Dehydroepiandrosterone sulfate stimulates expression of blood-testis-barrier proteins claudin-3 and -5 and tight junction formation via a Gna11-coupled receptor in Sertoli cells

Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 µM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gna11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gna11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility

Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

Transforming growth factor-βs (TGF-βs) signal after binding to the TGF-β receptors TβRI and TβRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-β2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-β1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-β1 as well as TGF-β2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-β signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-β2 but not TGF-β1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-β1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-β2 increased it. Silencing of BG as well as TIMP3 reduced TGF-β2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-β2 signaling. In contrast, this effect was not observed with TIMP3/TGF-β1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-β2 signaling in Sertoli cells

Silencing expression of Gnα11 by means of siRNA.

<p>Cells were treated for 3 days with OptiMem plus Lipofectamine RNAiMAX alone (1) or OptiMem plus Lipofectamine RNAiMAX plus the siRNA negative control (2) or OptiMem plus Lipofectamine RNAiMAX plus the Gnα11-specific siRNA (3). Cells were then used to either isolate mRNA for RT-PCR, for immunofluorescence or for western blot experiments. (A) RT-PCR for the detection of GAPDH-, Gnα11- or Gnαq-specific mRNA. Effects on GAPDH mRNA/cDNA (430 bp) are shown on the left, results for Gnα11-specific mRNA/cDNA (917 bp) are shown in the center, and effects on Gnαq (688 bp) are shown on the right. (B) Detection of Gnα11 by immunofluorescence. The green fluorescence indicates Gnα11, and the blue refers to DAPI-stained nuclei. In both western blotting (A) and immunofluorescence (B) experiments, treatment of cells with Gnα11-specific siRNA (panel labeled “3”) abrogates Gnα11 expression. (C) Results of western blotting using an antibody against Gnα11 on lysates from cells treated with either nc-siRNA or Gnα11-siRNA. Effects on expression of actin (left panel) and Gnα11 (right panel, upper band, 42 kDa). The lower band also recognized by the antibody is Gnαq (40 kDa). All results shown are representative of n = 3 similar experiments.</p

DHEAS-stimulated expression of claudin-3 and claudin-5 proteins as detected by immunofluorescence.

<p>TM4 Sertoli cells were cultured to 80% confluence and were further incubated for 2 days in the absence or presence of 1 μM DHEAS. Nuclei were labeled with DAPI and appear blue; the Alexa fluor 488-labeled secondary antibody shows the localization of claudin-3 or -5. (A and D) Fluorescence in the absence of DHEAS; (B and E) Fluorescence after exposure of cells to 1 μM DHEAS. (C) Quantification and statistical analysis of results shown in panels (A) and (B); (F) Quantification and statistical analysis of results shown in panels (D) and (E) (in each case n = 45; means ±SEM; **p≤0.01).</p

DHEAS-induced activation of Erk1/2, CREB and ATF-1 detected in western blots.

<p>TM4 cells were treated for 120 min with the indicated concentrations of DHEAS. Proteins in cell lysates were then separated on SDS polyacrylamide gels and subsequently probed in a western blot using a monoclonal antibody against either total Erk1/2 (t-Erk1/2) (A), as a loading control, or phosphorylated (activated) Erk1/2 (p-Erk1/2) (B). The western blots in (A) and (B) show typical results for the Erk1/2 bands of 42/44 kDa. (C) Statistical analysis of Erk1/2 activation as a function of DHEAS concentration in several identical experiments in which the chemiluminescence was quantified by gel image analysis software (n = 4; mean ±SEM; *p≤0.05; **p≤0.01). (D) Detection of total actin served as loading control in further western blots for the detection of either phosphorylated CREB or ATF-1 (E). The western blots in (D) and (E) show representative results from several identical experiments using the indicated concentrations of DHEAS; the quantification and statistical analysis of these results are shown in (F) and (G) (n = 4; mean±SEM; *p≤0.05; **p≤0.01).</p

DHEAS-induced phosphorylation of Erk1/2 after silencing AR expression by siRNA.

<p>(A) AR expression, indicated by the green fluorescence of the secondary antibody, in TM4 cells exposed to negative control siRNA (nc-siRNA). (B) Abrogation of AR expression after treatment of the cells with AR-specific siRNA (AR-siRNA). In both A and B, nuclei are stained blue. (C) Western blot showing that the expression of AR in the presence of AR-siRNA is reduced by 94 ± 5% (n = 3). (D) At the same time expression of actin is not affected by AR-siRNA, indicating that the reduction of AR in (C) is specific and not due to an overall suppression of protein expression. (E) Erk1/2 phosphorylation in response to DHEAS after abrogation of AR expression with AR-specific siRNA. (F) Total Erk1/2 levels after treatment of cells with either nc-siRNA or AR-siRNA. The western blot shown was generated from the western blot shown in (E) which was first stripped of the original antibodies and then reprobed with appropriate antibodies to detect total Erk1/2. (G) Statistical analysis of Erk1/2 activation in the presence of either nc-siRNA or AR-siRNA (n = 3; mean ±SEM; **p≤0.01).</p

Western blot analysis of phospho-Erk1/2 after silencing Gnα11 expression by siRNA.

<p>(A) Blots showing the amount of total Erk1/2 and (B) phospho-Erk1/2 in cells pre-treated with OptiMem plus Lipofectamine RNAiMAX and negative control siRNA (nc-siRNA) or Gnα11-specific siRNA and incubated subsequently in the presence or absence of 1 μM DHEAS. (C) For statistical analysis, data were corrected for the amount of total Erk1/2 as shown in panel (A) (n = 4; means ± SEM; * = p≤ 0.01).</p

DHEAS-induced stimulation of claudin-3 and claudin-5 expression after silencing AR expression.

<p>The conditions are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150143#pone.0150143.g006" target="_blank">Fig 6</a>, with the exception that the cells were incubated with either negative control siRNA (nc-siRNA) or with AR-specific siRNA (AR-siRNA) to prevent its expression before treatment with DHEAS. Nuclei of the cells were labeled with DAPI and appear blue. (A-F) Stimulation of claudin-3 expression by DHEAS after incubation of cells with either nc-siRNA (A, B, and C) or AR-siRNA (D, E and F). (G-L) Stimulation of claudin-5 expression by DHEAS after treatment of the cells with either nc-siRNA (G, H, and I) or with AR-siRNA (J, K, and L). For the statistics shown in C, F, I, and L: n = 45; means ±SEM; **p≤0.01.</p

Detection of phospho-Erk1/2, phospho-CREB, and phospho-ATF-1 by immunofluorescence after silencing Gnα11 expression by siRNA.

<p>In all photomicrographs the green fluorescence indicates activated Erk1/2 or transcription factors CREB and ATF-1, and the blue fluorescence shows DAPI-stained nuclei. (A) All cells were treated with OptiMem plus Lipofectamine RNAiMAX and negative control siRNA (nc-siRNA). Cells in the leftmost three panels were treated with vehicle only (no DHEAS). The cells of the right-hand three panels were treated with 1 μM DHEAS. (B) Quantification and statistical analysis of results shown in panels in (A) (n = 90; means ±SEM; **p≤0.01). (C) All cells were treated with OptiMem, Lipofectamine RNAiMAX, and Gnα11-specific siRNA (Gnα11-siRNA). Cells in the leftmost three panels were treated with vehicle only (no DHEAS), and cells in the right-hand three panels were treated with 1 μM DHEAS. (D) Quantification and statistical analysis of results shown in panels in (C) (n = 90; means ±SEM).</p

Antibodies used in the study and their providers.

<p>Antibodies used in the study and their providers.</p