin A signals via SMAD3. Insufficient exposure of Sertoli cells to activin during testis development delays the onset of fertility and reduces the expression of activin target genes, associated in vitro with insufficient SMAD3 nuclear accumulation. Excessive exposure to activin A, associated with nuclear accumulation of both SMAD2 and SMAD3, results in premature expression of activin target genes preceding the development of Sertoli cell-derived stromal tumours and infertility.